bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4588: Estrogen receptor alpha mediates HPV E6/E7 oncoproteins: Induced breast cells proliferation
Authors: McGhee Eva , Tran Lucy , Li Mengtao , Wu Yong , Chung Seyung , Araniego Cheryl , Tate Karen , Baker Melanie , Evans Kamilah , Rouse Mechelle , Vadgama Jay
Published: July 2016

The association of human papillomavirus (HPV) with cervical cancer, head and neck cancer is well established, but the involvement of HPV E6/E7 oncoproteins in breast cancer is more contentious. Previous studies suggest that high risk HPV E6/E7 oncoproteins inactivate two tumor suppressor proteins p53 and pRb signaling and increase cell proliferation. There is cumulative evidence that high risk HPV 16 E6/E7 oncoproteins may have causal roles in some human breast cancers. Moreover, some studies have identified HPV DNA in breast tissue and breast cancer specimens. Nevertheless, the route of transmission of HPV in breast cells has not been determined, and the mechanisms by which HPV involvement in breast cancer are not known. We used a non-tumorigenic (MCF12A) epithelial breast cell line transfected with HPV 16 to elucidate the role of oncoproteins E6/E7 in pro-proliferative and anti-apoptotic signaling pathways. We also used Tamoxifen treatment, which is an antagonist of the estrogen receptor in breast tissues through its active metabolite, 4hydroxytamoxifen. We demonstrate that HPV E6/E7 transfection activates the extracellular signal-regulated kinase (ERK) MAPK pathway and Akt/PKB kinase signaling pathways through activating estrogen receptor alpha in MCF12A cells. E6/E7 oncoproteins transfection alone dramatically increases estrogen receptor alpha protein expression. Treatment with tamoxifen, an antagonist of the ER, significantly abolishes E6/E7-increase in ERK and PKB phosphorylation without altering their protein levels, suggesting that the estrogen receptor is a key mediator of HPV E6/E7 mediated ERK and Akt/PKB activation. Our current work show an insightful understanding of the molecular mechanisms of HPV-elicited Akt/PKB activation and its roles in cell proliferation and survival in normal breast epithelial cells. Therefore, these findings indicate that HPV E6/E7 oncoproteins may promote the transforming activities of high-risk HPV E6/E7 proteins and cell proliferation in breast cells. Therefore, suppression of estrogen receptor signaling networks may be used as a therapeutic strategy for HPV associated lesions and cancers such as breast metastasis.

Citation Format: Eva McGhee, Lucy Tran, Mengtao Li, Yong Wu, Seyung Chung, Cheryl Araniego, Karen Tate, Melanie Baker, Kamilah Evans, Mechelle Rouse, Jay Vadgama. Estrogen receptor alpha mediates HPV E6/E7 oncoproteins: Induced breast cells proliferation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4588.
Abstract 4593: Analysis of transforming growth factor β receptor trafficking on different signaling transduction pathways
Authors: Ng Evelyn , Di Guglielmo John
Published: July 2016

Transforming growth factor beta (TGFβ) is a cytokine that regulates cellular adhesion, proliferation and apoptosis. Its canonical downstream effectors include receptor-regulated Smad2/3 proteins, which are phosphorylated and then translocated to the nucleus to alter transcription. Additionally, other atypical pathways are simultaneously initiated by TGFβ, including that of p38 (a mitogen activated protein kinase).

TGFβ acts as a tumour suppressor in its normal epithelial environment, but in tumor cells, it promotes epithelial-mesenchymal transition and cell migration. The manner in which tumor cells overcome the growth suppressive effects of TGFβ is not well-understood, especially since downstream TGFβ signaling is still apparent in these cells. This may point to selective pathway activation as a reason for its dual roles. Thus, it is important to consider membrane trafficking—a central process that directs signaling between various cascades.

Previous studies from our lab demonstrate that perturbation of aPKC signaling alters TGFβ receptor trafficking and Smad signal transduction. Furthermore, we have also observed increased access to the p38 MAPK pathway in cells that have been silenced for aPKC expression. We are currently assessing the trafficking of the TGFβ receptor complex in order to investigate the mechanism(s) responsible differential access to specific signaling pathways. This will be addressed using techniques such as co-immunoprecipitation, immunofluorescence microscopy, subcellular fractionation and western blotting.

Determining these mechanisms will further our understanding on cancerous TGFβ pathway regulation, and may lead to the discovery of promising therapeutic targets.

Citation Format: Evelyn Ng, John Di Guglielmo. Analysis of transforming growth factor β receptor trafficking on different signaling transduction pathways. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4593.
Abstract 4594: Involvement of receptors tyrosine kinase in the modulation of store-operated calcium entry
Authors: Emeriau. Noémie
Published: July 2016

A multitude of studies show that alterations in Ca2+ signalling initiate or support the development of hallmarks of cancer. In particular, the role of store-operated Ca2+ entry (SOCE) in tumorigenesis and tumour progression has been subject to intense investigation. SOCE is the main mechanism by which external Ca2+ enters into the cell. It is initiated by depletion of ER Ca2+ stores and mediated by several proteins such as STIM1, an ER transmembrane protein sensing Ca2+ within the ER and ORAI1, a plasma membrane Ca2+ channel. We recently showed that SOC inhibition interfered with EGFR-dependent signalling in non-small cell lung carcinoma cells.

In this study, we observed that STIM1 depletion reduced neuregulin-dependent proliferation of breast cancer cells. Since neuregulin binds to ErbB3 and/or ErbB4 and therefore activates ErbB2, this result prompted us to investigate whether ErbB proteins might modulate SOCE. We observed that lapatinib, a dual inhibitor of EGFR and ErbB2, dramatically inhibited SOCE. As expected, lapatinib also inhibited phosphorylation of EGFR, ErbB2 and downstream pathways of both receptors. Specific inhibition of EGFR by erlotinib or gefitinib had no effect on SOCE. In contrast, specific inhibition of ErbB2 by CP724714 mimicked the effects of lapatinib. We also investigated the role of downstream pathways of ErbB2 in the modulation of SOCE. Inhibition of the MAPK and the JAK-STAT pathways does not modify the amplitude of SOCE. Contrariwise, LY294002 and MK2206, two inhibitors of the PI3K pathway, dramatically reduced SOCE. Interestingly, in silico analysis showed that STIM1 might be phosphorylated by Akt.

All these results suggest that SOCE is important in the proper function of ErbB2-dependent signalling. This raises the possibility to target molecular mediators of SOCE in order to interfere with the proliferation of cancer cells.

Citation Format: Noémie Emeriau. Involvement of receptors tyrosine kinase in the modulation of store-operated calcium entry. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4594.
Abstract 4595: Development and validation of a novel EGF receptor-neutralizing monoclonal antibody
Authors: Zuberek Hincman Krystyna , Manning Christopher A. , Nelson Michael R. , Lewis Michael , Scialdone Roy , Darce Jaime , Lutz Stephen R. , Burford Evans , Haack Herbert , Silver Matthew R.
Published: July 2016

The epidermal growth factor receptor (EGFR) is a cell-surface receptor for members of the epidermal growth factor family (EGF-family). EGFR dimerizes upon ligand binding, which leads to autophosphorylation, downstream signaling and ultimately internalization. In cancer, EGFR mutations and amplification have been linked to cell proliferation and survival. Two EGFR neutralizing monoclonal antibodies are currently FDA approved, which includes Cetuximab (Erbitux, BMS; mouse/human chimeric) and Panitumumab (ABX-EGF, Amgen; fully humanized). However, these antibodies are often unavailable to researchers studying this important pathway. Here we describe the development and validation of a new rabbit monoclonal EGFR neutralizing antibody (clone D1D4J). D1D4J was validated using cell-based models and immunofluorescence imaging. D1D4J binds EGFR and neutralizes EGF-induced activation and internalization. Compared to other Research Use Only (RUO) neutralizing antibodies on the market, D1D4J shows superior performance and is a useful research tool in the EGFR signaling space.

Citation Format: Krystyna Zuberek Hincman, Christopher A. Manning, Michael R. Nelson, Michael Lewis, Roy Scialdone, Jaime Darce, Stephen R. Lutz, Evans Burford, Herbert Haack, Matthew R. Silver. Development and validation of a novel EGF receptor-neutralizing monoclonal antibody. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4595.
Abstract 4599: Bile acids modulate estrogen receptor β signaling through nongenomic actions in colorectal cancer
Authors: Trujillo Jesse
Published: July 2016

Bile acids deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA) have contrasting effects on tumor development. DCA is measurable at increased levels in the serum of colorectal adenocarcinoma patients and is associated with disease progression. DCA is also a well-established tumor promoter. On the other hand, UDCA is a chemopreventative agent revealed to suppress the recurrence of highly dysplastic polyps in a phase III clinical trial. Remarkably, retrospective investigation of the data had shown that UDCA's efficacy as a chemopreventive mediator was gender specific. UDCA significantly lowered the recurrence of advanced lesions in males, but caused a significant increase in the recurrence of advanced lesions in females. Our lab is in pursuit of understanding the gender specific chemopreventive role of UDCA. Estrogen receptor β (ERβ) has been shown to play a significant role in controlling the development of colorectal cancer. Therefore, we hypothesized that the altered regulation of the ERβ pathway by UDCA might explain the gender specificity of this molecule. In order to assess this, we analyzed the effects of UDCA on ERβ to those of the known tumor promoter DCA in both male and female human adenocarcinoma cell lines. Female HT-29 colorectal adenocarcinoma cells induced the phosphorylation of ERβ, as well as up-regulated gene expression of estrogen response elements (EREs), showing stimulated activity of this hormonal receptor. Moreover, we found that pre-treating the cells with PD98059, a MEK 1/2 inhibitor, UDCA and DCA-induced phosphorylation of ERβ, along with increased ERE gene expression, was significantly reduced. Collectively, this data suggests that UDCA and DCA can stimulate ERβ signaling through the non-genomic MAPK signaling cascade; suggesting that UDCA's gender-specific effects may be due, in part, to signaling through hormone receptors.

Citation Format: Jesse Trujillo. Bile acids modulate estrogen receptor β signaling through nongenomic actions in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4599.
Abstract 4601: Regulation of Hes1 expression by the Wnt transcription factor T-cell factor-4
Authors: Koga Hironori , Imamura Yasuko , Ikezono Yu , Wada Fumitaka , Nakamura Toru , Iwamoto Hideki , Masuda Atsutaka , Sakaue Takahiko , Yano Hirohisa , Torimura Takuji
Published: July 2016

Background: The Notch effector Hes1 plays a critical role in stemness of cancer stem cells (CSCs). T-cell factor (TCF)-4 is a key transcription factor in Wnt signaling, which is suggested to be linked with Notch signaling. Among our TCF-4 isoforms cloned previously, the TCF-4J and K pair have been characterized based on the presence (K) or absence (J) of SxxSS motif (Exp Cell Res 2010). TCF-4J was highly expressed in poorly differentiated human hepatocellular carcinoma (HCC) tissues, and the TCF-4J-overexpressing HCC cells (J cells) exhibited high tumor-initiating potential, which is one of the features of CSCs (PLoS One 2013). Thus, the AIM of this study was to investigate whether the SxxSS motif of TCF-4 was involved in the regulation of Hes1 expression in HCC cells. Methods: TCF-4K mutants were prepared by site-directed mutagenesis. Sphere formation assay was used to condense CSC-like cells. Results: Hes1 was strikingly expressed in spheres of J cells and K-mutant cells in both protein and mRNA levels, while its expression level was 70% inhibited in K cells. Consistently, protein expression levels of Jagged1 and Notch1 were highest in J cells under both attached and floating conditions. The Notch inhibitor DAPT, a γ-secretase inhibitor, clearly decreased the expression levels of Hes1, suggesting that the Notch-Hes1 axis was functional. Conclusion: Lack of SxxSS motif in TCF-4 robustly upregulated Hes1 expression in HCC cell spheres. The finding strongly suggests that TCF-4 directly regulates Hes1 in HCC spheres, where CSCs abundantly exist.

Citation Format: Hironori Koga, Yasuko Imamura, Yu Ikezono, Fumitaka Wada, Toru Nakamura, Hideki Iwamoto, Atsutaka Masuda, Takahiko Sakaue, Hirohisa Yano, Takuji Torimura. Regulation of Hes1 expression by the Wnt transcription factor T-cell factor-4. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4601.
Abstract 4604: Activities of in vitro prepared recombinant wnt inhibitory factor-1 (Wif-1)
Authors: Babaer Duaa A. , Wang Xiaofei
Published: July 2016

Wnt Inhibitory Factor-1 (Wif-1) is a secreted modular protein that inhibits the activities of Wnt signaling pathway. The latter is involved in many aspects of cancer and embryonic development. Many studies have shown that reduced WIF-1 expression is associated with the development of cancer. Since WIF-1 acts in extracellular space, it is possible to use in vitro expressed WIF-1 as a therapeutic agent for cancer treatment. We tested whether in vitro expressed recombinant WIF-1 could elicit inhibitory effects on Wnt pathway in 3T3 cells by examining the expression of insulin-like growth factor II (Igf2), Wnt1 inducible signaling pathway protein 2 (Wisp2), (C/EBPδ), and peroxisome proliferator-activated receptor γ (PPARγ) which has been implicated as a suppressor in numerous cancer types including human breast, lung, and colon cancer. 3T3 cells were treated with 0, 1, 5 and 20 ng/ml of recombinant mouse Wif-1 protein. Total RNA was isolated after 24-48 hours of treatment. The levels of Igf2, Wisp2, C/EBPδ, and PPARγ mRNA were analyzed with reverse transcription quantitative polymerase chain reaction. Results are being analyzed to determine whether the in vitro expressed recombinant Wif-1 protein can modulate the expression of these genes and regulate cancer to have a significant inclusion in the field of cancer biology.

Citation Format: Duaa A. Babaer, Xiaofei Wang. Activities of in vitro prepared recombinant wnt inhibitory factor-1 (Wif-1). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4604.
Abstract 4608: Survivin downregulation by α-santalol is not mediated through PI3K-Akt pathway in human breast cancer cells
Authors: BOMMAREDDY AJAY , CRISAMORE KARRYN , FILLMAN SARAH , BROZENA SARAH , STEIGERWALT JAMES , LANDIS TERRA , VANWERT ADAM L. , DWIVEDI CHANDRADHAR
Published: July 2016

α-Santalol, a terpenoid found in sandalwood oil, has been shown to inhibit cancer cell growth in vitro by inducing apoptosis. This study was performed to investigate the anticancer properties of α-santalol associated with the induction of apoptosis in cultured MCF-7 (estrogen receptor (ER) positive, and wild type p53) and MDA-MB-231 (ER-negative and mutant p53) breast cancer cells. Expression of major proteins examined in the study were determined using standard Western blot protocol and analyzed by LICOR-Odyssey infra-red scanner. Total protein levels of survivin were confirmed by survivin ELISA kit. Cell viability was assessed by trypan blue dye exclusion assay, and caspase-3 activity was determined by caspase-3 (active) ELISA kit. Treatment of breast cancer cells for 6 and 9 hour time intervals with α-santalol (20, 40 μM) resulted in statistically significant concentration-dependent downregulation of survivin. pAkt levels were found to be slightly upregulated despite the downregulation of survivin. Pharmacological inhibition of the PI3K-Akt pathway did not result in a synergistic/additive increase in cell death or caspase-3 activity caused by α-santalol. The study reveals that survivin downregulation by α-santalol in breast cancer cells is not mediated through the PI3K-Akt pathway.

Citation Format: AJAY BOMMAREDDY, KARRYN CRISAMORE, SARAH FILLMAN, SARAH BROZENA, JAMES STEIGERWALT, TERRA LANDIS, ADAM L. VANWERT, CHANDRADHAR DWIVEDI. Survivin downregulation by α-santalol is not mediated through PI3K-Akt pathway in human breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4608.
Abstract 461: Combined analysis of circulating epithelial cell count and serum thyroglobulin for differentiating disease status of the patients with papillary thyroid carcinoma
Authors: Tseng Ching-Ping , Lin Jen-Der , Lin Hung-Chih , Cheng Ju-Chien
Published: July 2016

Papillary thyroid carcinoma (PTC) is one type of thyroid cancer and accounts for about 80% of the cases. Routine surveillance by serum thyroglobulin (Tg) and medical imaging is the current practice to monitor disease progression of the patients. However, the presence of anti-Tg antibody or other influence factors may discount the value of serum Tg in disease monitoring. Whether enumeration of circulating epithelial cells (CECs) and its combined analyses with serum Tg help to define disease status of PTC patients was investigated. CECs were first enriched from the peripheral blood of the healthy control subjects (G1, n = 17) and the patients at disease-free status (G2, n = 26) or with distant metastasis (G3, n = 22). The number of CECs expressing epithelial cell adhesion molecule (EpCAM) was determined by immunofluorescence microscopy analyses. The medium number of EpCAM+-CECs was 6 (intequartile range 1-11), 12 (interquartile range 7-16) and 91 (interquartile range 31-206) cells/ml of blood for G1, G2 and G3, respectively. EpCAM+-CEC counts were significantly higher in G3 than in G1 (p<0.05) and G2 (p<0.05). Our data further revealed that combined analysis of serum Tg with EpCAM+-CEC defined the disease status (disease-free vs. distant metastasis) in 46 (95.8%) of the 48 patients with the AUC equivalent of 0.962 (p<0.0001) in the ROC analysis. The sensitivity and specificity of the assay was 100% and 92.3%, respectively. Moreover, all 22 patients (100%) with metastatic status were defined accordingly. These data demonstrate that combined analysis of serum Tg with EpCAM+-CECs is suitable to distinguish the patients at disease-free status from the patients with distant metastasis. CEC testing thereby can supplement the current standard methods for monitoring disease status of PTC.

Citation Format: Ching-Ping Tseng, Jen-Der Lin, Hung-Chih Lin, Ju-Chien Cheng. Combined analysis of circulating epithelial cell count and serum thyroglobulin for differentiating disease status of the patients with papillary thyroid carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 461.
Abstract 4611: Interrogation of PI3K signaling via multiplex detection of differential phosphorylation of specific Akt isoforms
Authors: Saporita Anthony J. , Schluter Melissa , MacIvor Debra , Mistry Jehangir , Hwang Joseph
Published: July 2016

The serine/threonine protein kinase Akt is a key node in the PI3K pathway, one of the primary signaling cascades hyperactivated in human cancer. Emerging evidence demonstrates that the three Akt isoforms (Akt1, Akt2, and Akt3) may have unique, isoform-specific roles in key cellular processes such as differentiation and proliferation. We have developed 2-plex assays for each of the Akt isoforms in order to measure the relative levels of phospho- and total Akt1, Akt2, and Akt3. Differential regulation of Akt isoforms was characterized in multiple human cancer cell lines, including SH-SY5Y neuroblastoma cells and MCF-7 breast cancer cells. SH-SY5Y cells were used to study Akt phosphorylation in the context of differentiation. Briefly, SH-SY5Y cells were treated with retinoic acid (RA) for 3 days to induce neuronal differentiation before cells were collected, lysed, and evaluated by Luminex assay. RA-induced differentiation of SH-SY5Y cells stimulated phosphorylation of all three Akt isoforms, with Akt2 displaying the greatest induction. Multi-pathway analysis demonstrated co-induction of phospho-JNK in the RA-treated SH-SY5Y cells, consistent with the role of JNK in RA-mediated differentiation. To study differential regulation of the Akt isoforms in response to growth-stimulatory and growth-inhibitory signals, we used MCF-7 cells. In contrast to SH-SY5Y cells which express all three isoforms, MCF-7 cells only expressed Akt1 and Akt2. Serum-starved MCF-7 cells were cultured in the presence or absence of the PI3K inhibitor LY294002 prior to stimulation with insulin growth factor (IGF). Akt1 showed a greater induction of phosphorylation in response to IGF relative to Akt2. Similarly, whereas LY294002 pre-treatment reduced phospho-Akt1 to baseline levels, it only partially inhibited the IGF-dependent induction of phospho-Akt2. This suggests that Akt1 may be more sensitive to PI3K inhibition than Akt2 in certain human breast cancer cells. Collectively, our results demonstrate that Akt1, Akt2, and Akt3 are differentially regulated in human cancer cells at both the level of phosphorylation and of total protein expression.

Citation Format: Anthony J. Saporita, Melissa Schluter, Debra MacIvor, Jehangir Mistry, Joseph Hwang. Interrogation of PI3K signaling via multiplex detection of differential phosphorylation of specific Akt isoforms. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4611.
Abstract 4612: In breast cancer cells IGF-I induces upregulation of DDR1 by suppressing miR-199a-5p via the PI3K/Akt pathway
Authors: Palladino Chiara , Matà Roberta , Nicolosi Maria Luisa , Lo Presti Anna Rita , Malaguarnera Roberta , Ragusa Marco , Sciortino Daniela , Morrione Andrea , Maggiolini Marcello , Vella Veronica , Belfiore Antonino
Published: July 2016

Discoidin Domain Receptor 1 (DDR1) is a collagen receptor tyrosine-kinase that is implicated in tumor progression by contributing to epithelial mesenchymal transition (EMT) and by enhancing tumor cell migration and invasion.

We previously demonstrated that, in breast cancer cells, DDR1 functionally cross-talks with IGF-I receptor (IGF-IR), by affecting IGF-IR expression and trafficking. In these cells, DDR1 was able to enhance the stimulatory effects of IGF-I on mitogenesis and invasion suggesting that the DDR1-IGF-IR cross-talk may play a role in cancer progression.

Herein, we evaluated whether IGF-I may in turn affect DDR1 expression. Indeed, we demonstrated that in breast cancer cells (MCF-7 and MDA-MB-231), IGF-I was able to upregulate DDR1 protein expression in a time and dose dependent manner. In contrast, DDR1 mRNA levels showed non-significant changes after stimulation with IGF-I.

Moreover, we found that DDR1 protein upregulation requires the activation of the PI3K/Akt pathway, while the Erk1/2, the p70/mTOR and the PKC pathways are not involved.

Indeed, IGF-I induced DDR1 protein upregulation through a post-trascriptional mechanism involving miR-199a-5p suppression via Akt. We also found that autocrine IGF-II, induced by stable IGF-II cDNA transfection, is also able to elicit significant DDR1 upregulation in breast cancer cells. Similar effects were induced by IGF-II secreted by human cancer associated fibroblasts.

Finally, we showed that miR-199a-5p transfection in breast cancer cells, decreased DDR1 expression and affected the response to IGF-I in terms of intracellular signaling through the PI3K/Akt and Erk1/2 pathways, and biological effects such as cell proliferation and migration.

These results demonstrate that the protumoral effects of IGF-I include downregulation of miR-199a-5p and consequent upregulation of DDR1 protein. Both these molecules may represent important targets for breast cancers with overactivation of the IGF-IR axis.

Citation Format: Chiara Palladino, Roberta Matà, Maria Luisa Nicolosi, Anna Rita Lo Presti, Roberta Malaguarnera, Marco Ragusa, Daniela Sciortino, Andrea Morrione, Marcello Maggiolini, Veronica Vella, Antonino Belfiore. In breast cancer cells IGF-I induces upregulation of DDR1 by suppressing miR-199a-5p via the PI3K/Akt pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4612.
Abstract 4613: SMYD3-mediated lysine methylation is critical for the activation of AKT1 in human cancer
Authors: Yoshioka Yuichiro , Nakamura Yusuke , Hamamoto Ryuji
Published: July 2016

v-Akt Murine Thymoma Viral Oncogene Homolog 1 (AKT1), a serine/threonine-protein kinase also known as protein kinase B Alpha (PKB Alpha), is a key mediator of a signaling pathway that governs various cellular processes regulating cell growth, survival, glucose metabolism, genome stability and neovascularization. It is known that dysregulation of AKT1 activity is one of the critical factors for the development and/or progression of a variety of human cancers. Posttranslational modifications of AKT1, including phosphorylation, ubiquitination and glycosylation, are well-analyzed, and it is known that the phosphorylation status of AKT1 is strongly correlated with its oncogenic activity. However, biological functions of methylation on AKT1 have never been analyzed so far. In the present study, we demonstrated that the protein lysine methyltrasnferase SMYD3 methylates lysine 14 in the PH domain of AKT1, which is known to play a critical role in the regulation of AKT1 activity through binding to phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3) or phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2), in vitro and in vivo. Lysine 14-substituted AKT1 shows significantly lower levels of phosphorylation at threonine 308, which is the major indicator of AKT1 activity, than wild-type AKT1, and knockdown of SMYD3 as well as treatment with a SMYD3 inhibitor significantly attenuates this phosphorylation in cancer cells. Furthermore, substitution of lysine 14 diminishes the plasma membrane accumulation of AKT1, and cancer cells overexpressing lysine 14-substiuted AKT1 shows lower growth rate than those overexpressing wild-type AKT1. These results imply that SMYD3-mediated methylation of AKT1 at lysine 14 plays a central role in the oncogenic activity of AKT1. Although Phosphatidylinositide 3-kinase (PI3K)-dependent AKT1 phosphorylation by Pyruvate Dehydrogenase Kinase, Isozyme 1 (PDK1) and mTOR Complex 2 (mTORC2) has long been considered to be the primary mechanism accounting for AKT1 activation, these members are not sufficient enough to explain how AKT1 hyperactivation can occur in tumors with normal levels of PI3K/Phosphatase And Tensin Homolog (PTEN) activity. Our findings unveiled the novel mechanism of constitutive hyperphosphorylation on AKT1 in cancer cells, mediated by SMYD3-mediated methylation. The inhibition of this methylation pathway appears to be a promising strategy to develop anti-cancer drugs.

Citation Format: Yuichiro Yoshioka, Yusuke Nakamura, Ryuji Hamamoto. SMYD3-mediated lysine methylation is critical for the activation of AKT1 in human cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4613.
Abstract 4622: Identification of p62/SQSTM1 as a component of non-canonical WNT VANGL2-JNK signaling in breast cancer
Authors: Borg Jean-Paul , Tania Puvirajesinghe , Bertucci François , Birnbaum Daniel , Jain Ashish , Johansen Terje , Scerbo Pierluigi , Kodjabachian Laurent
Published: July 2016

Planar cell polarity (PCP), an evolutionarily conserved developmental process originally described in Drosophila melanogaster, is essential to the control of tissue morphogenesis and development of polarized structures in metazoans. Its importance in human diseases is best demonstrated in polycystic kidney disease, hearing loss, Bardet Biedl syndrome, neural tube closure defects and cancer. PCP involves a set of evolutionarily conserved PCP genes encoding WNT ligands, transmembrane (vangl2, frizzled, fat, dachsous) and cytoplasmic (scribble, prickle, dishevelled, diego) molecules triggering a genetically well-defined non-canonical WNT-JNK pathway. Recent work has linked defects of this pathway to breast cancer aggressiveness and proposed non-canonical WNT/PCP signaling as a therapeutic target. Here we show that the archetypal non-canonical WNT/PCP protein VANGL2 is overexpressed in basal breast cancers, associated with poor prognosis and implicated in tumor growth. We identify the scaffold p62/SQSTM1 protein as a novel VANGL2-binding partner and show its key role in an evolutionarily conserved VANGL2-p62/SQSTM1-JNK pathway in breast cancer cells and in Xenopus. This proliferative signaling cascade is upregulated in breast cancer patients with shorter survival, and can be inactivated in patient-derived xenograft cells by inhibition of the JNK pathway or by disruption of the VANGL2-p62/SQSTM1 interaction. VANGL2-JNK signaling is thus a potential target for breast cancer therapy.

Puvirajesinghe T.M., et al. Identification of p62/SQSTM1 as a component of non-canonical Wnt VANGL2-JNK signaling in breast cancer. Nature Communications, in press. Sebbagh M. and Borg J.-P. Insight into Planar Cell Polarity. (2014) Invited review in Experimental Cell Research, 328:284-295. Belotti E., et al. The human PDZome: a gateway to PDZ mediated functions. (2013) Mol Cell Proteomics, 12: 2587-603.

Citation Format: Jean-Paul Borg, Puvirajesinghe Tania, François Bertucci, Daniel Birnbaum, Ashish Jain, Terje Johansen, Pierluigi Scerbo, Laurent Kodjabachian. Identification of p62/SQSTM1 as a component of non-canonical WNT VANGL2-JNK signaling in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4622.
Abstract 4628: The RSK inhibitor SL0101 impairs the proliferation of glioblastoma cell lines
Authors: Roffe Martín , Lupinacci Fernanda C. , Soares Luana C. , Hajj Glaucia N. , Martins Vilma R.
Published: July 2016

Glioblastoma (GBM) is the most common and lethal type of brain tumor. The most frequently altered signaling pathway in this tumor is the RTKs-PI3K-Akt-mTORC1 (88% of cases). However, clinical trials for GBM using inhibitors of this pathway exhibited poor clinical responses. One of the main responsibles for this resistance is the compensatory activation of the Ras-ERK1/2 pathway. In fact, GBMs can also present alterations in the Ras-ERK1/2 pathway, such as mutation of NF1 in 18% of the cases. RSK is a direct target of ERK1/2 and has been shown to regulate important functions in different types of tumors, such as prostate and breast. RSK is a family of kinases comprised by four highly-homologous isoforms in humans (RSK1-4) and nothing is known about their functions in GBMs. Here we show that RSK1 levels are highly variable between cells, the levels of RSK2 are less variable, and RSK3 and RSK4 were not detected. To gain insight into the function of RSK, we studied the effects of the RSK inhibitor, SL0101, on the proliferation of four different GBM cell lines (LN18, LN229, U87MG and A172). A suboptimal concentration of SL0101 (50 μM) was able to inhibit basal and EGF-stimulated proliferation, and it was more potent than 100 nM rapamycin in some of the cells. In 3 of the cell lines, SL0101 inhibited serum-stimulated proliferation, and in 2 of them, it was more potent than rapamycin. We have previously shown that SL0101 can inhibit mTORC1 signaling in an ERK1/2-RSK independent manner. However, SL0101 showed a synergistic interaction with rapamycin in all of the cell lines, which suggests that other effects besides RSK-mTORC1 inhibition might be involved in the effect of SL0101. This was further suggested by the modest synergistic interaction between rapamycin and the RSK inhibitor, fmk. Altogether, these results indicate that SL0101 is a potent inhibitor of proliferation in a manner not related with the RSK1 status of GBM cells. Further studies to define the molecular mechanism of the effects of SL0101 are necessary to understand which pathways are indeed modulated.

Citation Format: Martín Roffe, Fernanda C. Lupinacci, Luana C. Soares, Glaucia N. Hajj, Vilma R. Martins. The RSK inhibitor SL0101 impairs the proliferation of glioblastoma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4628.
Abstract 4631: SCFFbxo7 ubiquitin ligase targets cereblon to limit the anti-myeloma activity of IMiDs
Authors: Liu Jiye , Zhou Wenrong , Feng Yan , Song Tianyu , Gao Shaobing , Wu Junfeng , Peng Zhenggang , Cang Yong
Published: July 2016

Multiple myeloma (MM) patients have witnessed significantly improved survival after the approved treatment with the proteasome inhibitor bortezomib and immunomodulatory drugs (IMiDs) including thalidomide and its derivatives lenalidomide and pomalidomide. IMiDs directly bind cereblon (CRBN), an adaptor of the Cul4-DDB1 (CRL4) ubiquitin ligase, and activate CRL4CRBN to target B cell-specific transcription factors IKZF1 and IKZF3 for ubiquitination. IKZF1 and IKZF3 are essential for MM cell survival and their proteasomal degradation induced by IMiDs provides insights into how these drugs work. Paradoxically, IMiDs achieve better clinical response in MM patients when combined with bortezomib, which blocks IKZF1 and IKZF3 turnover and would in principle counteract IMiDs therapy. We performed a CRISPR-based genome-wide knockout screening for regulators of pomalidomide sensitivity in MM cells, and found that CRBN is inherently unstable and its level is controlled by the COP9 signalosome (CSN) to sustain IMiDs cytotoxicity. When unbound to CRL4, CRBN is recruited by the F-box protein Fbxo7 to the Cul1-Skp1 (SCF) ubiquitin ligase for ubiquitination and degradation, a process suppressed by CSN-mediated de-neddylation of Cul1. Deletion of SCFFbxo7 components, or treatment with bortezomib or neddylation inhibitor MLN4924 stabilizes CRBN, and IMiDs further promote the loading of CRBN to CRL4 by inhibiting SCFFbxo7-targeted CRBN degradation, leading to enhanced turnover of IKZF1 and IKZF3 in MM cells and synergistic inhibition of MM cell proliferation. Our findings unravel a dual role of IMiDs in activating CRL4CRBN E3 ligase in MM cells and provide a framework to select patients most susceptible to bortezomib and IMiDs combination therapy.

Citation Format: Jiye Liu, Wenrong Zhou, Yan Feng, Tianyu Song, Shaobing Gao, Junfeng Wu, Zhenggang Peng, Yong Cang. SCFFbxo7 ubiquitin ligase targets cereblon to limit the anti-myeloma activity of IMiDs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4631.
Abstract 4638: Targeted therapy of precancerous fields in the mucosal linings of the head and neck
Authors: de Boer D Vicky , Martens-de Kemp Sanne R. , Buijze Marijke , Stigter-van Walsum Marijke , Bloemena Elisabeth , Leemans C René , Braakhuis Boudewijn J.M. , Brakenhoff Ruud H.
Published: July 2016

Primary head and neck squamous cell carcinomas (HNSCC) and local relapses develop in preneoplastic fields in the mucosal linings of the upper aerodigestive tract. These fields are characterized by tumor-associated genetic changes, show frequently dysplastic features and are occasionally macroscopically visible as lesions. Currently, there are no adequate treatment options for these fields to prevent tumor development. To develop a targeted therapeutic approach we established four unique preneoplastic cell lines, derived from fields adjacent to HNSCCs. Next, we performed a screen with a selective panel of small interfering RNAs (siRNAs) that are lethal for HNSCC cells, to identify genes essential for survival of preneoplastic cells and not for normal cells. One of these selective essential genes was Polo-like kinase 1 (PLK1). Inhibition of PLK1, both with siRNAs as well as small molecule inhibitors, caused cell death of preneoplastic and HNSCC tumor cells, while primary fibroblasts and keratinocytes were hardly affected. Targeting PLK1 caused a strong G2/M cell cycle arrest in preneoplastic cells accompanied by formation of monopolar spindles during mitosis. Hence, PLK1 is a promising therapeutic target for chemopreventive eradication of preneoplastic fields in patients at high risk of developing HNSCC and local relapses.

Citation Format: D Vicky de Boer, Sanne R. Martens-de Kemp, Marijke Buijze, Marijke Stigter-van Walsum, Elisabeth Bloemena, C René Leemans, Boudewijn J.M. Braakhuis, Ruud H. Brakenhoff. Targeted therapy of precancerous fields in the mucosal linings of the head and neck. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4638.
Abstract 4644: Inhibition of the androgen receptor at two drug-targetable sites on the DNA-binding domain protein surface
Authors: Dalal Kush , Sharma Aishwariya , Roshan-Moniri Mani , Borgmann Hendrik , Lallous Nada , Awrey Shannon , Li Huifang , Ban Fuqiang , LeBlanc Eric , Cherkasov Artem , Rennie Paul S.
Published: July 2016

The androgen receptor (AR) is a hormone inducible transcription factor that continues to be an important drug-target to prevent or slow the progression of prostate cancer. Current small molecule inhibitors, such as Enzalutamide (anti-androgens), compete with naturally occurring steroids that bind to the hormone binding pocket of the AR ligand binding domain (LBD). In advanced or castration resistant prostate cancer (CRPC), mutations in the LBD confer drug-resistance by converting anti-androgens into agonists, prompting research to develop small molecule inhibitors that target different sites on the AR protein surface. Recently, we characterized a set of small molecules that could interact with the DNA binding domain (DBD) of the AR and block its transcriptional activity. Here, we extend the pioneering observations by clarifying the mechanism of two classes of compounds that either block AR-chromatin interactions or reduce AR-dimerization in the cell nucleus. Compound efficacy is demonstrated across multiple prostate cancer cells lines, including enzalutamide resistant forms, with respect to AR transactivation, cell viability and expression of downstream genes. We also characterize the pharmacological properties of the lead compound and its effects on tumour xenografts in mice. Collectively, these results lay the foundation for the development of alternative prostate cancer drugs that interfere with the biochemical function of the activated, nuclear localized AR.

Citation Format: Kush Dalal, Aishwariya Sharma, Mani Roshan-Moniri, Hendrik Borgmann, Nada Lallous, Shannon Awrey, Huifang Li, Fuqiang Ban, Eric LeBlanc, Artem Cherkasov, Paul S. Rennie. Inhibition of the androgen receptor at two drug-targetable sites on the DNA-binding domain protein surface. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4644.
Abstract 4660: Inhibition of p38 enhances ERK inhibitor efficacy in pancreatic ductal adenocarcinoma
Authors: Ryan Meagan B. , Bryant Kirsten L. , Hayes Tikvah K. , Kher Swapnil , Wood Kris C. , Samatar Ahmed A. , Cox Adrienne D. , Der Channing J.
Published: July 2016

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths in the United States, with a poor prognosis and limited treatment options. Oncogenic mutation of KRAS in greater than 90% of PDAC leads to aberrant activation of multiple effector pathways including the extra cellular related kinase (ERK)/mitogen activated protein kinase (MAPK) cascade. Hyperactivation of the ERK MAPK cascade has been correlated with poorer prognosis in PDAC patients. We recently showed that direct pharmacological inhibition of ERK1/2 kinases with the ERK1/2-selective inhibitor SCH772984 inhibits the growth of PDAC cell lines both in vitro and in vivo. However, much like the response to ERK/MAPK pathway inhibitors acting at upstream nodes RAF or MEK, resistance to direct inhibition at the level of ERK will also inevitably arise. We performed a novel gain-of-function “Cancer Toolkit” (CTK) genetic screen to identify mechanisms of resistance to the ERK inhibitor SCH772984 in a panel of KRAS-mutant PDAC cell lines. Our CTK screen revealed that expression of either MAPK14 (p38α) or its upstream activator MKK6 could cause resistance to this and other ERK inhibitors. Because we identified activation of the p38 pathway as a mechanism of ERK inhibitor resistance, we tested whether inhibition of p38 would enhance sensitivity to SCH772984. We observed a >2 fold shift to a lower GI50 of SCH772984 upon cotreatment of a panel of established and patient-derived xenograft (PDX) PDAC cell lines with SCH772984 and the clinical candidate p38 inhibitor LY2228820. Consistent with this, we found that SCH772984 induced p38 signaling, marked by phosphorylation of the downstream substrate HSP27, and that combination treatment of SCH772984 and LY2228820 both reversed p38 pathway activation and enhanced PARP cleavage. Results of in vivo testing of the combination treatment as well as the mechanistic basis for p38-driven resistance will be reported. We propose that p38 is a mechanism of resistance to ERK inhibition in PDAC and that p38 inhibitors such as LY2228820 can overcome that resistance to enhance ERK inhibitor efficacy.

Citation Format: Meagan B. Ryan, Kirsten L. Bryant, Tikvah K. Hayes, Swapnil Kher, Kris C. Wood, Ahmed A. Samatar, Adrienne D. Cox, Channing J. Der. Inhibition of p38 enhances ERK inhibitor efficacy in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4660.
Abstract 4661: Loss of LKB1 in NSCLC confers sensitivity to MEK inhibition by regulating activation of AKT-FOXO3 pathway
Authors: Yamada Tadaaki , Kaufman Jacob M. , Amann Joseph M. , Yano Seiji , Carbone David P.
Published: July 2016

The use of MEK inhibitors for non-small cell lung cancer (NSCLC) has shown little efficacy in clinical trials, even in the case of tumors with mutant KRAS where one might predict good outcomes. From these data, it is clear that the success of MEK inhibitors is going to rely on finding a biomarker that predicts sensitivity to this type of therapy. Our area of interest is finding a way to treat LKB1 mutant tumors and, surprisingly, an in silico screen of drug sensitivity data for NSCLC cell lines determined that four MEK inhibitors were among the top drugs that were significantly associated with LKB1 loss. We confirmed the effects MEK inhibition by evaluating 23 lung cancer cell lines with known LKB1 status. The results of the study showed that MEK inhibition with trametinib led to a significant reduction in cell viability in LKB1 mutant cell lines when compared to cell lines with wild type LKB1. In addition, we investigated MEK sensitivity by restoring wild-type (Wt) LKB1 in lung cancer cell lines with LKB1 loss, or by silencing LKB1 in lung cancer cell LKB1-Wt lines. Transduction of LKB1 resulted in significant MEK resistance in three of the five LKB1 add-back lines, while silencing LKB1 induced MEK sensitivity in all five LKB1-Wt lines tested. The mechanism behind these observed results appears to be through phosphorylation of AKT and its downstream target FOXO3, which are important determinants of the apoptotic response to MEK inhibition. With LKB1 transduction into mutant cell lines we see an increase in the activating phosphorylation of AKT, a protein involved in survival mechanisms, and an increase in the deactivating phosphorylation of FOXO3, a transcription factor implicated in increased levels of apoptosis. Our data suggest that the identification of LKB1 activity may be promising biomarker for the sensitivity to MEK inhibition by regulating activation of AKT-FOXO3 pathway in NSCLC.

Citation Format: Tadaaki Yamada, Jacob M. Kaufman, Joseph M. Amann, Seiji Yano, David P. Carbone. Loss of LKB1 in NSCLC confers sensitivity to MEK inhibition by regulating activation of AKT-FOXO3 pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4661.