bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2290: The serine-threonine kinase Mink1 as a potential therapeutic target in triple negative breast cancer
Authors: Daulat Avais , Wagner Monica Silveira , Kowalczewska Malgorzata , Finetti Pascal , Castellano Rémy , Audebert Stéphane , Bertucci François , Camoin Luc , Borg Jean-Paul
Published: July 2021

The developmental Wnt/planar cell polarity (PCP) pathway is the most recently described branch of the Wnt signaling pathways and is strongly implicated in cancer development at early and late stages (PMID: 28718442 and PMID: 30952630). Upregulation of the Wnt/PCP components is observed in many cancers and is associated with cancer progression. However, how their molecular functions are regulated remains an open question. Recent data from our laboratory showed that Prickle1 and Vangl2, two core Wnt/PCP components, are overexpressed in Triple Negative Breast Cancers (TNBC) and associated with poor prognosis (PMID: 27184734 and 26754771). Prickle1 is a cytoplasmic protein phosphorylated by the serine/threonine kinase Mink1, which triggers Prickle1 localization at the plasma membrane and regulates its activity (PMID: 22037766). Activation of this axis contributes to breast cancer cell motility and metastatic spreading in vitro and in vivo (PMID: 27184734). To extend our knowledge of the Mink1 substrates, we carried out a phosphoproteomic strategy and identified LL5β. LL5β is a membrane scaffold molecule that anchors microtubules (MTs) at the cell cortex through its association with CLASP, a plus-end MT protein, to trigger focal adhesion disassembly. We found that LL5β is a prominent member of the Prickle1-associated protein complex and harbors a consensus motif for Mink1 phosphorylation. At the molecular level, we demonstrated a two-step phosphorylation cascade carried out by Mink1 that phosphorylates sequentially Prickle1 to mediate its association with LL5β, then LL5β to promote its interaction with CLASP. Finally, analysis of gene expression data suggests that the concomitant up-regulation of Prickle1 and LL5β mRNA levels is associated with a poor metastasis-free survival for TNBC patients. In an effort to counteract the Mink1-Prickle1-LL5β prometastatic pathway, we selected a Mink1 inhibitor that recapitulates all the phenotypes observed following the knockdown of Mink1 expression. This chemical compound is currently under investigation in preclinical assays using xenografted TNBC cell lines and patient-derived xenografts in nude mice.

Citation Format: Avais Daulat, Monica Silveira Wagner, Malgorzata Kowalczewska, Pascal Finetti, Rémy Castellano, Stéphane Audebert, François Bertucci, Luc Camoin, Jean-Paul Borg. The serine-threonine kinase Mink1 as a potential therapeutic target in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2290.
Abstract 2298: A potent and selective EGFR/HER2 exon 20 mutations inhibitor NIP142 induced tumor regression
Authors: Yin Huijun , Yan Xu , Tian Weixue , Chu Guobiao , Wang Zhuo , Zhao Xin , zhang lu , Liu Xile , Ding Charles Z. , Hu Lihong , Li Chengyuan , Bai Gang , Xia Yuanfeng , Chan Chi-Chung , Chen Shuhui
Published: July 2021

Objective: Approximately 1.6-3.4% and 2-4% of NSCLC patients harbor EGFR or HER2 exon20 insertion mutations, respectively. No targeted therapy has been approved for these patients yet. NIP142 is a novel EGFR/HER2 inhibitor with potent inhibitory activities against above mentioned mutations. Purpose of this study is to provide evidence to support NIP142 as a clinical candidate for treatment of NSCLC driven by EGFR or HER2 exon20 insertion mutations.

Method: Enzymatic activities of NIP142 were evaluated in EGFR/HER2 exon20 insertion mutants, including EGFR (D770_N771insNPG) and HER2 (V777_G778ins>CG). Anti-proliferative activities of NIP142 were evaluated in Ba/F3 cell lines with exon20 mutants (such as EGFR(D770_N771insSVD), EGFR(H773_V774insNPH), EGFR(V769_D770insASV), HER2(A775_G776insYVMA)), LU0387(EGFR-H773_V774insNPH), NCI-H1975 (EGFRL858R/T790M) and A431 (EGFRWT) cell lines. In vivo efficacy was evaluated in genetically engineered Ba/F3 (EGFR-D770_N771insSVD) mouse model, and LU0387 (EGFR-H773_V774insNPH) PDX model.

Result: NIP142 displayed potent enzymatic inhibition activities in exon20 mutants, such as EGFR (D770_N771insNPG) (IC50 = 0.5 nM), HER2 (V777_G778ins>CG) (IC50 = 0.67 nM) and potent anti-proliferative activities in Ba/F3 cell lines with exon20 mutants, EGFR-D770_N771insSVD (IC50 = 32 nM), EGFR-H773_V774insNPH (IC50 = 32 nM), EGFR-V769_D770insASV (IC50 = 22 nM), HER2 A775_G776insYVMA (IC50 = 30 nM) respectively. NIP142 also exhibited potent anti-proliferative activities in NCI-H1975 (EGFRT790M\L858R) (IC50 = 9.4 nM), LU0387 (EGFR-H773_V774insNPH) (IC50 = 25 nM), and moderate anti-proliferation activity in EGFRWT A431cell line (IC50 = 37 nM). NIP142 was investigated in various CDX models. In particular, NIP142 significantly inhibited tumor growth in the LU0387 (EGFR-H773_V774insNPH) PDX tumor model of NSCLC, and tumor regression was achieved at 15 mg/kg.

Conclusion: NIP142 is a potent EGFR/HER2 exon20 insertions inhibitor and showed excellent activities in preclinical in vitro assays and relevant tumor models. These results together with its preclinical safety data (not shown) support NIP142 to enter the clinical research to explore its potential for treating NSCLC with EGFR/HER2 exon20 insertions.

Citation Format: Huijun Yin, Xu Yan, Weixue Tian, Guobiao Chu, Zhuo Wang, Xin Zhao, lu zhang, Xile Liu, Charles Z. Ding, Lihong Hu, Chengyuan Li, Gang Bai, Yuanfeng Xia, Chi-Chung Chan, Shuhui Chen. A potent and selective EGFR/HER2 exon 20 mutations inhibitor NIP142 induced tumor regression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2298.
Abstract 2300: Overcoming TKI-resistance in NSCLC using SphK2 inhibitors
Authors: Khan Nabiha Haleema , Puri Neelu
Published: July 2021

In 2020, 228,820 lung cancer cases and 135,720 deaths have been anticipated. NSCLC is currently treated with Tyrosine Kinase Inhibitors (TKIs) such as Erolitinib (ER; second generation) and Osimertinib (OR; third generation). However, patients often develop resistance to these drugs overtime. This acquired resistance to TKIs is due to a secondary T790M mutation in the kinase domain which could be responsible for inducing Epithelial Mesenchymal Transition (EMT). EMT is a vital process in development of metastasis and occurs, by downregulation of E-cadherin, and upregulation of proteins like N-cadherin and vimentin. Therefore, it is imperative to find other molecularly targeted therapies to combat TKI resistance. Sphingosine kinase (SphK) is a type of lipid kinase that phosphorylates sphingosine to sphingosine-phosphates. There are two functional SphK isoforms (SphK1 and SphK2) that have been identified and characterized in mammalian cells. As a member of the SphK family, SphK2 activity can be stimulated by a series of cytokines such as EGF and VEGF, and acts as an oncogenic enzyme in tumor cells. Overexpression of SphK2 has been demonstrated in many cancer types and is closely related with cancer progression and the poor survival of patients. Currently minimal studies have been done on lung cancer and SphK2. This study investigated the roles of SphK2 in OR-resistant wild-type EGFR H2170 and H358, EGFR-mutated and TKI-sensitive H3255 cells (L858R mutation), and EGFR mutated TKI-resistant H1975 cells (L858R and T790M mutation). The modulation of EMT biomarkers was determined by qPCR. Key EMT regulators such as vimentin and N-cadherin were upregulated by 2.5 and 1.5-fold, respectively, while E-cadherin was downregulated by 1.5-fold. Immunofluorescence studies with Vimentin showed that H1975OR cells were elongated and have a mesenchymal phenotype compared to the H3255OR and their respective parental counterparts. MTT studies done using inhibitors of SphK2 in NSCLC OR-resistant cells showed a 3.0-fold decrease in cell viability in comparison to a 2.0-fold decrease in their parental counterparts. In wound closure assays at 24 hours the wound closure was 1.5-fold less in cells treated with SphK2 inhibitor as compared to parental cells treated with SphK2 inhibitors. Currently studies are being done to determine expression of SphK2 in late and early stage NSCLC tumors. In conclusion SphK2 was found to be a crucial positive regulator of different modes of cell migration and a key biomarker in combating TKI-resistance in NSCLC.

Citation Format: Nabiha Haleema Khan, Neelu Puri. Overcoming TKI-resistance in NSCLC using SphK2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2300.
Abstract 2324: A novel oncogene mediated metabolic gene signature predicts breast cancer outcome
Authors: Aslan Merve , Hsu En-Chi , Marques Fernando J. Garcia , Bermudez Abel , Shen Michelle , Rice Meghan A. , Liu Shiqin , West Robert , Pitteri Sharon J. , Győrffy Balázs , Stoyanova Tanya
Published: July 2021

Introduction and Objectives: Breast cancer remains the second most lethal cancer among women in the US, with triple negative breast cancer (TNBC) as the most aggressive subtype with limited treatment options. Trop2, a cell membrane glycoprotein, is overexpressed nearly all epithelial cancers including triple negative breast cancer. In this study, we demonstrate that loss of Trop2 derives downregulation of metabolic genes. Trop2 oncogene-driven metabolic gene signature is highly elevated in breast cancer patients and predicts poor outcome.

Methods: Trop2 expression levels in two independent patient cohorts were analyzed with immunohistochemistry. Trop2 was knockdown in TNBC cell line HCC1806 with shRNA and knock-out in HCC1806 with CRISPR Cas9 system. Trop2 downregulation or gene deletion effect was tested in vitro with colony formation and 3D Matrigel invasion assays. The effect of Trop2 downregulation in tumorigenesis was evaluated in vivo by subcutaneous implantation of control and Trop2 knockdown xenografts. Proteomic changes were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Trop2 led to downregulation of TALDO1 associated metabolic cluster genes which expression levels were further investigated in four independent breast cancer cohort. Finally, the correlation of Trop2 oncogene-driven metabolic gene signature with breast cancer outcome was analyzed in 13 independent breast cancer patient datasets.

Results: We demonstrate Trop2 is overexpressed in TNBC, and downregulation of Trop2 disrupts TNBC cell and tumor growth. Through whole proteomic profiling, we discovered a novel metabolic cluster that was downregulated in Trop2-depleted breast cancer tumors. The identified oncogene-driven metabolic gene signature is significantly upregulated in TNBC patients across multiple clinical datasets. Our study further reveals that the metabolic gene signature reliably predicts poor survival of breast cancer patients with early stage of the disease.

Conclusion: Our study identifies a new five gene metabolic oncogene-driven signature as a prognostic predictor of breast cancer.

Citation Format: Merve Aslan, En-Chi Hsu, Fernando J. Garcia Marques, Abel Bermudez, Michelle Shen, Meghan A. Rice, Shiqin Liu, Robert West, Sharon J. Pitteri, Balázs Győrffy, Tanya Stoyanova. A novel oncogene mediated metabolic gene signature predicts breast cancer outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2324.
Abstract 2364: MicroRNA-1205 directly targets ONECUT2 in neuroendocrine prostate cancer cells
Authors: Dieujuste Bachelard , Naidoo Michelle , Ogunwobi Olorunseun
Published: July 2021

One of six deaths globally is due to cancer. In 2018, prostate cancer (PCa) represented 1.28 million cases worldwide, the second most frequent cancer diagnosis and fifth leading cause of death in men. Currently, the mortality rate of PCa has experienced a steady increase due to the progression of metastatic castration-resistant prostate cancer (mCRPC). As an androgen driven disease, androgen deprivation therapies (ADTs) have been employed for treatment. However, despite ADTs, mCRPC develops by maintaining AR signaling, which can eventually lead to progression of AR-independent neuroendocrine PCa (NEPC). NEPC is observable in 1 in 5 men with mCRPC and associated with poor outcome. Therefore, further understanding the mechanisms of progression to mCRPC and NEPC is required. The 8q24 human chromosomal region associated with Increased PCa incidence and aggressiveness contains the PVT1 (Plasmocytoma Variant Translocation 1) locus. The PVT1 locus encodes six microRNAs, including microRNA-1205 (miR-1205) in PCa. Our laboratory previously confirmed that miR-1205 is underexpressed in PCa tissue in comparison to normal prostatic tissue and in CRPC cells in comparison to non-CRPC cells. To understand the molecular mechanism of miR-1205, we performed RNA pull down and RNA sequencing analysis to identify miR-1205 molecular targets in non-tumorigenic RWPE-1 prostate epithelial cells and PCa cells (MDA PCa 2b (metastatic PCa), PC-3 (NEPC), and C4-2B (CRPC)). We identified one cut homeobox 2 (ONECUT2 or OC2) as a direct molecular target of miR-1205. ONECUT2 is a master regulator of transcriptional factors that drives NEPC by through regulating hypoxia signaling or inducing neuroendocrine markers. ONECUT2 enrichment was approximately 3-fold higher in RWPE-1 (P=0.26), 2-fold in PC-3 (P=0.06), 1-fold in C4-2B (P=0.05) and MDA PCa 2b (P=0.02) cells. The significant binding of miR-1205 to ONECUT-2 in the PC-3 NEPC cell line suggests that miR-1205 regulation of ONECUT2 may be an important molecular mechanism in NEPC. Consequently, further study of miR-1205 and ONECUT2 in NEPC is required.

Citation Format: Bachelard Dieujuste, Michelle Naidoo, Olorunseun Ogunwobi. MicroRNA-1205 directly targets ONECUT2 in neuroendocrine prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2364.
Abstract 2369: Tumor-suppressive efficacy of let-7b microRNA against lung carcinogenesis is mediated by modulating the tumor microenvironment
Authors: Zhang Qi , Pan Jing , Xiong Donghai , Wang Yian , Miller Mark S. , Izzotti Alberto , You Ming
Published: July 2021

Lung cancer is the leading cause of mortality worldwide. MicroRNAs (miRNAs) are potential candidates for lung cancer therapy. However, a major limitation is the lack of an efficient delivery system to directly deliver miRNA to cancer cells while limiting exposure to healthy cells. In our previous studies on aerosolized let-7b in lung cancer prevention, let-7b showed good inhibition of B[a]P-induced lung adenoma with no side effects. In this study, we found that aerosolized let-7b decreased tumor growth in the LKR13 (KRAS mutant) syngeneic mouse model. Let-7b post-transcriptionally suppresses PD-L1 and PD-1 expression in the tumor immune microenvironment, suggesting that let-7 microRNAs may promote antitumor immunity in vivo. Single cell RNA sequencing (scRNAseq) data showed that let-7b treatment decreased the expression of PD-1 in CD8+ T cells and reduced PD-L1 expression in lung tumor cells. Let-7b treatment also significantly changed the percentages of distinct CD8+ tumor-infiltrating lymphocytes (TIL) states. The proportion of CD8+ T cells mediating anti-tumor functions (EM-like CD8+ TILs) was increased significantly by let-7b treatment compared to control. In contrast, the CD8+ T cell subpopulation that has negative effects on anti-tumor immune response, exhausted CD8+ TILs, was significantly decreased by let-7b. Flow cytometry data showed that Let-7b treatment led to the accumulation of CD8+ T cells, granzyme B+ CD8+ T cells and IFN-γ+ CD8+ T cells in tumors, and a decrease of intratumoral Granulocyte-like myeloid derived suppressor cells (G-MDSC) cells. Our results suggest that the in vivo tumor-suppressive efficacy of let-7 is mediated, at least in part, by immune-promoting effects via down-regulating PD-L1 in tumors and/or PD-1 on CD8+ T cells.

Citation Format: Qi Zhang, Jing Pan, Donghai Xiong, Yian Wang, Mark S. Miller, Alberto Izzotti, Ming You. Tumor-suppressive efficacy of let-7b microRNA against lung carcinogenesis is mediated by modulating the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2369.
Abstract 2376: Diagnostic and predictive biomarkers of radiation pneumonitis
Authors: Yadav Marshleen , Liu Joseph , Qamri Zahida , Jacob Naduparambil K.
Published: July 2021

Increased rates of treatment failure in lung cancer patients necessitate treatment with high doses of radiation, which often cause toxicities. However, normal tissue injury in lung may manifest as acute toxicities or as delayed radiation pneumonitis. Identification of early indicators of delayed toxicities will allow timely management and mitigation. In present study, using mouse model, we identified a set of circulating microRNAs as biomarkers that potentially allow early detection of radiation induced lung injury such as inflammation and pneumonitis. Longitudinal analysis of serum samples collected from mice exposed to whole thorax lung irradiation (WTLI) allowed discovery of a panel of evolutionary conserved miRNAs that are potential predictors of radiation pneumonitis. Among these, miR21 and miR29a, miR130a, and miR19a were found peaked between 2-4 weeks after radiation, while miR146a exhibited a peculiar biphasic expression. Some of these changes were found to be associated with concomitant changes in anti- and pro-inflammatory serum cytokines. Several of the biomarkers identified in WTLI model, showed overlapping response in a bleomycin mouse model of acute lung injury. We further tested the translational utility of these biomarkers in single drop blood collected from mice using a validated internal normalizer, miR23a (Yadav et al., Science Translational Medicine, 2020, PMID: 32669422). The response noted for several of the candidate biomarkers identified in mouse model are conserved in specimens collected from human patients who received radiation therapy. In conclusion, our study has identified a panel of microRNA biomarkers with potential to develop as early predictor of pulmonary toxicities in patients receiving therapeutic radiation, and in victims of radiation accidents for timely mitigation.

Citation Format: Marshleen Yadav, Joseph Liu, Zahida Qamri, Naduparambil K. Jacob. Diagnostic and predictive biomarkers of radiation pneumonitis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2376.
Abstract 2384: Hypoxia-induced exosomal miR-376c-3p promotes metastatic potential of ovarian cancer cells
Authors: Jo HyunA , Wang Wenyu
Published: July 2021

Background: Hypoxia significantly impacts cancer progressions, in various types of solid tumors including ovarian cancer. Exosomes (30-150nm) secreted from cells in the hypoxic tumor microenvironment play a critical role in cancer progression through transferring functional proteins, mRNAs, and miRNAs as mediators of intercellular communication. However, the underlying mechanisms how cancer exosomes induced by hypoxia regulate cancer progressions remain elusive. This study aims to assess the effect of exosomes secreted from hypoxic cancer cells on the metastatic ability of ovarian cancer cells.

Methods: Two ovarian cancer cell lines, Kuramochi and SKOV-3, were used. Exosomes from cancer cells cultured under normoxic or hypoxic conditions (normoxic or hypoxic exosomes) were isolated using Exoquick-TC kit and identified by nanoparticle tracking analysis, transmission electron microscopy, and western blotting. BCA assay was done to evaluate the concentration of exosomes. Normoxic or hypoxic exosomes were treated on ovarian cancer cells. Cell migration and invasion abilities were examined by wound healing assay and MatriGel invasion assay respectively. Furthermore, miRNA sequencing was performed to explore the miRNA profiling of hypoxic and normoxic exosomes.

Results: The size of exosomes was ranging from 52nm to 121nm (mean diameter = 85nm). The quantity of hypoxic exosomes was significantly more than that of normoxic exosomes. Hypoxic exosomes increased the migration and invasion ability of kuramochi and SKOV-3 cells. In addition, hypoxic exosomes treatment on ovarian cancer cells up-regulated the expression of MMP-2 and Snail-1 at the protein level. MiRNA sequencing showed that miR-376c-3p was significantly upregulated in hypoxic exosomes in both ovarian cancer cell lines. Moreover, miR376c-3p enhanced migration and invasion ability of ovarian cancer cells, validated by miR376c-3p inhibitor and mimics.

Conclusions: Taken together, our study suggests that miR376c-3p transferred by hypoxic exosomes could promote the metastatic potential of ovarian cancer cells by inducing MMP-2 and Snail-1.

Citation Format: HyunA Jo, Wenyu Wang. Hypoxia-induced exosomal miR-376c-3p promotes metastatic potential of ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2384.
Abstract 2403: Pyruvate dehydrogenase mediates chemo-resistance in pancreatic cancer
Authors: Zayou Fouzia , Chheda Chintan , Lim Adrian , Elmadbouh Omer H. , Pandol Stephen J. , Edderkaoui Mouad
Published: July 2021

Background: Resistance of pancreatic ductal adenocarcinoma (PDAC) to current treatment regimens contributes to the dismal prognosis for patients diagnosed with the disease. Accumulated evidence indicates that metabolic alterations may play a role in chemo-resistance. In this project, we investigated the role of pyruvate dehydrogenase Alpha1 (PDHA1) in chemo-resistance.

Methods: Wild type and Gemcitabine-resistant pancreatic cancer cell lines MIA PaCa-2, HPAF-II, and UN-KPC-961 were used. Mitochondrial respiration, ATP production and glycolytic activities were measured by seahorse. PDHA1 was overexpressed or its activity inhibited by over-expressing PDHA1 kinase in these cells and cancer cell survival was measured by MTT and cancer stemness measured by Western and RT-PCR. PDHA1 level was measured in human and mouse PDAC tissues.

Results: Seahorse analysis showed a significantly lower mitochondrial activity, ATP production, and increased glycolytic activity and lactate production in gemcitabine-resistant compared to non- resistant pancreatic cancer cells. PDHA1 mRNA and protein levels were less in gemcitabine-resistant cancer cells compared to non-resistant cells. PDHA1 levels were less in human PDAC tissues from patients and mice who developed resistance to chemotherapy. PDHA1 over-expression in gemcitabine-resistant cancer cells reversed resistance to chemotherapy, decreased cancer cell survival and decreased expression of cancer stemness markers. Whereas, PDHA1 inhibition by over-expressing PDHA1 kinase, in non-resistant cancer cells, induced resistance to chemotherapy, increased cancer cell survival and increased expression of cancer stemness markers. We found that inhibition of histone deacetylase (HDAC) (class 1 and 2) increased transcription level of PDHA1 and reversed chemo-resistance.

Conclusion: We show that PDHA1 is a regulator of chemo-resistance in pancreatic cancer cells. PDHA1is a novel target for treating chemo-resistant PDAC.

Citation Format: Fouzia Zayou, Chintan Chheda, Adrian Lim, Omer H. Elmadbouh, Stephen J. Pandol, Mouad Edderkaoui. Pyruvate dehydrogenase mediates chemo-resistance in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2403.
Abstract 2406: The essential role of ATP binding to the mitochondrial protein ATAD3A in driving oncogenesis of head and neck squamous cell carcinoma
Authors: Lang Liwei , Lam Tiffany , Chen Alex , Shay Chole , Teng Yong
Published: July 2021

Head and neck squamous cell carcinoma (HNSCC) represents only 4% of all cancers in the US, but the adverse impact of this disease on health quality is dramatic. There has been little improvement in HNSCC treatment outcomes in decades, and a better understanding of the molecular biology of HNSCC is urgently needed to facilitate the design of anti-HNSCC therapies. The mitochondrial ATPase family AAA-domain containing protein 3A (ATAD3A) has been demonstrated as an oncogene in breast and lung cancer. However, nothing has been reported regarding its role in HNSCC. We report here that loss of ATAD3A expression inhibits HNSCC cell growth as evidenced by reduced cell proliferation and decreased the ability of colony formation and anchorage-independent growth in soft agar, whereas gain of ATAD3A expression counteracts the observed effects. Overexpression of a Walker A dead mutant of ATAD3A (ATAD3AK358) leads to a suppression in HNSCC cell growth and lipids synthesis, suggesting a potent dominant-negative effect of defective ATP-binding of ATAD3A. In line with the in vitro data, overexpression of ATAD3A facilities tumor growth in an orthotopic mouse model of HNSCC. In contrast, inhibition of ATAD3A function by knockout of ATAD3A or expression of ATAD3AK358 induces significant repression of tumor growth. These findings indicate that the mitochondrial protein ATAD3A plays an oncogenic role in HNSCC, and abrogating its ATP-binding ability may represent a promising regimen for better treatment of patients with HNSCC.

Citation Format: Liwei Lang, Tiffany Lam, Alex Chen, Chole Shay, Yong Teng. The essential role of ATP binding to the mitochondrial protein ATAD3A in driving oncogenesis of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2406.
Abstract 2426: Effects of MEN2 mutations on RET receptor localization and function
Authors: Reyes-Alvarez Eduardo , Hyndman Brandy D. , Mulligan Lois M.
Published: July 2021

Multiple Endocrine Neoplasia type 2 (MEN2) is a cancer syndrome characterized by medullary thyroid carcinoma (MTC) and adrenal tumors. MEN2 is caused by activating point mutations of the REarranged during Transfection (RET) receptor tyrosine kinase, a protein essential for normal cell proliferation, migration, and differentiation in multiple tissues. MEN2 RET mutations can result in constitutive receptor dimerization (MEN2A) or in loss of receptor autoinhibition (MEN2B), which are associated with distinct disease courses and oncogenic potential. However, the specific cellular mechanisms contributing to these differences have not been well characterized. We have previously shown that RET maturation, cell surface localization and trafficking through the endolysosomal system modulate RET signaling and contribute to regulation of normal RET functions. Here, we have explored the contributions of these processes to MEN2A and MEN2B RET mutant activity. In preliminary studies using immunofluorescence (IF) microscopy and MTC cell lines endogenously expressing MEN2A (2ARET) or MEN2B (2BRET) RET mutant forms, we showed that RET mutants do not accumulate in pre-membrane compartments (ER, Golgi) at significant levels. Both 2ARET and 2BRET mutant receptors matured and reached the cell membrane efficiently. Using IF and cell surface protein labeling methods, we demonstrated that wildtype RET, 2ARET and 2BRET forms had differential patterns of plasma membrane distribution in the absence of ligand stimulation. Further, constitutive stimulation of wildtype RET receptors could not recapitulate the pattern observed for MEN2 RET mutants. We showed that, in the absence of ligand, 2ARET and 2BRET, but not wildtype RET, were constitutively phosphorylated, activated downstream signaling pathways and could localize to multiple endosomal compartments. We also showed different extents of protein turnover in 2ARET and 2BRET, which is important for sustained signals and suggests distinct endosomal sorting mechanisms for these receptors. Together, our data indicate that constitutive activation of MEN2 RET mutant receptors is not the only mechanism contributing to aberrant RET function in MEN2. Our results suggest that receptor localization at the membrane and trafficking through endosomal compartments is mutation-specific and may modulate RET signals that contribute to its roles in MEN2 oncogenicity.

Citation Format: Eduardo Reyes-Alvarez, Brandy D. Hyndman, Lois M. Mulligan. Effects of MEN2 mutations on RET receptor localization and function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2426.
Abstract 2446: IL4 receptor-induced proliferation is mediated via Glut1 activity in metastatic breast cancer
Authors: Hargrove-Wiley Ebony , Valent Daniel , Williams Demond , Bindeman Wendy , Fingleton Barbara
Published: July 2021

Breast cancer is the leading cause of cancer mortality in women. The breast cancer microenvironment supplies immune signals that influence tumor growth and metastatic behavior. The cytokine, Interleukin-4 (IL4), promotes increased survival, proliferation and invasion upon stimulation of the type II IL4 receptor alpha (IL4Rα) which is aberrantly expressed on epithelial cancer cells. Previously, we have shown that metastatic mouse mammary cancer cells have a strong dependence on IL4 signaling for colonization of metastatic sites in the lung and liver. Additionally, these cells uptake more glucose when treated with IL4 in vitro. Here, we assessed the importance of glucose consumption on pro-tumorigenic phenotypes induced by IL4/IL4Rα. In 4T1 cells, IL4/IL4Rα signaling increased protein expression of the glucose transporter, Glut1. IL4/IL4Rα activation also increased global glycosylation of Glut1. In human metastatic breast cancer cells (MDA-MB-231 and BT549), Crispr-mediated knockout (KO) of Glut1 significantly reduced glucose consumption when assayed by 2-NBDG uptake, a fluorescently labelled glucose analog. Additionally, Glut1 KO reversed the proliferative effects of IL4/IL4Rα stimulation on human cancer cells in vitro. Our data suggest that IL4-induced Glut1 expression supports the increased metabolic activity necessary for cancer cell proliferation. Targeting the IL4/IL4Rα signaling axis could potentially be a therapeutic strategy to impede tumor-associated metabolism and reduce tumor burden at primary and metastatic sites.

Citation Format: Ebony Hargrove-Wiley, Daniel Valent, Demond Williams, Wendy Bindeman, Barbara Fingleton. IL4 receptor-induced proliferation is mediated via Glut1 activity in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2446.
Abstract 2465: The opposing regulation of ALDOA and FBP1 in Glucose metabolism contribute to cancer progression
Authors: Li Chien Hsiu , Chang Yu-Chan , Hsiao Michael
Published: July 2021

Metabolism reprogramming and increased glycolysis of cancer are associated with rapid cell growth, motility, and resistance to drug treatment. Fructose-1, 6-bisphosphate (F-1,6-BP), an important product involvement in glucose metabolism, especially in cancer development. It is known that the essential enzymes of gluconeogenesis and glycolysis, fructose-1,6-bisphosphonates 1 (FBP1) and glycolytic enzyme aldolase A (ALDOA) expression are involved in the F-1,6-BP conversion process. Especially, increased expression of ALDOA has been identified to contribute to multiple human cancer development. However, the underlying molecular mechanism involvement of ALDOA and FBP1 on the switching of F-1,6-BP is still being uncovered. This study shows that the expression levels of ALDOA and FBP1 with an inversed correlation during the cancer progression, especially in lung adenocarcinoma and hepatocellular carcinoma. Immunoprecipitation and proteomics analyses revealed the candidates, including the anterior gradient 2 (AGR2), ubiquitin ligase gene cullin 4B (CUL4B), and telomeric repeat binding factor 1 (TERF1) are significantly correlated with ALDOA and FBP1 expression. We designed a specific peptide to interfere the interaction of candidates on both ALDOA and FBP1. Moreover, we evaluate the relative glucose metabolism events. Further, we raised the hypothesis that these candidates may serve as a modulator to regulate the conversion of F-1,6-BP and contribute to cancer progression, which can be used as a therapeutic target for further research.

Citation Format: Chien Hsiu Li, Yu-Chan Chang, Michael Hsiao. The opposing regulation of ALDOA and FBP1 in Glucose metabolism contribute to cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2465.
Abstract 2480: RE1-silencing transcription factor (REST) controls neuroendocrine gene programs in pancreatic ductal adenocarcinoma
Authors: MacPherson Kevin Alexander , Joly Meghan M. , Allen-Petersen Brittany , Pelz Carl , Thoma Mary C. , Torkenczy Kristof , Adey Andrew , Liefwalker Daniel , Worth Patrick J. , Sears Rosalie C.
Published: July 2021

Neuroendocrine (NE) differentiation features contribute to intratumoral heterogeneity and aggressive biology in a subset of pancreatic ductal adenocarcinoma (PDAC) tumors associated with poor disease outcome. We recently showed that treating pancreatic cancer cells with gemcitabine, a standard-of-care chemotherapy for PDAC, increases expression of NE markers, suggesting that ductal to neuroendocrine lineage plasticity could play a role in drug resistance. NE features in prostate cancer have been previously associated with loss of the neuronal gene repressor RE1-Silencing Transcription Factor (REST); in the present study, we explore a potential role of REST in regulating NE differentiation in PDAC. Our experiments show that loss of REST in PDAC cells increases NE gene expression, gemcitabine resistance, and colony formation. RNA sequencing data from gemcitabine-treated PDAC cells were enriched for REST target genes, suggesting gemcitabine relieves REST-mediated repression. Chromatin immunoprecipitation experiments corroborated this by demonstrating that gemcitabine reduces REST binding to the NE genes SYP and SNAP25. In addition, single cell ATAC sequencing also uncovered a heterogeneous response to gemcitabine treatment, revealing two gemcitabine-driven cell states with differing REST motif accessibility. Our study indicates that REST controls NE gene programs in PDAC and that loss of REST promotes gemcitabine resistance and oncogenic growth. Mirroring the heterogeneity of PDAC tumors, a subset of PDAC cells treated with gemcitabine have reduced REST motif accessibility, which may reflect a therapy resistant NE-like subpopulation.

Citation Format: Kevin Alexander MacPherson, Meghan M. Joly, Brittany Allen-Petersen, Carl Pelz, Mary C. Thoma, Kristof Torkenczy, Andrew Adey, Daniel Liefwalker, Patrick J. Worth, Rosalie C. Sears. RE1-silencing transcription factor (REST) controls neuroendocrine gene programs in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2480.
Abstract 2485: Metabolic implications of TIGAR in glioblastoma multiforme
Authors: Sahagun Joseph
Published: July 2021

The incidence of glioblastoma multiforme (GBM) has been rising throughout the last decade, yet interventional medical advances have not been able to keep up. With current literature focusing on hypoxia-inducible factor 1 and angiogenic properties of GBM, our study focuses on Tp53 glycolysis and apoptosis regulator (TIGAR) enzyme in order to supplement the limited amount of published literature. Recent literature implicates Tp53 glycolysis and apoptosis regulator (TIGAR) in tumor survival and proliferation by shunting glycolytic intermediates to generate NADPH and activation of AKT, respectively. In this study, we aimed at establishing TIGAR's dependence on p53 and whether pifithrin-α blocks transcription of TIGAR within the proneuronal subtype of glioblastoma. This cell line is of interest due to its frequent mutations and loss of heterogeneity within p53 and association with chemotherapy resistance in GBM. In this cell line, both p53 and TIGAR protein expression were confirmed using western blot. An MTT assay determined the minimal concentration of pifithrin-α required to inhibit p53 expression in the proneuronal cell line. A known p53 null line, SAOS-2, was used to monitor TIGAR expression using real-time PCR to determine the dependence of p53 expression. Through this study, we determined that TIGAR behaves independently of p53 expression, and a knockdown of p53 with pifithrin-α has no influence on the transcription of TIGAR. We hypothesize that p53 is required to act as a barrier against TIGAR overexpression in GBM.

Citation Format: Joseph Sahagun. Metabolic implications of TIGAR in glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2485.
Abstract 2503: Crispr/cas-9-mediated genome editing reveals that RBPMS acts as a tumor suppressor in ovarian cancer
Authors: Sharma Rohit K. , Santiago-Sánchez Ginette S. , Rabelo-Fernández Robert J. , Quiñones-Díaz Blanca I. , Valiyeva Fatima , Vivas-Mejia Pablo E.
Published: July 2021

RNA-Binding Protein with Multiple Splicing (RBPMS) is a member of a family of proteins that bind to the nascent RNA transcripts and regulate their processing, which includes pre-mRNA splicing and transport/localization/stability of the RNA molecule. Evidence indicates that RBPMS also binds to members of the AP-1 (c-Jun and c-Fos) transcription factor repressing his activity. Until now, little is known about the biological function of RBPMS in ovarian cancer. To understand the role of RBPMS in ovarian cancer we generated CRISPR (clustered regularly interspaced short palindromic repeats)-mediated RBPMS knock-out vectors that were stably transfected in the ovarian cancer cells OVCAR3. The Guide-it Genotype confirmation Kit was used to determine the type of mutations (monoallelic, biallelic, or unchanged) present in the developed clones. Additionally, resulting CRISPR-cleavage products were analyzed to establish the cleavage efficiency of the Cas9/sgRNA complex by densitometry analysis. We found that CRISPR clone cells grow faster and increase their invasiveness ability compared with control CRISPR cells. Furthermore, immunohistochemical (IHC) analysis in a tissue array showed that RBPMS staining was significantly lower in high-grade serous ovarian cancer (HGSOC) tissues as compared to normal ovarian tissues. RBPMS was also decreased in platinum-resistant ovarian cancer cells as compared to platinum-sensitive cancer cells. Senescence-associated β-galactosidase (β-Gal) levels were higher in CRISPR-clones as compared with empty vector and ovarian cancer cells, which suggest RBPMS knockdown induces senescence. These studies along with the data of proteomics and RNAseq suggest that RBPMS acts as a tumor suppressor gene and lower levels of RBPMS are associated with the cisplatin resistance of ovarian cancer cells.

Citation Format: Rohit K. Sharma, Ginette S. Santiago-Sánchez, Robert J. Rabelo-Fernández, Blanca I. Quiñones-Díaz, Fatima Valiyeva, Pablo E. Vivas-Mejia. Crispr/cas-9-mediated genome editing reveals that RBPMS acts as a tumor suppressor in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2503.
Abstract 2506: Smarca4 inactivation promotes lineage-specific transformation and early metastatic features in the lung
Authors: Concepcion Carla P. , Ma Sai , LaFave Lindsay M. , Bhutkar Arjun , Liu Manyuan , DeAngelo Lydia P. , Kim Jonathan Y. , Priore Isabella Del , Schoenfeld Adam J. , Kartha Vinay K. , Westcott Peter M. , Sánchez-Rivera Francisco J. , Meli Kevin , Gupta Manav , Riely Gregory J. , Rekhtman Natasha , Rudin Charles M. , Kim Carla F. , Regev Aviv , Buenrostro Jason D. , Jacks Tyler
Published: July 2021

SMARCA4 (BRG1) encodes for one of two mutually-exclusive ATPases present in SWI/SNF chromatin remodeling complexes, and is among the most frequently mutated genes in human lung adenocarcinoma. Despite its prevalence, the functional consequences of SMARCA4 alterations on lung cancer initiation and progression remain poorly understood. Using genetically engineered mouse models, patient-derived xenografts, and single-cell epigenomic profiling, we demonstrate that loss of Smarca4 sensitizes CCSP+ cells within the lung to malignant transformation and tumor progression, resulting in highly advanced dedifferentiated tumors and increased metastatic incidence. Consistent with these phenotypes, Smarca4-deficient tumors lack lung lineage transcription factor activities and instead show activation of embryonic stem cell-like programs, resembling a metastatic cell state. Thus, the SWI/SNF complex - via Smarca4 - acts as a gatekeeper for lineage-specific cellular transformation and metastasis during lung cancer evolution.

Citation Format: Carla P. Concepcion, Sai Ma, Lindsay M. LaFave, Arjun Bhutkar, Manyuan Liu, Lydia P. DeAngelo, Jonathan Y. Kim, Isabella Del Priore, Adam J. Schoenfeld, Vinay K. Kartha, Peter M. Westcott, Francisco J. Sánchez-Rivera, Kevin Meli, Manav Gupta, Gregory J. Riely, Natasha Rekhtman, Charles M. Rudin, Carla F. Kim, Aviv Regev, Jason D. Buenrostro, Tyler Jacks. Smarca4 inactivation promotes lineage-specific transformation and early metastatic features in the lung [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2506.
Abstract 2520: Detection of transcriptional active HPV 16/18 in patients with oropharyngeal squamous cell carcinoma by dual immunohistochemistry p16INK4 and in situ hybridization E6/E7 mRNA in archival material older than 25 years
Authors: Tornaas Stian , Fromreid Siren , Aarstad Hans Jørgen , Vintermyr Olav Karsten , Costea Daniela Elena , Dongre Harsh Nitin
Published: July 2021

Introduction: Determination of Human Papillomavirus (HPV) status in oropharyngeal squamous cell carcinomas (OPSCC) is important because it influences the therapeutic decision. A dual protocol combining immunohistochemistry (IHC) for detection of p16INK4 overexpression with in situ hybridization (ISH) for mRNA E6/E7 detection may improve the accuracy of determining the HPV status in OPSCC patients. The aim of this study was to optimize a multiplex method for simultaneous detection of p16 and mRNA E6/E7 on the same formalin-fixed paraffin-embedded (FFPE) tissue samples.

Methods: IHC for the detection of p16INK4 and chromogenic ISH for mRNA E6/E7 were combined in different sequences and with different intermediate steps for fixation on OPSCC tissue samples from primary tumors and lymph node metastases. Archival tissue samples stored at Department of Pathology at Haukeland University Hospital since 1990 has been used for tests.

Results: A combined method for simultaneous detection of p16INK4 and mRNA E6/E7 transcripts on the same tissue slides were successfully developed. Active HPV status was detected in OPSCC tissue samples from both primary and metastatic lymph nodes for all cases tested, including tissue samples stored for 30 years. The result was correlated to DNA HPV status.

Conclusion: This study confirms that a more precise detection of active HPV infection is possible on FFPE tissue slides. This method could be easily implemented in clinical settings for more specific stratification of OPSCC patients. At the same time, the method is also robust and sensitive on retrospective archival material, and this could be used for answering important research questions on HPV-related OPSCC.

Citation Format: Stian Tornaas, Siren Fromreid, Hans Jørgen Aarstad, Olav Karsten Vintermyr, Daniela Elena Costea, Harsh Nitin Dongre. Detection of transcriptional active HPV 16/18 in patients with oropharyngeal squamous cell carcinoma by dual immunohistochemistry p16INK4 and in situ hybridization E6/E7 mRNA in archival material older than 25 years [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2520.
Abstract 2521: Race specific differences in DNA damage repair dysregulation in breast cancer and association with outcome
Authors: Mazumder Aloran , Jimenez Athena , Ellsworth Rachel , Freedland Stephen , George Sophia , Bainbridge Matthew , Haricharan Svasti
Published: July 2021

African American (AA) estrogen receptor positive (ER+) breast cancer patients have worse outcomes than Caucasian Americans (CA) being 42 % more likely to die from the disease. However, AAs are severely underrepresented in currently available datasets of patient tumors, which precludes comprehensive approaches to identify race-specific molecular drivers of these poor outcomes. Endocrine therapy (ET) is the first line standard of care for ER+ breast cancer. Nevertheless, 1 in 4 patients develop ET resistance. Specific DNA damage/repair (DDR) defects have been shown to associate with poor outcome in CA patients, and to induce ET resistance. Whether these or other DDR defects contribute to poor outcomes observed in AA patients remains unknown. For the purpose of this investigation, we assessed the DDR dysregulation landscape of AA ER+ tumors using three independent tumor datasets (1) GSE78958 (2) GSE18229 (3) The Cancer Genome Atlas (TCGA) and two normal breast datasets (1) GSE43973 (2) GSE50939. This analysis identified a distinct set of AA ER+ tumors with simultaneous dysregulation of genes from multiple DDR pathways, rarely seen in CA tumors. This simultaneous dysregulation also associated with worse patient outcomes in all three datasets analyzed. This work constitutes the first systematic analysis of race-specific DDR dysregulation in ER+ breast cancer, and identifies DDR as a potential predictive biomarker for worse outcome seen in AA patients.

Citation Format: Aloran Mazumder, Athena Jimenez, Rachel Ellsworth, Stephen Freedland, Sophia George, Matthew Bainbridge, Svasti Haricharan. Race specific differences in DNA damage repair dysregulation in breast cancer and association with outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2521.