FOXJ1 mediates taxane resistance through regulation of microtubule dynamics

Domain: neuroscience
Published: 2026-02-14
Editorial outcome: minor_revision

Abstract
Docetaxel is the first-line chemotherapy for metastatic prostate cancer (PC), but clinically meaningful mechanisms of resistance remain to be established. Here we show, in an in vivo model of docetaxel resistant PC patient-derived xenografts, increased expression of genes that drive development of multi-ciliated cells including FOXJ1 and its effectors, many of which regulate microtubules (MTs). Mechanistically, FOXJ1 overexpression confers docetaxel resistance in vitro and in vivo, which is associated with decreased docetaxel-mediated MT bundling. Overexpression of a MT-associated FOXJ1-regulated gene (TPPP3) has similar effects. Conversely, FOXJ1 knockdown impairs basal MT function, enhances taxane binding to MTs, and increases docetaxel sensitivity. These results establish mechanistic causality between the FOXJ1 signaling axis, MT biology, and taxane resistance. Clinically, FOXJ1 gene amplification is increased in taxane-treated PC patients. Moreover, in the CHAARTED clinical trial of docetaxel combined with androgen deprivation for metastatic PC, higher baseline FOXJ1 is predictive of decreased survival in PC patients treated with docetaxel, further supporting clinical relevance. Together, these findings identify a previously unrecognized clinically impactful mechanism of taxane resistance whose exploitation could stratify patients who will not benefit from taxane treatment.

Introduction
Taxanes used in the clinic (docetaxel, paclitaxel, and cabazitaxel), either as single agents or in combination therapies, have efficacy in multiple solid tumors. In prostate cancer (PC), taxanes (docetaxel or cabazitaxel) are the only class of cancer chemotherapeutics that prolong survival as single agents in men with metastatic castration-resistant PC (mCRPC), defined as patients who have progressed after standard androgen deprivation therapy (ADT, surgical or medical castration)1,2. Moreover, a subset of men who become resistant to docetaxel may subsequently respond to the related taxane, cabazitaxel, consistent with the clinical efficacy of taxanes in this disease. In addition, in men with metastatic castration-sensitive PC, the combination of docetaxel with androgen receptor (AR) targeted therapy has shown unprecedented clinical efficacy, causing a paradigm shift in clinical practice3–6. Thus, docetaxel is not only used as standard-of-care treatment in men with mCRPC but is also increasingly used earlier, in combination with ADT, or with ADT plus androgen receptor (AR) pathway inhibitors (triple therapy), as the initial treatment in men with castration-sensitive PC. However, while docetaxel clearly enhances responses to ADT, the extent to which it enhances responses to ADT plus an androgen receptor (AR) pathway inhibitor in all or subsets of men remains unclear. Moreover, despite this central role of taxanes in PC and other cancers, there is a major gap in our understanding of clinically relevant mechanisms of taxane resistance7,8.

Taxanes act by binding to the \beta-subunit of the tubulin \alpha\beta heterodimer, stabilizing MTs and suppressing their dynamic properties, which are integral to their function during interphase and mitosis. Thus, taxanes cause aberrant mitotic arrest in cycling cells while also disrupting myriad other MT-dependent functions in interphase, such as translocation of nuclear proteins, including the AR in PC cells9,10. Mechanisms underlying taxane resistance, which inevitably emerge across all clinical settings, have been extensively studied in vitro. However, none of the multiple reported mechanisms of resistance have been shown to be clinically relevant. These include overexpression of drug efflux transporters such as ABCB111–15, tubulin mutations at the drug’s binding site, or overexpression of the TUBB3 tubulin isoform. They also include alterations in pathways downstream of MT stabilization that allow cells to survive prolonged mitotic arrest or disruption of MT dynamics during interphase, such as increased expression of PLK1, BCL2, or MCL1, lack of functional p53, upregulation of Notch, NF\kappaB activation, or increases in a GATA2-IGF2 network16–19. In PC, this may also include expression of ERG or of MT-independent AR splice variants20–22.

The absence of clinically faithful in vivo models of taxane resistance that recapitulate patients’ initial response followed by relapse has contributed to our inability to identify clinically relevant mechanisms of taxane resistance. Herein, we developed an in vivo docetaxel-resistant model using castration-resistant PC patient-derived xenografts (PDXs). Molecular characterization of the resistant tumors identified upregulation of FOXJ1 as a driver of taxane resistance, associated with decreased docetaxel-mediated MT bundling and mitotic arrest. FOXJ1 overexpression caused resistance to docetaxel in vitro and in vivo, while FOXJ1 knockdown impaired basal MT dynamics and sensitized to docetaxel, establishing causality. FOXJ1 has been well-established as a master transcription factor regulating the development of multiciliated cells, where it controls expression of multiple cilia-associated genes23–25, but previous studies have not implicated FOXJ1 in the regulation of MT dynamics in epithelial cells or in the mechanism of action of taxanes. The findings in this study support a broader role for FOXJ1 in non-ciliated cells, specifically in taxane resistance in PC and beyond.

Results
FOXJ1 expression is increased in docetaxel-resistant tumors
Docetaxel is the first-line chemotherapy used for PC treatment, but the mechanisms underlying clinical treatment resistance that inevitably occurs are poorly elucidated. To decipher in vivo molecular pathways mediating docetaxel resistance, we used two PDX models of castration-resistant PC (LuCaP35CR and LuCaP70CR). The PDXs were established in castrated male scid mice and then treated with cycles of docetaxel, using a clinically used schedule for PC (30 mg/kg, i.p., every 21 days). Both the LuCaP35CR and LuCaP70CR PDXs responded initially to docetaxel but relapsed after several cycles of treatment (Fig. 1A, B). We harvested resistant tumors 2 weeks after their last dose of docetaxel, along with control tumors of approximately the same volume that were never treated. We then performed RNA-seq and low-pass whole-genome sequencing (LP-WGS) on docetaxel-resistant and -sensitive tumors.

Analysis of differentially expressed genes in the LuCaP70CR PDXs showed a dramatic increase in mRNA encoding the drug exporter protein ABCB1/MDR1 (Fig. S1A). Previous studies have shown that ABCB1 gene amplification or overexpression can confer resistance to multiple large lipophilic drugs, including taxanes, which provided validation for our approach. However, the importance of this mechanism in patients remains to be established, and examination of genomic data from tumor biopsies from taxane-treated PC patients did not show an increased frequency of ABCB1 amplification as compared to tumors from patients who were not taxane treated (Fig. S1B). Interestingly, while increased expression of ABCB1 is generally mediated by gene amplification, by LP-WGS we found that the ABCB1 gene was already amplified in the initial LuCaP70CR PDX prior to docetaxel treatment (Fig. S1C). This amplification was presumably driven by prior therapy in the patient and indicates that the increased gene expression in the docetaxel-resistant PDXs was due to an epigenetic mechanism.

Analysis of differentially expressed genes in the second docetaxel-resistant PDX, LuCaP35CR, did not show increased ABCB1 (Fig. 1C). Instead, the most significantly increased gene was GMNC (geminin coiled-coil domain-containing), which encodes the transcription factor GEMC1 that initiates the development of multiciliated cells24–26. Along these lines, FOXJ1, a downstream target of GEMC1 and master regulator of ciliogenesis that controls expression of multiple MT-associated proteins23,25, was also significantly enriched. Consistent with these findings, examination of Gene Ontology Biological Process (GOPB) gene sets showed enrichment for gene sets directly related to the MT cytoskeleton, including Microtubule Bundle Formation and Motile Cilium Assembly (Fig. 1D, E). Amongst the enriched genes common to these gene sets was FOXJ1, as well as multiple genes encoding cilia-related proteins that are positively regulated by FOXJ1 in multiciliated cells (TPPP3, DNAAF3, SPEF1, ZMYND10, SPAG17, and SPAG1)23,25. Taken together, these results suggested that activation of a ciliogenesis program through the GEMC1 and FOXJ1 axis in the docetaxel-resistant LuCaP35CR xenografts contributed to docetaxel resistance. However, the functions of FOXJ1 have been most extensively studied in multiciliated cells and in cells that develop mono-motile cilia, and have not been well-studied in nonciliated cells23–25,27,28. To address this, we next assessed for correlations between FOXJ1 and these cilia-related genes by mining transcriptomic data from patients with localized PC in TCGA. We found significant correlations between FOXJ1, its upstream regulator GEMC1, and multiple of the FOXJ1 target genes that we also found increased in the docetaxel-resistant LuCaP35CR PDXs, such as ZMYND10, SPEF1, TPPP3, DNAAF3, SPAG17, and SPAG1 (Fig. 1F, Figs. S2, S3A). GEMC1 expression was also correlated with a subset of cilia-related genes, but the correlations were greater with FOXJ1, consistent with FOXJ1 directly regulating these genes (Fig. S3B, C). Together, these data support FOXJ1 regulation of MT-associated proteins in non-ciliated cells, including in PC cells.

To further assess FOXJ1 as a mediator of taxane resistance across tumor types we examined DepMap drug response data on approximately 300 cancer cell lines, which showed that higher FOXJ1 expression was correlated with docetaxel resistance across multiple tumor types (Fig. 1G, Fig. S4A). To assess the potential relevance of FOXJ1 and downstream genes in docetaxel resistance in PC patients, we examined the SU2C dataset of patients with advanced PC whose metastatic castration-resistant tumors were biopsied and analyzed by whole-exome sequencing29. Notably, tumors from patients who had been treated with a taxane (primarily docetaxel) had a significantly higher frequency of FOXJ1 gene amplification (10.88%) compared with tumors from patients who were not exposed to a taxane (5.45%) (Fig. 1H). FOXJ1 mRNA levels were also increased in the taxane-exposed patients, although the increase was not significant, which may reflect biopsies being obtained at variable times after treatment was discontinued (Fig. S4B).

FOXJ1 overexpression decreases docetaxel-induced MT bundling and confers docetaxel resistance in vitro
To further assess whether FOXJ1 plays a functional role in docetaxel resistance, we generated FOXJ1 stably over expressing LNCaP cells (Fig. 2A). Colony formation assays showed that FOXJ1 overexpressing (OE) LNCaP cells were less sensitive to docetaxel treatment than LNCaP cells containing empty vector control (EV) (Fig. 2B, C, S5A). A major effect of taxanes in vitro is impairment of mitotic spindle function, with subsequent mitotic arrest and apoptosis. Flow cytometry further showed that there was less accumulation of cells in G2/M in FOXJ1_OE than in EV control cells after docetaxel treatment, indicating that FOXJ1 overexpression could attenuate taxane-induced mitotic arrest (Fig. 2D, E). To further determine effects on mitosis, we examined phosphorylation of serine 10 on histone 3 (pH3S10) as an indicator of mitotic cells. Treatment with 10 nM docetaxel for 6 h caused a marked increase in pH3S10 in the control cells, but not in the FOXJ1 overexpressing cells, where 100 nM docetaxel was required to obtain comparable levels of pH3S10 (Fig. 2F).

Taxanes act by promoting tubulin polymerization leading to the formation of stable MT bundles in interphase cells. Based on our previous finding that docetaxel-resistant cells had decreased docetaxel-induced MT bundling30, we hypothesized that FOXJ1 overexpression might be acting by impairing docetaxel-driven MT polymerization. To test this hypothesis, we examined effects on soluble versus polymerized tubulin after docetaxel treatment. Interestingly, the fraction of polymerized tubulin (%P) under basal conditions was modestly increased in the FOXJ1 overexpressing cells (Fig. 2G, H). As expected, polymerized tubulin was greatly increased after docetaxel treatment in the control LNCaP EV cells. In contrast, docetaxel-driven tubulin polymerization was significantly impaired in FOXJ1-overexpressing cells. We also examined another taxane, cabazitaxel, which has efficacy in a subset of PC that becomes resistant to docetaxel. Notably, FOXJ1 overexpression also reduced MT aggregation in response to cabazitaxel (Fig. 2I). Together these results demonstrate that FOXJ1 overexpression confers docetaxel resistance by impairing the ability of the drug to effectively engage with and alter its target.

We also assessed the effects of FOXJ1 overexpression on the response to other agents that target tubulin by binding to distinct sites. FOXJ1 overexpression conferred resistance to each of these agents (colchicine, vincristine, epothilone A, and maytansine) (Fig. S6A). Notably, FOXJ1 overexpressing cells also had decreased sensitivity to olaparib, cisplatin, and carboplatin, although the effect was less marked than for the tubulin-targeted agents (Fig. S6B). The basis for this effect is not clear, but could be due to increased MT dynamics (see below) and subsequent increased drug export, increased response to DNA damage, or other mechanisms.

As noted above, FOXJ1 has been most studied as a driver of multiciliated cells. Therefore, we also assessed for cilia in FOXJ1 overexpressing cells. As a positive control, cilia could be readily detected by immunofluorescence of acetylated tubulin and centrin, a component of the centriole, in BEAS-2B lung bronchial epithelium cells (Fig. S7). In contrast, we did not find cilia or evidence of centriole amplification in LNCaP cells overexpressing FOXJ1.

FOXJ1 downregulation sensitizes to docetaxel in vitro
We then examined whether downregulation of endogenous FOXJ1 expression affects responses to docetaxel. We used two independent shRNAs to suppress FOXJ1 expression in LNCaP cells (Fig. 3A). Colony formation assays showed that the FOXJ1 knockdown (KD) LNCaP cells were more sensitive to docetaxel treatment than the control cells (Fig. 3B, Fig. S5C). We next examined FOXJ1 expression in a series of PC cell lines, and found the highest expression in DU145 cells (Fig. S8A). Interestingly, we also found that DU145 xenografts were relatively resistant to docetaxel (Fig. S8B). Knockdown of FOXJ1 in DU145 cells (Fig. 3C) increased their sensitivity to docetaxel in colony formation assays (Fig. 3D, Fig. S8C). Consistent with this result, the G2/M fraction was greater in the FOXJ1 KD cells than in the control cells after 24-h docetaxel treatment (Fig. 3E, F). Confocal microscopy of the MT network in the FOXJ1 KD versus the control shNeg cells indicated an increase in docetaxel-induced MT bundling in the FOXJ1 KD cells (Fig. 3G). To quantify this response to docetaxel, we used an algorithm that was developed and clinically validated to assess effective drug-target engagement (DTE) between taxanes and MTs, and subsequent increases in MT bundling, by measuring the microtubule fluorescence intensity (MFI) in mitotic and interphase MTs30. This confirmed increased MT bundling in the docetaxel-treated FOXJ1 KD cells (Fig. 3H). Together, these results show that the downregulation of FOXJ1 expression sensitizes cells to docetaxel.

One basis for this sensitization could be increased taxane binding in the FOXJ1 KD cells. To test this hypothesis, we used Flutax, a fluorescent taxane derivative, to directly assess taxane binding on interphase MTs. Permeabilized cells were treated for 5 minutes with Flutax, which was then washed away and Flutax-MT binding was followed by time-lapse live cell imaging (Fig. S9A). Flutax binding was greater at time 0 and in all subsequent times in the FOXJ1 KD cells, while the rate of Flutax dissociation from MTs was similar (Fig. 3I, J, Fig. S9B). The pattern of the MT cytoskeleton decorated by bound Flutax was also strikingly distinct, with the FOXJ1 KD cells displaying a more extensive labeling of the MT network. These findings indicate that FOXJ1 depletion increases the availability of taxane binding sites, likely increasing the drug’s association rate, but does not have a clear effect on the drug’s dissociation rate.

FOXJ1 regulates basal MT dynamics
Tubulin acetylation at lysine 40 (K40ac) in \alpha-tubulin is the sole post-translational modification that marks the MT lumen, accumulates on long-lived stable MTs, and alters the conformation of MTs to enhance their flexibility and prevent structural damage31,32. A hallmark of taxane activity is an increase in tubulin acetylation, consistent with taxane-mediated suppression of MT dynamics and an increase in MT stability. Notably, we found that acetylated-\alpha-tubulin (K40ac) was decreased upon FOXJ1 depletion, indicating that FOXJ1 and its transcriptional network may regulate MT polymerization dynamics and stability under basal conditions (Fig. 4A). As expected, docetaxel treatment caused a dose-dependent increase in acetylated \alpha-tubulin across all conditions. However, docetaxel caused a dramatic fold increase in acetylated-\alpha-tubulin in the FOXJ1 KD cells, up to 8.7 fold higher than their untreated counterparts, compared to only an 1.8-fold increase in cells expressing endogenous FOXJ1. These results are consistent with the increased taxane binding and sensitivity in the FOXJ1 KD cells, but also point to a previously unrecognized role of FOXJ1 in basal MT dynamics in non-ciliated cells.

To further assess the potential effects of FOXJ1 on MT dynamics, we transfected the FOXJ1 KD or control cells with fluorescently labeled EB1, a protein that binds to the plus ends of growing MTs. This generates distinct “comet tails” that represent the visible length of time an EB1 molecule remains bound to a growing MT plus end, and is used as a read-out of MT dynamics. Live-cell imaging of untreated versus docetaxel-treated cells, and tracking of the number of EB1 comets bound at the tips of individual growing MTs, did not reveal any changes caused by FOXJ1 depletion under basal conditions (Fig. 4B, C, Movie S1). As expected, taxane treatment decreased the number of EB1 comets in the control cells, consistent with taxane’s ability to suppress MT dynamics resulting in EB1 dissociation from MTs. Notably, this taxane-mediated decrease was more pronounced in the FOXJ1 KD cells. This result indicates that the taxane is targeting a greater fraction of growing MTs in these cells, and is consistent with the Flutax binding studies above.

While the baseline number of EB1 comets was not detectably altered by FOXJ1 depletion, there was a decrease in EB1 comet length, suggesting an effect on MT growth rates, as the comet length is directly related to the speed at which the MT is growing (Fig. 4D, Fig. S10)33. Next, we assessed MT growth rates, using live cell time-lapse microscopy to calculate EB1 comet speed. The FOXJ1 KD cells had a decrease in EB1 comet speed relative to control cells under basal conditions and in response to taxane (Fig. 4E, Movie S2). In agreement with the decrease in MT growth rates in FOXJ1 KD cells, we also observed a concomitant decrease in the MT path/trajectory length calculated by quantifying the length of time that EB1 remained bound at MT plus ends, marking growing MTs (Fig. 4F, G, Movie S3). Taxane treatment markedly decreased the MT trajectory length, with a more pronounced effect in FOXJ1 KD cells, consistent with their enhanced taxane sensitivity. Together, these results indicate that FOXJ1 depletion modulates basal MT dynamics by decreasing MT growth rates while also augmenting MT stabilization in response to taxane treatment.

Conversely, these data indicate that increased FOXJ1 may enhance basal MT dynamics in interphase cells. To test this, we examined the androgen-stimulated nuclear import of AR, which, similar to other nuclear proteins, is MT-dependent. As expected, AR nuclear localization in LNCaP cells was increased by treatment with a potent agonist (R1881) (Fig. S11). Notably, this nuclear localization was increased in LNCaP cells overexpressing FOXJ1, consistent with enhanced MT dynamics. However, the taxane-mediated decrease in AR nuclear localization was not mitigated in the FOXJ1 overexpressing cells. One basis for this persistent suppression of AR nuclear localization by taxane may be dependence on a distinct subset of MTs and MT-associated proteins that have greater sensitivity to taxanes, but further studies are needed to assess this or other mechanisms.

FOXJ1 overexpression confers resistance to docetaxel in vivo
To determine if increased FOXJ1 can confer docetaxel resistance in vivo, we used the FOXJ1 OE versus EV control LNCaP cells to establish subcutaneous xenografts. When tumors reached approximately 500 mm3, mice were treated with 30 mg/kg docetaxel. FOXJ1 overexpression did not alter growth prior to docetaxel treatment, but markedly impaired the response to the initial dose of docetaxel on day 9, and the second dose on day 30 (Fig. 5A). Immunoblotting of tumors confirmed FOXJ1 overexpression in the OE versus EV control tumors (Fig. 5B). To assess for mechanisms of resistance, additional FOXJ1 OE and EV control xenografts were established and harvested 3 days after a single treatment with docetaxel or vehicle control. Examination of representative sections stained for tubulin indicated that the number of abnormal mitotic figures in response to docetaxel was decreased in the FOXJ1 OE tumors (Fig. 5C).

We then again measured MFI to quantify effective drug-target engagement (DTE) between taxanes and MTs in mitotic and interphase MTs30. Interestingly, MFI under basal conditions was higher in the FOXJ1 overexpressing tumors (Fig. 5D), consistent with the higher basal tubulin acetylation levels in control versus FOXJ1 depleted cells (Fig. 4A). In contrast, while docetaxel significantly increased DTE (higher MFI) in the control tumors, the increase in the FOXJ1 overexpressing cells was less marked and did not reach statistical significance. We also analyzed tumors from OE and EV mice sacrificed at ~3 days and ~10 days after the second dose of docetaxel. This analysis similarly showed increased DTE in the EV control tumors at 3 days versus 10 days after treatment, but not in the FOXJ1 overexpressing tumors (Fig. 5E). These findings further indicate that while FOXJ1 overexpression may increase MT stability under basal conditions, it mitigates taxane-induced MT stabilization.

We further examined another group of FOXJ1_OE versus EV xenografts by RNA-seq prior to therapy to identify pathways that may be increased by FOXJ1. The most significantly enriched Hallmark gene set was Mitotic Spindle (FDR 16%) (Fig. S12A), suggesting that a subset of FOXJ1-regulated genes may contribute to spindle formation. Analysis of the Gene Ontology Biological Process gene sets showed enrichment for one cilium-related gene set (Regulation of Cilium Assembly, FDR 59%), but no highly enriched gene sets (FDR<25%) (Fig. S12B). Notably, a substantial fraction of the genes (229 genes, 29%), that were increased in the docetaxel-resistant LuCaP35CR xenografts (791 genes total) were also increased in the FOXJ1_OE xenografts (Fig. 5F). This overlap supports the conclusion that increased FOXJ1 was modulating the transcriptome and contributing to docetaxel resistance in the LuCaP35CR xenografts. Consistent with the established function of FOXJ1 as a regulator of ciliogenesis, these 229 genes were most associated with positive regulation of cilium assembly (Fig. 5G).

TPPP3 is regulated by FOXJ1 and its overexpression confers resistance to docetaxel
We hypothesized that taxane resistance is mediated by FOXJ1 through the downstream genes it regulates. We recently reported that expression of an alternatively spliced short isoform of CLIP170 contributed to taxane resistance in gastric cancer34, but did not find expression of this isoform in cells overexpressing FOXJ1 (Fig. S13). TPPP3 (tubulin polymerization promoting protein 3) is a microtubule-associated protein and downstream target of FOXJ1 in multiciliated cells, and was increased in the docetaxel-resistant LuCaP35CR PDXs. Moreover, we confirmed that TPPP3 protein was increased by FOXJ1 in the FOXJ1_OE LNCaP cells (Fig. 6A). To further assess the potential role of TPPP3 in docetaxel resistance, we generated TPPP3 overexpressing LNCaP cells (Fig. 6B) and performed in vitro functional studies. Flow cytometry showed that TPPP3 overexpression mitigated the docetaxel-mediated increase in G2/M fraction (Fig. 6C). Tubulin polymerization assays showed decreased accumulation of polymerized tubulin (%P) in TPPP3_OE LNCaP after docetaxel treatment compared to EV control (Fig. 6D, E). These in vitro results indicated that TPPP3, similar to FOXJ1, could impair docetaxel-driven MT polymerization.

To determine whether increased TPPP3 could confer docetaxel resistance in vivo, mice bearing TPPP3_OE, FOXJ1_OE, or EV control xenografts were treated with 30 mg/kg docetaxel on day 0 and day 14. Consistent with the results in Fig. 3A (carried out in an independent cohort), overexpression of FOXJ1 impaired the response to docetaxel (Fig. 6F). The overexpression of TPPP3 similarly diminished the response to docetaxel, although to a lesser degree, suggesting that other FOXJ1 downstream targets also contribute to drug resistance. To assess the basis for resistance, we established additional LNCaP_TPPP3 and EV xenografts and harvested them on day 3 after an initial docetaxel treatment, and did fluorescence imaging for tubulin (Fig. 6G). This indicated a decrease in docetaxel-driven MT bundling and multipolar mitotic spindle formation in the LNCaP_TPPP3 versus EV control cells, which was confirmed by measurement of MFI (Fig. 6H). Together these findings indicate that FOXJ1 mediates docetaxel resistance, at least in part, through increased TPPP3.

FOXJ1 expression increases early in response to docetaxel
We initially examined the LP-WGS data to determine the basis for increased FOXJ1 expression in the docetaxel-resistant LNCaP35CR PDXs, which did not show FOXJ1 gene amplification (Fig. S14A). To determine whether taxane-mediated disruption of MT dynamics may feedback to directly alter FOXJ1 expression, we examined FOXJ1 expression in response to docetaxel in vitro. Notably, treatment with 10 nM docetaxel caused a time dependent increase in FOXJ1 mRNA after ~24 h that continued for up to 96 h, and treatment with various doses of docetaxel for 96 h caused a dose dependent increase in FOXJ1 mRNA in LNCaP cells (Fig. S14B, C). Similar results were also observed in CWR22Rv1 cells (Fig. S14D). Docetaxel treatment similarly increased FOXJ1 protein in LNCaP and CWR22Rv1 cells cultured under standard 2D conditions (Fig. S14E) or under 3D conditions (Fig. S14F). Consistent with induction of FOXJ1, docetaxel also increased the expression of TPPP3 in LNCaP and CWR22Rv1 cells (Fig. S14G, H).

The time course for FOXJ1 induction suggested that its expression may be cell cycle regulated and that the increased FOXJ1 may be indirect and due to docetaxel-mediated mitotic arrest. To test this hypothesis, we initially assessed whether FOXJ1 was increased in subconfluent versus confluent cultures. Significantly, FOXJ1 (and TPPP3) was higher in confluent cultures, indicating it is not increased in cycling cells (Fig. S15A). We next used FACS to isolate untreated DU145 cells in G0/G1 and G2/M. Notably, FOXJ1 mRNA and protein were expressed at comparable levels in G0/G1 versus G2/M cells (Fig. S15B, C). We similarly analyzed LNCaP cells and found comparable levels of FOXJ1 mRNA in G0/G1 and G2/M fractions (Fig. S15D). These observations indicated that the docetaxel-induced increase in FOXJ1 expression was not due to the accumulation of cells in G2/M.

Notably, FOXJ1 is induced by GEMC1 (encoded by GMNC) in multiciliated cells, and GEMC1 was increased in the docetaxel-resistant LuCaP35CR cells. Moreover, FOXJ1 expression in PC clinical samples is correlated with GEMC1 (see Fig. S3). Therefore, we next assessed GEMC1 expression in response to docetaxel and found it was induced within 12 h by docetaxel, indicating that its expression is responsive to disruption of microtubule dynamics and is consistent with GEMC1 driving the subsequent expression of FOXJ1 (Fig. S16A). GEMC1 expression in multiciliated cells is initiated by a decrease in Notch signaling. Notably, we found in the TCGA PC gene dataset that GEMC1 expression was highly negatively correlated with expression of the Notch-induced genes HES1 and HEY1, suggesting that docetaxel may be inducing GEMC1 by decreasing Notch signaling (Fig. S16B). However, we did not find a decrease in HES1 or HEY1 expression in response to docetaxel, indicating that the mechanism is not decreased Notch (Fig. S16C). Overall, these results support a feedback mechanism through which cells respond to docetaxel by increasing GEMC1 and downstream FOXJ1. However, while this may contribute to intrinsic resistance in some tumors, acquired resistance is presumably dependent on further adaptations in the expression of a subset of genes downstream of FOXJ1.

Higher FOXJ1 in patient tumors is predictive of docetaxel resistance
We next addressed whether there was an association between FOXJ1 expression and tumor behavior. In the TCGA cohort of untreated primary PC, there was no difference in survival in men with tumors expressing FOXJ1 above versus below the median (Fig. S17A). There was also no significant difference when the analysis was limited to higher-grade tumors (Gleason 8 or above) (Fig. S17B), although there was a trend toward decreased survival in the upper versus lower quartile of FOXJ1 expression (p=0.098) (Fig. S17C). Conversely, another primary PC cohort showed a trend toward increased survival in the tumors with above the median FOXJ1 expression (Fig. S18A), and in tumors in the upper versus lower quartile of FOXJ1 expression (Fig. S18B) (p=0.094 and 0.30, respectively) (33). We also analyzed the SU2C cohort of metastatic castration resistant PC (32), which showed a trend towards decreased survival in tumors with above the median (Fig. S19A) or upper quartile of FOXJ1 expression (Fig. S19B) (p=0.14 and 0.15, respectively). Consistent with FOXJ1 not being prognostic, its expression was not correlated with genomic alterations in the tumor suppressor genes RB1, TP53, or PTEN (Fig. S20).

To determine whether FOXJ1 expression may be predictive of response to docetaxel we examined data from the CHAARTED clinical study, in which patients with metastatic castration-sensitive PC were randomized to androgen deprivation therapy (ADT) versus ADT combined with six cycles of docetaxel3. Whole transcriptome data from pretreatment tumors, in conjunction with treatment and survival data, were available for analysis from a subset of patients35. Similar to the overall cohort, this subset of men showed highly significant improvement in progression-free survival (PFS) and overall survival (OS) from the addition of docetaxel, indicating that this subset of patients is representative of the entire patient cohort (Fig. S21A, B). Interestingly, analysis of the overall cohort (independent of treatment) showed that higher FOXJ1 expression (above the median or upper quartile) was weakly associated with improved PFS and OS, although the differences were not significant (Fig. S22A–D). In the ADT alone cohort, FOXJ1 expression above the median had a stronger trend toward improved PFS and OS (p=0.18 and 0.19, respectively) (Fig. S23A, B). In contrast, the trend was reversed in the combination arm, with FOXJ1 expression above the median associated with decreased PFS and OS (p=0.17 and 0.40) (Fig. S23C, D).

We similarly assessed outcomes in tumors with FOXJ1 expression in the upper versus lower quartiles. Higher FOXJ1 expression was again associated with improved PFS and OS in the ADT alone arm (p=0.067 and 0.024) (Fig. S24A, B), and with decreased PFS and OS in the combination arm (p=0.13 and 0.13) (Fig. S24C, D). A final comparison was between tumors with FOXJ1 expression in the upper 25% versus the bottom 75%. Higher FOXJ1 in the ADT alone arm was again associated with improved PFS and OS, which was significant for OS (p=0.036) (Fig. 7A, B). High FOXJ1 in the combination arm of ADT plus docetaxel was significantly associated with decreased PFS and OS (p=0.0053 and 0.052) (Fig. 7C, D). Notably, comparison of the arms showed dramatic PFS and OS benefits to addition of docetaxel in tumors with lower FOXJ1 expression, but no evident benefit in the tumors with high FOXJ1 (Fig. 7E, F).

We carried out a similar analysis for correlations between OS in CHAARTED and expression of RB1, PTEN, and TP53, which did not reveal any correlation (Fig. S25). We similarly did not find an association between OS and expression of ABCB1. These findings are consistent with our mechanistic studies and strongly support the conclusion that increased FOXJ1 is a clinically significant mediator of taxane resistance, and suggest that high FOXJ1 expression may have value as a biomarker to identify patients who are less likely to benefit from the addition of docetaxel to ADT.

Methods
Study approval
All animal experiments were approved by the BIDMC Institutional Animal Care and Use Committee (IACUC Protocol #051-2022-25) and were performed in accordance with institutional and national guidelines. All mice used were male as PC only occurs in males. All mice were sacrificed if tumors exceeded 2000 mm3.

Reagents
Docetaxel (T1034), cabazitaxel (T2543), vincristine (T6721), colchicine (T0320), maytansine (T21351), epothilone (T6490), olaparib (T3015), cisplatin (T1058), and carboplatin (T1564) were purchased from TargetMol for in vitro cell culture use. For the in vivo study, docetaxel was obtained from Selleck Chem USA and prepared in saline containing 5% DMSO, 30% PEG300, and 5% Tween 80 at a concentration of 2.0 mg/mL.

Plasmids and cell lines
FOXJ1 overexpression (FOXJ1_OE), TPPP3 overexpression (TPPP3_OE), and empty vector (EV) plasmids, which were constructed on a lentivirus-based vector (pReceiver-Lv241), were purchased from GeneCopoeia, Rockville, MD. The set of FOXJ1 shRNA plasmids, including TCRN0000420413 (shRNA-01), TCRN0000015305 (shRNA-02), and TCRN0000015305 (shRNA-03), with a non-target scramble, was purchased from Sigma-Aldrich. LNCaP, DU145, CWR22Rv1, and 293T cells were obtained from ATCC (Manassas, VA), authenticated by short tandem repeat profiling, and were free of mycoplasma.

Generation of cell lines with stable EB1
Approximately 1 \times 106 cells of the DU145 shNeg and DU145 FOXJ1 KD (shRNA_01) cell lines were infected with EB1-EGFP lentiviral particles (VectorBuilder Cat. # VB230531-1518prn) at a multiplicity of infection of 1. Clones were picked using cloning cylinders, grown individually, and screened by live microscopy. Specific clones showing EB1-EGFP expression were subsequently used with the Tubulin Tracker Deep Red (Invitrogen, Cat. # T34077) experiment to measure the speed, length, and trajectory of EB1 comets.

Patient-derived xenograft generation and treatment
Subcutaneous castration-resistant LuCaP35CR and LuCaP70CR PDX tumors were from Dr. Eva Corey (University of Washington, Seattle, WA)39. They were propagated in castrated male mice (6-8 week old ICRSC-M, IcrTac:ICR-Prkdcscid mice from Taconic) to generate an in vivo model of docetaxel resistance. Tumor size was assessed by caliper measurement. Treatment was initiated when the tumor volume reached 500 mm3. Mice were treated with 30 mg/kg docetaxel every 3 weeks by i.p. injection until they reached ~1500–2000 mm3. One last dose was then administered, and tumors were harvested two weeks after this last dose. For all experiments the light cycle was a 14-h light/10-h dark cycle. Temperature was 65–75 °F (~18–23 °C) with 40–60% humidity.

LNCaP xenograft generation and treatment
Male nude mice (Taconic, NCRNU, age ~6 weeks) were injected subcutaneously with ~2 \times 106 cells from LNCaP cell lines stably overexpressing FOXJ1 or EV, which were suspended in PBS containing 50% Matrigel (Corning Basement Membrane Matrix, CLS 354234). Tumor size was assessed by caliper measurements. Treatment was initiated when tumors reached 500 mm3. Mice were treated with vehicle or 30 mg/kg docetaxel, i.p., on Day 9 and Day 21. Three tumors from the EV and FOXJ1_OE groups were harvested 3 days after the first treatment (Day 9). The remaining tumors were harvested at 3 days after the second treatment (Day 21). A portion of the tumors was frozen and embedded in Tissue-Tek O.C.T. compound for RNA sequencing, while the rest of the tumors were fixed in formalin and preserved in paraffin for immunofluorescence. A similar protocol was used to assess xenografts generated from LNCaP cells overexpressing TPPP3.

RNA sequencing/low pass whole genome sequencing
Tumor tissue frozen blocks were sliced into 30 \mu m thick sections, totaling seven sections, and the first and seventh sections were stained with H&E. Once tumor quality was confirmed through H&E staining, the tissue sections were processed to extract RNA and DNA using the Allprep DNA/RNA mini kit (Qiagen, Q33238). RNA samples underwent RNA sequencing at GENEWIZ or Novogene, ensuring a minimum of 20 million read pairs per sample. DNA samples were sent to BGI America for low-pass whole-genomic sequencing.

Western blot
Cells were lysed on ice using RIPA buffer containing phosphate and proteasome inhibitors (Roche). Samples were run on SDS-PAGE gel 4–15% (BioRad), and transferred to nitrocellulose membrane using Bio-Rad Trans-Blot Turbo Transfer System. Primary antibodies used include anti-GAPDH (Cell Signaling Technology, #2118, 1:3000), anti-Vinculin (Santa Cruz, sc-73614, 1:5,000), anti-Acetyl-\alpha-Tubulin (Cell Signaling Technology, #5335, 1:3000), anti-\alpha-Tubulin (Abcam, ab7291, 1:4000), anti-FOXJ1 (R&D Systems, AF3619, 1:100), anti-TPPP3 (Thermo Fisher, PA5-24925, 1:100), anti-phospho-Histone3 (ser10) (Cell Signaling Technology, #9701, 1:1000), anti-CLIP-170 (clone: E-8, Santa Cruz, 1:500). Secondary antibodies used include Anti-Rabbit-HRP conjugated antibody (Promega, W401B, 1:5000), Anti-Mouse-HRP conjugated antibody (Promega, W402B, 1:5000), and Anti-Goat-HRP conjugated antibody (R&D systems, A15999, 1:2000).

qPCR
RNA was extracted using RNeasy mini kit (Qiagen). The qPCR reactions were prepared using TaqMan™ RNA-to-CT 1-Step Kit and run on ABI’s step one plus 96-well real-time qPCR system. Taqman probes were ordered from Thermo Fisher including FOXJ1 (Hs00230964_m1), TPPP3 (Hs00372228_g1), GMNC (Hs04970928_m1), HES1 (Hs00172878_m1), HEY1 (Hs05047713_s1), and GAPDH control (4326317E).

Tubulin polymerization assay
Tubulin polymerization assay was performed as described22,40. Briefly, cells were plated in 6-well plates and then treated with different concentrations of either docetaxel or cabazitaxel overnight. After the treatment, cells were washed with pre-warmed PBS and lysed at 37 °C for 10 min with 0.1 ml Low Salt buffer (20 mM Tris-HCl pH 6.8, 1 mM MgCl2, 2 mM EGTA, 0.5% NP-40) supplemented with protease inhibitor cocktail (Roche). The lysates were centrifuged at 15,000 r.p.m. for 5 min at room temperature. The supernatants containing soluble tubulin (S) were transferred to another tube, separating them from the pellets containing polymerized tubulin (P). The pellets were resuspended into Low Salt Buffer and sonicated. Both supernatants and sonicated pellets were subjected to a Western blot.

Flow cytometry
PC cells were collected and fixed with ethanol at 4 °C, followed by PBS washing and Propidium Iodide (BioLegend) staining. Data were acquired with CytoFLEX LX (Beckman), and results were analyzed with FlowJo.

Colony formation
A total of 5 \times 105 PC cells were allocated into each well of 6-well plates and cultured overnight to allow attachment. The cells were then treated with DMSO vehicle or the indicated drugs for 7 days, after which the drugs were washed out and the cells were cultured in regular media for another 1-3 weeks until colonies were visible. The colonies were washed with PBS, fixed by pre-chilled methanol at -20 °C for 10 minutes, and followed by staining with crystal violet (Sigma Aldrich) solution (0.5% in 25% methanol) for 4 h at room temperature. After removing the crystal violet solution, the colonies were washed with distilled water 10 times until the background became clear. The colonies containing over 50 cells were counted. Individual colonies were quantified using ImageJ. For wells with confluent colonies, colony area and intensity were measured instead, using the Image J plugin ColonyArea41.

Immunofluorescence
Formalin-fixed, paraffin-embedded (FFPE) xenograft tumor sections were heated at 60 °C for 30 min prior to staining. Sections were deparaffinized in xylene, rehydrated through a graded ethanol series, and rinsed in phosphate-buffered saline (PBS). Heat-induced antigen retrieval was performed for 30 min using EnVision FLEX Target Retrieval Solution (High pH; Dako, DM828), followed by permeabilization with 0.3% Triton X-100 in PBS. Non-specific binding was blocked by incubation in 10% goat serum prepared in Tris-buffered saline (TBS). Microtubules were immunolabeled using a rat monoclonal anti-beta-tubulin antibody (clone YL1/2; EMD Millipore, Mab1864-I) diluted 1:100 in blocking buffer. After primary antibody incubation, sections were washed in TBS and incubated with Alexa Fluor 488–conjugated goat anti-rat secondary antibody (Thermo Fisher Scientific, A-11006) at a dilution of 1:500 for 1 h at room temperature. Slides were subsequently washed in TBS and counterstained with DAPI. Confocal images were acquired using a Zeiss LSM700 microscope equipped with a \times 63/1.4 NA oil-immersion objective (Zeiss, Germany). For assessment of taxane–microtubule interactions, Flutax residence time assays were performed on native cytoskeletons34. Cells were incubated with 1 \mu M FITC-conjugated paclitaxel (Flutax-2; Thermo Fisher Scientific, P22310) for 5 min at 37 °C, followed by rapid drug washout. Samples were immediately rinsed with PEMP buffer, and time-lapse imaging was conducted using a Zeiss spinning disk confocal microscope and a Nikon super-resolution microscope.

Native cytoskeleton imaging
DU145 shNeg and DU145 FOXJ1 KD (shRNA_01) PC cells were permeabilised with 0.5% Triton X-100 for 60 seconds, washed, and then treated with FITC-conjugated paclitaxel (Flutax-2) at a concentration of 1 \mu M for 5 minutes to label the microtubules, as previously described42. After treatment, the cells were washed to remove excess Flutax-2, and imaging was performed over a total duration of 90 minutes using a Zeiss spinning disk confocal microscope. Representative images of Flutax-2-labeled microtubules in native cytoskeletons from these cells were taken at time points 0, 15, 30, 60, and 90 minutes after washout. The fluorescence intensity of tubulin tracker was quantified using Fiji software43.

Immunofluorescence staining and imaging for cilia
BEAS-2B cells, a human bronchial epithelial cell line, were used as a positive control for cilia staining and were processed in parallel with LNCaP cells stably overexpressing FOXJ1 (LNCaP FOXJ1 OE). Cells were initially plated in complete medium containing 10% fetal bovine serum (FBS). After allowing the cells to attach overnight, the medium was replaced with serum-free medium to induce serum starvation, which was maintained for an additional three days prior to fixation. Following serum starvation, cells were fixed and stained for acetylated \alpha-tubulin, a well-established marker of stabilized microtubules in motile cilia. In addition, cells were stained with an anti-centrin antibody, which labels centrioles and basal bodies and was used in this context to identify the ciliary base and assess centriole localization. Nuclear DNA was counterstained with DAPI. Immunofluorescence imaging was performed using a Nikon structured illumination super-resolution microscope.

EB1 imaging and quantification
DU145 shNeg and DU145 FOXJ1 KD (shRNA_01) PC cells stably expressing EB1-EGFP cells were washed and then treated with Tubulin Tracker Deep Red (Invitrogen, Cat. # T34077) at a concentration of 1 \mu M for 5 minutes to label the microtubules. After treatment, the cells were washed to remove excess tubulin tracker, and movies were acquired every 0.5 sec using a 60X lens in a Nikon Eclipse Ti2-E Inverted Microscope Yokogawa Spinning Disk Confocal System CSU-W1 SoRa. The fluorescence intensity of tubulin tracker and EB1 comets was quantified using the Nikon Elements software.

The speed of EB1 comets was analyzed using Nikon Elements software. EB1 movies were captured at a rate of one frame every 0.5 seconds for a total duration of 10 seconds. To accurately assess EB1 dynamics, areas within the leading edge of the cell were selected for quantification. These regions exhibited a relatively low density of EB1-EGFP staining and low background fluorescence. After selecting an area in the leading edge, the denoise function of the software was applied to reduce noise. Following this, the rolling ball algorithm was utilized to further enhance image quality. The defined threshold function was then set up to ensure consistent detection of EB1 comets. Using the bright spot function of the software, EB1 comets were identified and marked. The following parameters were measured: speed, number of comets, and EB1 trajectory. These measurements were conducted on hundreds of cells for each experimental condition.

The length of EB1-EGFP comets was measured in the leading edge of the cells, with a single time frame selected for this measurement. The same protocol was followed as previously described: the denoise function of the software was applied to reduce noise, followed by the application of the sharpen and rolling ball algorithms to enhance image quality. Subsequently, the threshold was defined to select all visible EB1 comets in the field. The border segmentation function was used to separate individual comets. The length of each EB1 comet was then computed and quantified using the software. To generate a bar graph representing the number of comets for each EB1 comet length, the automated measurement results function was utilized.

Image analysis and quantitative assessment of effective drug-target engagement
We previously developed a quantitative measure of effective drug-target engagement (DTE) as a marker of taxane response or resistance, by developing a scoring algorithm to quantify MT fluorescent intensity following indirect immunofluorescence and confocal microscopy imaging30. Briefly, tubulin immunofluorescence microscopy in cells or tissues was performed under identical conditions and the extent of a tubulin fluorescence intensity was quantified by the number of pixels in the top quartile of integrated intensity and normalized by the total cell count, as determined by the DAPI nuclear stain. This scoring algorithm was validated in biopsies from patients with PC following taxane treatment, where we found that high DTE correlated with taxane sensitivity and vice versa30.

Statistics and reproducibility
All the experiments were randomised, and no data were excluded from the analyses. Mice were randomly allocated into treatment groups, and tumor proliferation were displayed as curves with each point representing mean \pm SEM. Two-way ANOVA tests with were applied to the animal experiments. Student’s t-test were applied to colony formation, cell cycle experiments, and tubulin polymerization assay. Microtubule fluorescence intensity (MFI) quantitation, as a read out of DTE, is displayed as a dot plot with mean \pm SEM values. Mann–Whitney tests were applied to the details of each analysis, and significance thresholds are listed in each figure. Nuclear AR single-cell intensity measurements were analyzed using one-way ANOVA with Dunnett’s post hoc test for within-cell-line comparisons and two-tailed independent-samples t-tests for comparisons between LNCaP EV and LNCaP FOXJ1 OE cells. GraphPad Prism 10 was used for the analyses. Overall significance threshold is p<0.05. Gene expression analysis of TCGA primary PC samples (Firehose Legacy) were analyzed on cBioPortal44. Whole genome sequencing data were visualized using the Integrative Genomics Viewer45. Correlations between FOXJ1 and docetaxel sensitivity across cell lines was determined using the Cancer Dependency Map (DepMap)46.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is an innovative manuscript coming from Dr. Balk's group, focusing on the mechanistic dissection of therapeutic resistance to microtubule-targeting (taxane) chemotherapy in in-vivo relevant models of prostate cancer. The authors report novel results establishing the mechanistic link between the FOXJ1 signaling axis, microtubule dynamics and taxane resistance. The focus on FOXJ1 as the protagonist takes significant translational dimensions as clinically FOXJ1 gene amplification was elevated in taxane-treated prostate cancer patients. Moreover, in the CHAARTED clinical trial of docetaxel combined with (androgen deprivation) ADT for metastatic prostate cancer higher baseline FOXJ1 was predictive of decreased survival in patients treated with docetaxel (1st line taxane chemotherapy). The findings validated in pre-clinical model of advanced prostate cancer identify a new clinically impactful mechanism of taxane resistance, exploitation of which will lead to a potential stratification of patients likely to develop resistance to taxane treatment, thus should be considered for alternative therapeutic modalities.\nThe study is well-designed built on a compelling rationale and the results significantly impactful at the mechanistic, translational and clinical level. The work is beautifully executed supporting new evidence on mechanisms of resistance to taxane chemotherapy in advanced prostate cancer.\nI only have a few minor points to improve presentation of the manuscript, as follows:\n1) The length of the manuscript can be shortened, in order to allow for a more succinct flow. Experimental details of standard methods can be deleted for clarity and space.\n2) A schematic diagram indicating the FOXJ1 signaling interactions with other major effectors of the microtubule dynamics and the androgen receptor (AR) signaling mechanism, will benefit the interpretation of the results by the reader. It will be a significant addition.\n3) Can the authors comment on the role/contribution of FOXJ1 to therapeutic resistance in response to other chemotherapeuric strategies ( e.g. PARP inhibitorS?_", "reviewer_2": "Key results:\nBiomarkers to predict docetaxel sensitivity for men with prostate cancer are lacking. The authors of this manuscript use multiple methodologies to demonstrate the FoxJ1 expression increases intrinsic resistance to docetaxel through modulation of MT function. In general, this is a well-developed manuscript using sound experimental approaches of potential great importance to clinicians treating men with metastatic prostate cancer.\nValidity:\nThe data and conclusions are robust and valid. I would like to see more integration of FoxJ1 expression status with the currently available prognostic and predictive biomarkers Rb, PTEN, and p53 based on the CHAARTED data.\nSignificance:\nFoxJ1 expression may eventually become an important predictive biomarker for taxane benefit in prostate and potentially fulfills an unmet need for men with metastatic prostate cancer.\nData and methodology:\nAll figures were reviewed including supplementary information were reviewed and deemed appropriate.\nThe authors begin by developing two castration-resistant PDXs. In one of these, LuCaP35CR PDX, GEMC was the most significantly enriched gene. Given that downstream FOXJ1 controls expression of MT associated protein and is enriched in GOPB gene sets, it was further evaluated. TCGA Mining was performed and consistent with the PDX. DepMap drug response data indicated that FOXJ1 expression was positively correlated with docetaxel resistance in cancer cell lines and gene amplification was increased two-fold in taxane-treated patients.\nNext, docetaxel treated LNCaP cells stably overexpressing FOXJ1 vs vector were subjected to colony formation (showing less growth) and cell sorting (less G2/M indicative of less docetaxel induced mitotic arrest). pH3S10 was not increased also indicating less mitosis in docetaxel treated FOXJ1 overexpressing cells. MT aggregation was impaired in docetaxel treated LNCaP cells stably overexpressing FOXJ1 but basal levels were increased.\nLNCaP, DU145 FOXJ1 KD cells demonstrated increased docetaxel sensitivity and induced MT bundling as well as more extensive labeling of MT network, measured by BFI. To understand the mechanism of bundling, docetaxel induced alpha-tubulin acetylation in KD vs parental cells was examined and found to be dramatically enhanced (8.7 x vs 1.8 x) upon docetaxel treatment. MT dynamics were evaluated by EB1 comet assay in FOXJ1 KD vs control cells with comets demonstrating more pronounced taxane-mediated decrease in KD cells. EB1 length but not number was decreased indicative of slower MT growth rate in KD cells and confirmed cinematograpahically for resting and taxane treated cells. LNCaP xenografts overexpressing FOXJ1 cells exhibited docetaxel resistance and decreased mitotic figures.\nDownstream Pathways were assessed with mitotic spindle being most enriched gene set and findings were congruent with LuCaP35CR. TPPP3 overexpressing LNCaP cells were generated with findings similar to those of FOXJ1.\nTime courses of docetaxel treatment in cultured cells indicated induction of FOXJ1 and TPPP3 from 24-96 hours with authors showing it independent of cell cycle dependent upregulation. FOXJ1 induction was shown to be preceded 12 hours by upstream GEMC expression upon docetaxel treatment\nFinally, using CHAARTED data, quartile analysis of FOXJ1 expression was shown to inversely predict addition of docetaxel benefit with benefit seen more pronounced in ADT alone arm. It was not overall prognostic except possibly in high grade disease.\nSuggested improvements:\nLine 86: Docetaxel is not the only chemotherapy used in PC treatment. Would say it is primary first line chemotherapy.\nThe ABCB1/MDR PDX results were identified but not at all discussed- are the ABC1/MDR overexpression and GEMC/FOXJ1 pathways mutually exclusive resistance pathways? Which is predominant in patients. Is ABCB1 at all altered by FOXJ1 expression.\nWould like more discussion of potential mechanisms downstream of GEMC/FOXJ1/TPPP3 axis- How exactly does this lead to resistance. Is there a connection with AR translocation or classic downstream targets such as bcl-2, or is it an off-target effect. Experimentally, would make your study more compelling to include the above targets.\nWould like a discussion how FOXJ1 compares to other established prognostic alterations such as PTEN, Rb and p53, which are increasingly being used by clinicians to select patients for taxane-based therapy. Was there any evidence of any correlation of FOXJ1 expression with these more established markers?\nIs there any correlation between timing (synchronous vs metachronous) and volume in the CHAARTED study as these are both integral to NCCN guidelines for the use of docetaxel in the CSPC setting.\nDoes ABCB1 expression in the CHAARTED study have any predictive value- suggest including in discussion.\nReferences:\nAdequate and appropriate.", "reviewer_3": "In the paper by Balk and colleagues, data are presented that suggest an induction of the motile ciliated transcriptional program, notably that driven by the forkhead transcription factor FOXJ1 in docetaxel resistant prostrate cancer (PC) cells. The authors have also presented results that indicate FOXJ1 mediated alterations in microtubule dynamics as a likely cause of docetaxel resistance and implicate the FOXJ1 target gene and tubulin associated protein, tubulin polymerization promoting protein 3 (TPPP3) as one of the possible downstream effectors of tubulin dynamics that mediates docetaxel resistance.\nEctopic activation of the motile, and more specifically, the multiciliated cell transcriptional program in docetaxel-resistant PC is an intriguing observation and this pathway provides a mechanistic basis of how PC can become resistant to taxane treatment.\nCriticisms:\na) The authors state that not much is known about the function of FOXJ1 in non-ciliated cells. This is an erroneous belief. FOXJ1 is largely a motile ciliated cell-specific transcription factor. It is expressed not only in motile multiciliated cells as the authors repeatedly reference, but it be noted that FOXJ1 is also expressed in cells that differentiate monomotile cilia such as in the vertebrate left-right organizer and spermatozoa. Besides these motile cilia bearing cell-types, there are very few other cells where FOXJ1 is expressed in vivo. So, the notion that not much is known about this protein in non-ciliated cells is wrong.\nb) It is not clear how the multiciliated cell transcriptional program gets activated on docetaxel administration. The authors invoke attenuation of Notch signaling but the data presented are rather superficial. In normal development, Notch signaling acts cell non-autonomously to inhibit the multiciliated cell program. It is not clear in what capacity the authors envisage docetaxel could be influencing Notch signaling. In my view, this is a key area that needs more detailed investigation.\nc) Even though key multiliciliated cell transcription factors like GMNC and FOXJ1 are over-expressed, the data do not indicate any sign of cilia formation or multiciliation. Have the authors looked at these issues carefully? Are centrioles amplified these cells as it happens in multiciliated cells undergoing differentiation? Do they see any kind of ciliation – mono or multiple?"}, "author_response": "RESPONSES TO REVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis is an innovative manuscript coming from Dr. Balk's group, focusing on the mechanistic\ndissection of therapeutic resistance to microtubule-targeting (taxane) chemotherapy in in-vivo\nrelevant models of prostate cancer. The authors report novel results establishing the mechanistic\nlink between the FOXJ1 signaling axis, microtubule dynamics and taxane resistance. The focus\non FOXJ1 as the protagonist takes significant translational dimensions as clinically FOXJ1 gene\namplification was elevated in taxane-treated prostate cancer patients. Moreover, in the\nCHAARTED clinical trial of docetaxel combined with (androgen deprivation) ADT for metastatic\nprostate cancer higher baseline FOXJ1 was predictive of decreased survival in patients treated\nwith docetaxel (1st line taxane chemotherapy). The findings validated in pre-clinical model of\nadvanced prostate cancer identify a new clinically impactful mechanism of taxane resistance,\nexploitation of which will lead to a potential stratification of patients likely to develop resistance to\ntaxane treatment, thus should be considered for alternative therapeutic modalities.\nThe study is well-designed built on a compelling rationale and the results significantly impactful\nat the mechanistic, translational and clinical level. The work is beautifully executed supporting\nnew evidence on mechanisms of resistance to taxane chemotherapy in advanced prostate\ncancer. I only have a few minor points to improve presentation of the manuscript, as follows:\nResponse: We thank the reviewer for the positive feedback and the constructive suggestions,\nwhich we address below.\n1) The length of the manuscript can be shortened, in order to allow for a more succinct flow.\nExperimental details of standard methods can be deleted for clarity and space.\nResponse: Thank you for the suggestion. We have done editing to remove standard methods\nand make the manuscript more succinct.\n2) A schematic diagram indicating the FOXJ1 signaling interactions with other major effectors of\nthe microtubule dynamics and the androgen receptor (AR) signaling mechanism, will benefit the\ninterpretation of the results by the reader. It will be a significant addition.\nResponse: Thank you also\nfor this suggestion. We have\nadded a diagram showing\nFOXJ1 acting as a\ntranscription factor to increase\nexpression of TPPP3 and\nadditional microtubule\nassociated proteins, with a\nsubsequent increase in basal\nmicrotubule dynamics\n(including increased AR\nnuclear import) and\ndecreased taxane binding\n(Figures S26).\n1\n3) Can the authors comment on the role/contribution of FOXJ1 to therapeutic resistance in\nresponse to other chemotherapeuric strategies ( e.g. PARP inhibitors?\nResponse: We had\nshown that increased\nFOXJ1 decreases the\nresponses to cabazitaxel\nas well as to docetaxel.\nHowever, it is an\nexcellent suggestion to\nexamine other agents\nincluding those that\ninteract with microtubules\nor act by other\nmechanisms. Hence, we\nhave carried out colony\nformation assays to\nassess responses to a\nseries of agents that\ntarget microtubules by\ndistinct mechanisms\n(colchicine, vincristine,\nepothilone, and\nmaytansine). We also\nexamined agents that act\nby other mechanisms\n(olaparib, cisplatin, and\ncarboplatin). As shown in\nthe figure (new Figure\nS6), FOXJ1\noverexpression\nconferred resistance to\neach of the microtubule\ntargeted agents. Notably,\nthe binding sites for these\nare distinct from the taxane site, suggesting that the alteration in microtubules in response to\nFOXJ1 overexpression does not specifically affect the taxane binding site. Interestingly, cells\noverexpressing FOXJ1 also had decreased sensitivity to cisplatin, carboplatin, and olaparib,\nalthough the effect was less substantial than for the MT targeted agents. Further studies are\nneeded to determine whether the basis for this is related to increases in microtubule dynamics\n(possibly through increased drug efflux or other mechanisms), or is through distinct mechanisms.\nReviewer #2 (Remarks to the Author):\nKey results:\nBiomarkers to predict docetaxel sensitivity for men with prostate cancer are lacking. The authors\nof this manuscript use multiple methodologies to demonstrate the FoxJ1 expression increases\nintrinsic resistance to docetaxel through modulation of MT function. In general, this is a well-\ndeveloped manuscript using sound experimental approaches of potential great importance to\nclinicians treating men with metastatic prostate cancer.\nResponse: We thank the reviewer for their overall positive evaluation of the work.\nThe data and conclusions are robust and valid. I would like to see more integration of FoxJ1\nexpression status with the currently available prognostic and predictive biomarkers Rb, PTEN,\nand p53 based on the CHAARTED data.\nResponse: This is an\nexcellent suggestion. One\nway we have addressed it is\nto determine whether there\nis a correlation between\nexpression of FOXJ1 and\nRB1, PTEN, or TP53. As\nshown in the figure (new\nFigure S20), analysis of the\nTCGA primary PCa data\n(which reflects the\npopulation treated in the\nCHAARTED trial) does\nshow a weak negative\ncorrelation between\nexpression of FOXJ1 and\neach of these tumor\nsuppressor genes. While it\nis possible that they are\ndirectly or indirectly\nregulated by FOXJ1, it is\nmore likely that decreased\nexpression of each of these\ngenes can increase FOXJ1\nindirectly through effects on\ncell cycle. In any case, the\npresence of genomic\nalterations in these tumor\nsuppressor genes are more\nprognostic and predictive\nthan levels of expression.\nTherefore, the figure also\nshows mutations (left\npanels) and copy number alterations in PTEN, TP53, and RB1 (right panels). This analysis does\nnot reveal an association between genomic alterations in these genes and increased FOXJ1.\nOverall these findings do not indicate that increased FOXJ1 expression is driven by oncogenic\nalterations in these tumor suppressor genes.\nA second approach we have taken is to analyze the gene expression data from CHAARTED to\ndetermine whether expression of RB1, PTEN, or TP53 are associated with response to docetaxel.\nAs shown in the figure (new Figure S25), in contrast to the results for FOXJ1, expression of these\ngenes was not associated with survival. However, one caveat is that mutations or indels may also\nresult in decreased activity for these tumor suppressor genes, which may not be reflected in\ndecreased transcript levels. Therefore, we cannot rule out entirely that decreased activity of these\ngenes has effects on the response to docetaxel (although the data above indicate that any such\n3\neffect would be\nindependent of\naltered FOXJ1\nexpression). We\nalso examined\nABCB1\nexpression, and\ndid not find a\nnegative\ncorrelation\nbetween increased\nexpression and\nresponse. Notably,\nthis does not argue\nagainst increased\nABCB1 expression\nas a basis for\nacquired\nresistance, as the\ngene expression\nanalyses were\ndone on tumor\nsamples taken\nprior to initiation of\ntherapy.\nSignificance:\nFoxJ1 expression may eventually become an important predictive biomarker for taxane benefit in\nprostate and potentially fulfills an unmet need for men with metastatic prostate cancer.\nResponse: We appreciate these comments.\nData and methodology:\nAll figures were reviewed including supplementary information were reviewed and deemed\nappropriate. The authors begin by developing two castration-resistant PDXs. In one of these,\nLuCaP35CR PDX, GEMC was the most significantly enriched gene. Given that downstream\nFOXJ1 controls expression of MT associated protein and is enriched in GOPB gene sets, it was\nfurther evaluated. TCGA Mining was performed and consistent with the PDX. DepMap drug\nresponse data indicated that FOXJ1 expression was positively correlated with docetaxel\nresistance in cancer cell lines and gene amplification was increased two-fold in taxane-treated\npatients.\nNext, docetaxel treated LNCaP cells stably overexpressing FOXJ1 vs vector were subjected to\ncolony formation (showing less growth) and cell sorting (less G2/M indicative of less docetaxel\ninduced mitotic arrest). pH3S10 was not increased also indicating less mitosis in docetaxel treated\nFOXJ1 overexpressing cells. MT aggregation was impaired in docetaxel treated LNCaP cells\nstably overexpressing FOXJ1 but basal levels were increased.\nLNCaP, DU145 FOXJ1 KD cells demonstrated increased docetaxel sensitivity and induced MT\nbundling as well as more extensive labeling of MT network, measured by BFI. To understand the\n4\nmechanism of bundling, docetaxel induced alpha-tubulin acetylation in KD vs parental cells was\nexamined and found to be dramatically enhanced (8.7 x vs 1.8 x) upon docetaxel treatment. MT\ndynamics were evaluated by EB1 comet assay in FOXJ1 KD vs control cells with comets\ndemonstrating more pronounced taxane-mediated decrease in KD cells. EB1 length but not\nnumber was decreased indicative of slower MT growth rate in KD cells and confirmed\ncinematograpahically for resting and taxane treated cells. LNCaP xenografts overexpressing\nFOXJ1 cells exhibited docetaxel resistance and decreased mitotic figures.\nDownstream Pathways were assessed with mitotic spindle being most enriched gene set and\nfindings were congruent with LuCaP35CR. TPPP3 overexpressing LNCaP cells were generated\nwith findings similar to those of FOXJ1.\nTime courses of docetaxel treatment in cultured cells indicated induction of FOXJ1 and TPPP3\nfrom 24-96 hours with authors showing it independent of cell cycle dependent upregulation.\nFOXJ1 induction was shown to be preceded 12 hours by upstream GEMC expression upon\ndocetaxel treatment. Finally, using CHAARTED data, quartile analysis of FOXJ1 expression was\nshown to inversely predict addition of docetaxel benefit with benefit seen more pronounced in\nADT alone arm. It was not overall prognostic except possibly in high grade disease.\nSuggested improvements:\nLine 86: Docetaxel is not the only chemotherapy used in PC treatment. Would say it is primary\nfirst line chemotherapy.\nResponse: Thank you for pointing this out. We did indeed mean to say it is first line. This has\nbeen changed.\nThe ABCB1/MDR PDX results were identified but not at all discussed- are the ABC1/MDR\noverexpression and GEMC/FOXJ1 pathways mutually exclusive resistance pathways? Which is\npredominant in patients. Is ABCB1 at all altered by FOXJ1 expression.\nResponse: The reviewer is correct that while we found increased ABCB1 in one docetaxel\nresistant model, we did not pursue this mechanism or discuss it further (except to say that its\nclinical significance remains to be established) as it has been extensively studied and we wanted\nto focus on the novel FOXJ1 results. Whether the FOXJ1 and ABCB1 pathways are mutually\nexclusive is an interesting question. There is no clear mechanistic link that would suggest mutual\nexclusivity, although it is not unlikely that cells with ABCB1 amplification would no longer be under\nstrong pressure to select for other alterations that cause taxane resistance. Similarly, cells with\nFOXJ1 upregulation or amplification may be under less selective pressure to increase ABCB1.\nNotably, analysis of the SU2C Dream Team data in cBioPortal (see below) shows that some\nmetastatic CRPC cases have amplification of both FOXJ1 and ABCB1.\nHowever, only two of the ABCB1 amplified tumors were taxane exposed, with one having low\nFOXJ1 and one having high FOXJ1 (see below), so it is difficult to draw any definite conclusions\nabout mutual exclusivity.\nso it is difficult to assess whether it or FOXJ1 is more prominent. However, while FOXJ1\namplification is more frequent in taxane exposed versus naïve CRPC cases (see Figure 1H),\nABCB1 amplification is comparable in both (4.36 in naïve and 5.44% in exposed, see below). This\nhas now been added as a new figure (Figure S1B).\nFinally, we have no data\nindicating that ABCB1 expression\nis regulated by FOXJ1. We noted\nin the results that ABCB1 was not\nincreased in the docetaxel\nresistant LuCaP35CR xenografts.\nMoreover, it was not increased in\ncells with ectopic FOXJ1\noverexpression. Finally, as shown\nin the figure on the right, there is\nno correlation between increased\nFOXJ1 and ABCB1 in clinical\nsamples.\nWould like more discussion of potential mechanisms downstream of GEMC/FOXJ1/TPPP3 axis-\nHow exactly does this lead to resistance. Is there a connection with AR translocation or classic\n6\ndownstream targets such as bcl-2, or is it an off-target effect. Experimentally, would make your\nstudy more compelling to include the above targets.\nResponse: The major conclusion we reach in the manuscript is that an increase in the\nGEMC/FOXJ1/TPPP3 axis confers resistance by altering microtubule dynamics. This is\nsupported by multiple lines of evidence including the direct demonstration that FOXJ1\noverexpression decreases docetaxel-mediated microtubule stabilization. Moreover, we directly\nshow FOXJ1 downregulation results in increased taxane binding to microtubules. However, as\nnoted by the reviewer, we do not discuss precisely why mitigating taxane-mediated microtubule\nstabilization confers resistance (or conversely why taxane-mediated microtubule stabilization kills\ncells). Our presumption (which is supported by the literature) is that multiple pathways are\nimpaired by disruption of microtubule dynamics. One is clearly mitosis due to effects on the mitotic\nspindle, and we show that FOXJ1 overexpression mitigates the taxane-mediated mitotic arrest.\nHowever, as noted by the\nreviewer, increased FOXJ1\nlikely mitigates other toxic\neffects of taxanes. We believe\nit is beyond the scope of this\nstudy to comprehensively\nexamine the many\ndownstream effects of\ninterfering with microtubule\nfunction. However, as\nsuggested by the reviewer, we\ndid examine effects of FOXJ1\noverexpression on AR nuclear\ntranslocation. As show in a\nnew figure (Figure S11), and\nconsistent with previous\nreports from us and others,\ntaxane treatment decreased\nthe androgen-stimulated\nnuclear localization of AR.\nNotably, FOXJ1\noverexpression significantly\nincreased this androgen-\nstimulated nuclear localization,\nwhich is consistent with our\nfinding that FOXJ1\noverexpression increases\nmicrotubule dynamics.\nHowever, docetaxel still\ndecreased AR nuclear\nlocalization in the FOXJ1\noverexpressing cells. One\ninterpretation of this result is\nthat the microtubule-dependent transport of AR is mediated by specific cofactors that are\nparticularly sensitive to the effects of taxanes, but further studies are clearly needed to test this\nor alternative hypotheses.\ntargeted therapies may directly or\nindirectly increase BCL2. Therefore, it\nis possible that FOXJ1 could be acting\nin part indirectly through AR to support\nits nuclear translocation and mitigate\ndecreased AR activity in response to\ntaxane. However, this would\npresumably result in decreased BCL2.\nFOXJ1 could certainly also have\neffects on BCL2 or other apoptosis\nrelated genes independent of AR.\nHowever, the expression of BCL2 is\nnot correlated with FOXJ1 expression\nin clinical data sets (see panel on\nright), and we did not find significantly\nincreased expression of BCL2 or other\nBCL2 family anti-apoptotic proteins in FOXJ1 overexpressing cells. Notably, while taxanes can\nimpair AR nuclear translocation, they clearly interfere with myriad other cellular functions, so\neffects on AR are probably not the major driver of their efficacy. Therefore, we have not carried\nout further AR focused studies.\nWould like a discussion how FOXJ1 compares to other established prognostic alterations such\nas PTEN, Rb and p53, which are increasingly being used by clinicians to select patients for\ntaxane-based therapy. Was there any evidence of any correlation of FOXJ1 expression with these\nmore established markers?\nResponse: This comment is related to the suggestion above under the topic “Validity”. As\ndescribed in that response, we did not find evidence that loss of these tumor suppressors was\ncorrelated with FOXJ1 expression. We also examined the CHAARTED data and did not find any\ncorrelations between response and expression of PTEN, RB1, or TP53 (see above).\nIs there any correlation between timing (synchronous vs\nmetachronous) and volume in the CHAARTED study as\nthese are both integral to NCCN guidelines for the use of\ndocetaxel in the CSPC setting.\nResponse: This is a great question. It is certainly possible\nthat FOXJ1 expression in CHAARTED may be associated\nwith tumor volume or other clinical features, and/or that\nintegrating these clinical data with FOXJ1 expression would\nenhance predictive value. Unfortunately we don’t have this\ndata for the CHAARTED study, and it would probably be\nunderpowered to address this point. However, as described\nin the manuscript, FOXJ1 expression does not appear to be\nprognostic in primary PC or CRPC. To further address this\nquestion we also examined biochemical recurrence (BCR) in\nTCGA primary tumors as a metric that that is presumably\nassociated with occult metastatic disease. Notably, there was\nno correlation between FOXJ1 and BCR (see panel on right).\nin discussion.\nResponse: This is a great question. As described in the response to the “Validity” section, we\nfound that ABCB1 expression did not have predictive value in CHAARTED. As noted above, this\ndata is included in a new Figure (Figure S25) and in the Discussion.\nReferences:\nAdequate and appropriate.\nReviewer #3 (Remarks to the Author):\nIn the paper by Balk and colleagues, data are presented that suggest an induction of the motile\nciliated transcriptional program, notably that driven by the forkhead transcription factor FOXJ1 in\ndocetaxel resistant prostrate cancer (PC) cells. The authors have also presented results that\nindicate FOXJ1 mediated alterations in microtubule dynamics as a likely cause of docetaxel\nresistance and implicate the FOXJ1 target gene and tubulin associated protein, tubulin\npolymerization promoting protein 3 (TPPP3) as one of the possible downstream effectors of\ntubulin dynamics that mediates docetaxel resistance.\nEctopic activation of the motile, and more specifically, the multiciliated cell transcriptional program\nin docetaxel-resistant PC is an intriguing observation and this pathway provides a mechanistic\nbasis of how PC can become resistant to taxane treatment.\nCriticisms:\na) The authors state that not much is known about the function of FOXJ1 in non-ciliated cells.\nThis is an erroneous belief. FOXJ1 is largely a motile ciliated cell-specific transcription factor. It is\nexpressed not only in motile multiciliated cells as the authors repeatedly reference, but it be noted\nthat FOXJ1 is also expressed in cells that differentiate monomotile cilia such as in the vertebrate\nleft-right organizer and spermatozoa. Besides these motile cilia bearing cell-types, there are very\nfew other cells where FOXJ1 is expressed in vivo. So, the notion that not much is known about\nthis protein in non-ciliated cells is wrong.\nResponse: We thank the reviewer for pointing this out. We agree that it was misleading to indicate\nthat not much is known about FOXJ1 in non-ciliated cells. While it has perhaps been most studied\nin the development of multi-ciliated cells, it is also expressed in cells that differentiate mono-motile\ncilia (as noted by the reviewer). Moreover, there have been many studies describing its\nexpression in nonciliated cells including tumor cells, although its functions in these cells have not\nbeen well studied. We have now clarified these points and added a reference in the revised\nmanuscript.\nb) It is not clear how the multiciliated cell transcriptional program gets activated on docetaxel\nadministration. The authors invoke attenuation of Notch signaling but the data presented are\nrather superficial. In normal development, Notch signaling acts cell non-autonomously to inhibit\nthe multiciliated cell program. It is not clear in what capacity the authors envisage docetaxel could\nbe influencing Notch signaling. In my view, this is a key area that needs more detailed\ninvestigation.\nResponse: We apologize for any confusion on this point. We investigated NOTCH because (as\nnoted by the reviewer) a decrease in NOTCH signaling in the microenvironment initiates GEMC1\nexpression in precursor multiciliated cells. Moreover, we found in the TCGA PC gene data set\n9\nthat GEMC1 expression was highly negatively correlated with expression of the Notch-induced\ngenes HES1 and HEY1, suggesting that docetaxel may be inducing GEMC1 by decreasing Notch\nsignaling (see previous Figure S13B). Therefore, we examined Notch signaling in response to\ndocetaxel. However, we did not find a decrease in HES1 or HEY1 expression in response to\ndocetaxel (see previous Figure S13C). Based on this we concluded that the initial increase in\nGEMC1 is not mediated by a decrease in Notch. Therefore, we have not explored further\nwhether, and by what mechanisms, docetaxel may influence Notch signaling. Nonetheless, we\nagree this area needs further investigation given the negative correlation between GEMC1 and\nHES1/HEY1. Indeed, it is possible that while short term docetaxel treatment does not directly\nsuppress Notch, its longer term effects in vivo may be to directly or indirectly suppress Notch and\ntherapy further induce GEMC1 and FOXJ1. We have now added these points to the Discussion.\nc) Even though key multiliciliated cell transcription factors like GMNC and FOXJ1 are over-\nexpressed, the data do not indicate any sign of cilia formation or multiciliation. Have the authors\nlooked at these issues carefully? Are centrioles amplified these cells as it happens in multiciliated\ncells undergoing differentiation? Do they see any kind of ciliation – mono or multiple?\nResponse: This is also a great question. Indeed, during the early stages of this study we did look\nfor evidence of cilia formation in cells overexpressing FOXJ1. We did not see any such evidence,\nbut did not have positive controls in these studies. Therefore, in response to the reviewer’s\ncomment we have gone back and repeated these experiments with positive controls. As shown\nin the figure below, cilia are clearly seen by staining for acetylated tubulin in BEAS-2B cells, a\nhuman bronchial epithelial cell line. In contrast, we could not detect cilia in LNCaP cells\noverexpressing FOXJ1 (which were process in parallel with the BEAS-2B cells). We also see no\nevidence of of centriole amplication. These results have been added to the revised manuscript\n(Figure S7).\n10"}, {"round": 2, "reviewer_comments": {"reviewer_1": "This is a novel revised manuscript, focusing on novel mechanisms driving therapeutic resistance to microtubule-targeting (taxane) chemotherapy in models of advanced prostate cancer. The authors provide an insightful response to the previous critique and new compelling data on mechanistic contribution of FOXJ1 signaling axis towards impacting microtubule dynamics and taxane resistance.\nThe findings are of major translational significance in defining novel biomarkers for resistance to taxane chemotherapy. The work is of high impact and relevance to the prostate cancer investigators.", "reviewer_3": "The authors have addressed my criticisms to the best of their abilities.", "reviewer_4": "RESPONSES TO REVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis is an innovative manuscript coming from Dr. Balk's group, focusing on the mechanistic\ndissection of therapeutic resistance to microtubule-targeting (taxane) chemotherapy in in-vivo\nrelevant models of prostate cancer. The authors report novel results establishing the mechanistic\nlink between the FOXJ1 signaling axis, microtubule dynamics and taxane resistance. The focus\non FOXJ1 as the protagonist takes significant translational dimensions as clinically FOXJ1 gene\namplification was elevated in taxane-treated prostate cancer patients. Moreover, in the\nCHAARTED clinical trial of docetaxel combined with (androgen deprivation) ADT for metastatic\nprostate cancer higher baseline FOXJ1 was predictive of decreased survival in patients treated\nwith docetaxel (1st line taxane chemotherapy). The findings validated in pre-clinical model of\nadvanced prostate cancer identify a new clinically impactful mechanism of taxane resistance,\nexploitation of which will lead to a potential stratification of patients likely to develop resistance to\ntaxane treatment, thus should be considered for alternative therapeutic modalities.\nThe study is well-designed built on a compelling rationale and the results significantly impactful\nat the mechanistic, translational and clinical level. The work is beautifully executed supporting\nnew evidence on mechanisms of resistance to taxane chemotherapy in advanced prostate\ncancer. I only have a few minor points to improve presentation of the manuscript, as follows:\nReviewer #2 (Remarks to the Author):\nKey results:\nBiomarkers to predict docetaxel sensitivity for men with prostate cancer are lacking. The authors\nof this manuscript use multiple methodologies to demonstrate the FoxJ1 expression increases\nintrinsic resistance to docetaxel through modulation of MT function. In general, this is a well-\ndeveloped manuscript using sound experimental approaches of potential great importance to\nclinicians treating men with metastatic prostate cancer.\nReviewer #3 (Remarks to the Author):\nIn the paper by Balk and colleagues, data are presented that suggest an induction of the motile\nciliated transcriptional program, notably that driven by the forkhead transcription factor FOXJ1 in\ndocetaxel resistant prostrate cancer (PC) cells. The authors have also presented results that\nindicate FOXJ1 mediated alterations in microtubule dynamics as a likely cause of docetaxel\nresistance and implicate the FOXJ1 target gene and tubulin associated protein, tubulin\npolymerization promoting protein 3 (TPPP3) as one of the possible downstream effectors of\ntubulin dynamics that mediates docetaxel resistance.\nEctopic activation of the motile, and more specifically, the multiciliated cell transcriptional program\nin docetaxel-resistant PC is an intriguing observation and this pathway provides a mechanistic\nbasis of how PC can become resistant to taxane treatment.\nCriticisms:\na) The authors state that not much is known about the function of FOXJ1 in non-ciliated cells.\nThis is an erroneous belief. FOXJ1 is largely a motile ciliated cell-specific transcription factor. It is\nexpressed not only in motile multiciliated cells as the authors repeatedly reference, but it be noted\nthat FOXJ1 is also expressed in cells that differentiate monomotile cilia such as in the vertebrate\nleft-right organizer and spermatozoa. Besides these motile cilia bearing cell-types, there are very\nfew other cells where FOXJ1 is expressed in vivo. So, the notion that not much is known about\nthis protein in non-ciliated cells is wrong."}, "author_response": ""}]

Decision letter