EMT Phenotype Research Articles

1949P - Cell plasticity and taxanes resistance in metastatic prostate cancer: ESRP1 as a predictive biomarker of taxane response

1949P - Cell plasticity and taxanes resistance in metastatic prostate cancer: ESRP1 as a predictive biomarker of taxane response

Overexpressed PKMYT1 promotes tumor progression and associates with poor survival in esophageal squamous cell carcinoma.

BackgroundEsophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors worldwide and the 5-year overall survival rate remains poor. Protein kinase, membrane associated tyrosine/threonine (PKMYT1) is overexpressed in several cancers and participate in tumor progression. However, the mechanism of PKMYT1 in ESCC is unclear.PurposeThe objective of our study was to demonstrate the the expression and role of PKMYT1 in ESCC.Patients and methods We detected the expression of PKMYT1 in ESCC patients and analysed the correlation with overall survival time and disease-free survival time. Then we detected PKMYT1 expression in ESCC cell lines and immortalized human esophageal epithelial cell line. Down-regulated PKMYT1 was carried out in KYSE70 and KYSE450 cells to invetigate the mechanism of PKMYT1 in ESCC cells.ResultsPKMYT1 was up-regulated in tumor tissues and ESCC cell lines, and higher expression of PKMYT1 correlated with poorer overall survival in ESCC patients. Besides, in ESCC cell lines KYSE70 and KYSE450, knocking down PKMYT1 allowed more cells to skip G2/M checkpoint to complete mitosis, which promoted cell apoptosis, inhibited cell proliferation, and prevented the EMT phenotype in vitro. Meantime, we also observed that down-regulated PKMYT1 in ESCC cells suppressed AKT/mTOR signaling pathway. These results demonstrated PKMYT1 may act as an oncogene in ESCC.ConclusionPKMYT1 plays an crutial role in ESCC progression, downregulated PKMYT1 might inhibit the development of ESCC by AKT/mTOR signaling pathway, and might be a novel target in the treatment of ESCC.

Elevated levels of hsa_circ_006100 in gastric cancer promote cell growth and metastasis via miR-195/GPRC5A signalling.

ObjectivesCircular RNAs (circRNAs) are non‐coding RNAs, some of which are thought to be involved in gastric cancer development. Here, we examined the functions of circRNA hsa_circ_006100 in gastric cancer cells and an animal model of gastric cancer.Materials and MethodsThe expression of hsa_circ_006100, miR‐195 and various functional genes was determined by quantitative RT‐PCR. Cell viability, clone formation, apoptosis and cell migration/invasion abilities were analysed by the CCK‐8 assay, crystal violet staining, Hoechst staining and Transwell assay, respectively. A tumour model was established by subcutaneously injecting tumour cells into nude mice. Levels of protein expression were analysed by Western blotting and immunohistochemistry.ResultsA bioinformatics analysis showed that miR‐195 was negatively co‐expressed with hsa_circ_006100. Patients with a high hsa_circ_006100 level or low miR‐195 level had tumours with a high TNM stage, poor cellular differentiation and lymph node metastasis. miR‐195 was targeted and inhibited by hsa_circ_006100. Overexpression of hsa_circ_006100 enhanced cellular viability and proliferation, while miR‐195 suppressed hsa_circ_006100‐enhanced cell growth and induced apoptosis in MGC‐803 and AGS cells. Forced hsa_circ_006100 expression promoted the migration and invasion of MGC‐803 and AGS cells, while those activities were inhibited by miR‐195. Mechanistically, GPRC5A was predicted as a target of miR‐195 and was upregulated in gastric cancer. A miR‐195 inhibitor restored cell viability, proliferation, migration and invasion, and repressed apoptosis via GPRC5A. In vivo studies showed that knockdown of hsa_circ_006100 delayed tumour growth, reduced PCNA expression and upregulated miR‐195 and BCL‐2 expression which was restored by miR‐195 inhibition due to GPRC5A/EGFR signalling, and changed the EMT phenotype in vivo.ConclusionsHsa_circ_006100 functions as an oncogene in gastric cancer and exerts its effects via miR‐195/GPRC5A signalling.

Gold nano-particles (AuNPs) carrying miR-326 targets PDK1/AKT/c-myc axis in hepatocellular carcinoma

Abnormal expression of microRNAs (miRNAs) contributes to tumour growth and invasion. MiR-326 expression often down-regulates in several kinds of cancer and low expression of miR-326 is linked with poor prognosis in cancer patients. In the present study, we aimed to explore the modulatory mechanism of miR-326 in hepatocellular carcinoma (HCC). miR-326 expression was significantly decreased in HCC cell lines and tissues. miR-326 decreased HCC cell growth by affecting cell-cycle progression and by promoting apoptosis. In addition, miR-326 inhibited HCC cell invasion by decreasing the EMT phenotype. We found that miR-326 functioned as a tumour suppressor by repressing its down-stream target PDK1. C-myc contributed to miR-326 down-regulation through binding at its promoter and inhibited its expression. Based on these results, we conducted a therapeutic experiment by using gold nano-particles (AuNPs) carrying miR-326. Restoration of miR-326 reduced tumour growth in vivo. Our findings suggest that miR-326 may be a candidate prognostic biomarker and a target for new therapies in HCC patients.

Arrestin Domain Containing 3 Reverses Epithelial to Mesenchymal Transition and Chemo-Resistance of TNBC Cells by Up-Regulating Expression of miR-200b.

Our previous studies demonstrated the importance of arrestin domain containing 3 (ARRDC3), a metastasis suppressor, in inhibiting invasive and metastatic potential of triple negative breast cancer (TNBC) in vitro and in vivo. However, little is known about ARRDC3 mediated transcriptional control and its target genes that are implicated in its metastatic suppressing activity. In this study, we used miRNA array and subsequent functional analyses to identify miRNAs whose expression are significantly regulated by ARRDC3 in TNBC cells. We identified miR-200b as a major target gene of ARRDC3. miR-200b played an essential role in mediating ARRDC3 dependent reversal of EMT phenotypes and chemo-resistance to DNA damaging agents in TNBC cells. Expression of miR-200b also increased the expression of ARRDC3 as well in TNBC cells, suggesting a positive feedback loop between these two molecules. In addition, we combined the therapeutic powers of miR-200b and 5-fluorourancil (5-FU) into a single compound (5-FU-miR-200b) to maximize the synergistic effects of these compounds. Chemically modified miR-200b (5-FU-miR-200b mimic) was more effective in inhibiting metastatic potentials of TNBC cells than unmodified miR-200b and does not require transfection reagents, implying its therapeutic potential in TNBC. Our studies showed the importance of therapeutic targeting ARRDC3/miR-200b pathway in TNBC.

Abstract 1887: MDM2 induces a partial/hybrid EMT phenotype switching through TGFβ1-Smad signaling pathway

Abstract Epithelial-Mesenchymal Transition (EMT) is a key step in the induction of metastasis, which is critical for the conversion of early-stage prostate tumors into metastatic malignancies. Therefore, identifying the signaling pathways promoting EMT is crucial for the prediction of disease progression and consequently treatment outcomes. The role of MDM2 proto-oncogene was investigated in regulating EMT in LNCaP prostate cancer cells. MDM2 over-expression in LNCaP-MST (MDM2 transfected prostate cancer cells) caused a reduction in epithelial characteristics, by altering the expression levels of proteins that are involved in cell adhesion, cell polarity and cell-ECM interactions. While forcing the loss of epithelial characteristics, the MDM2 oncogene seems to facilitate the gain of mesenchymal properties (such as increased motility, invasive properties, and a spindle-like morphology etc.). These phenotypic changes are generally due to cytoskeletal rearrangements and increased expression of mesenchymal markers, which was clearly identified through our western blotting and fluorescence microscopy imaging analysis in LNCaP-MST cells compared to the LNCaP cells. Nutlin-3, an MDM2 specific inhibitor was able to reverse the expression of mesenchymal markers, which further confirmed the role of MDM2 in phenotype switching. Analysis of cell culture supernatants using ELISA indicated that LNCaP-MST cells secreted two folds higher levels of TGFβ1 compared to LNCaP cells, which appears to cause the activation of downstream Smad signaling pathway leading to EMT. Activation of the TGFβ1-Smad pathway also results in the increased transcription of mesenchymal markers (N-cadherin, vimentin, and fibronectin) while retaining the epithelial characteristics as evidenced by the unaltered levels of E-cadherin. Recent reports have established that, while transitioning between the epithelial and mesenchymal phenotypes, cells can also attain a hybrid, partial, or intermediate EMT phenotypes. Cancer cells in this hybrid phenotype can migrate collectively as clusters (epithelial) or as a single cell (mesenchymal) and exhibit stemness and drug resistance. In addition, these migratory cells with hybrid characteristics can attain the necessary phenotype and growth depending on the microenvironment that is surrounding the metastatic sites. So far, our results have clearly demonstrated that MDM2 could serve as a key regulator to drive the EMT of prostate cancer cells, and this process seems to be aided by the elevation of TGFβ1 signaling. Thus, the pro-metastatic role of MDM2 oncoprotein has been steadily expanding in the last few years, and the newest knowledge acquired through our experiments is the confirmation of its ability to induce hybrid EMT phenotype in prostate cancer cells, (This project was supported by the generous financial support from The Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida) Citation Format: Priya Dondapati, Simran Agarwal, Deepthi Gangaram, Appu Rathinavelu. MDM2 induces a partial/hybrid EMT phenotype switching through TGFβ1-Smad signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1887.

Abstract 2006: Targeting AXL kinase with TP-0903 successfully reverses the mesenchymal phenotype and extends survival in preclinical models of advanced ovarian cancer

Abstract Epithelial-to-mesenchymal transition (EMT) is a critical step in the progression of ovarian cancer (OVCA), which drives local invasion, metastasis and drug resistance leading to poor survival. AXL, a receptor tyrosine kinase, functions as a molecular driver of EMT in multiple cancers, including OVCA. The high mortality of this tumor is explained by the fact that the majority (75%) of patients present at an advanced stage, with widely metastatic disease within the peritoneal cavity often associated with ascites. The extent of ascites is a prognostic factor for survival, impacts quality of life, and correlates with the EMT phenotype. Approaches to block or reverse EMT are needed to improve the prognosis of OVCA patients, particularly those with refractory or early relapse (<6 months) disease following debulking surgery and conventional adjuvant treatments. TP-0903 is a potent, small molecule AXL inhibitor that has activity in reversing EMT in multiple models of disease and embryonic development. The objective of this study was to explore the anti-tumor activity in preclinical mesenchymal OVCA-derived models, which may provide rationale to evaluate TP-0903 in treatment-resistant or refractory OVCA patients. TP-0903 showed potent effects on cell viability in multiple OVCA-derived cell lines. The IC50 values in cell viability studies ranged from 33 nM (OVTOKO cells) to 840 nM (OVMANA cells). Kuramochi cells represent the most common ovarian cancer subtype, high-grade serous ovarian cancer and was particularly platinum-resistant (>5 μM), yet sensitive to TP-0903 (210 nM). Baseline phospho-AXL (Y702) was exceptionally high in Kuramochi cells and TP-0903 inhibited this phosphorylation at concentrations as low as 100 nM. TP-0903 reversed EMT in OVCA cells as evident by the down-regulation of mesenchymal markers including Snail and Slug. Consistent with the reversal of the EMT, TP-0903 inhibited the migration of ES-2 OVCA cells in a scratch assay. TP-0903 suppressed ascites development in preclinical models of OVCA and inhibited metastatic intraperitoneal tumor dissemination. Treatment suppressed ascites development (ascites volume, vehicle: 3.34 mL, TP-0903: 0.28 mL; abdominal circumference, vehicle: 7.96 cm (day 15), TP-0903: 6.82 cm (day 15), p<0.05) in an orthotopic ES-2 model and extended overall survival compared to vehicle or cisplatin alone. Phospho-AXL modulation and changes in other mechanism-validating biomarkers were observed in the ES-2 model. Similar preclinical activities, including decreased ascites volume, were observed in A2780cis and OVCAR-3 xenograft models. These results support further clinical evaluation of TP-0903 in either single-agent or combination regimens treating recurrent or refractory OVCA. TP-0903 is investigated in a Phase I trial that includes an expansion cohort of this patient population (NCT02729298). Citation Format: Nozomi Tomimatsu, Ken Fujimura, Yuta Matsumura, Hiroki Umehara, Lars Mouritsen, Steven L. Warner, David J. Bearss. Targeting AXL kinase with TP-0903 successfully reverses the mesenchymal phenotype and extends survival in preclinical models of advanced ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2006.

Abstract 2834: Bacteroides fragilis: A potential pathogen orchestrating EMT and stemness in breast epithelial cells via concomitant activation of Notch and βcatenin axes

Abstract Background: Last decade established significant contributions of microbiome to organ specific cancers. A few recent studies suggested the existence of distinct breast microbiota and a shift in microbial community composition in diseased breast compared to normal breast. However, their functional impact and underlying mechanisms are unknown. Present study was designed to examine the contribution of pro-carcinogenic bacteria in breast cancer initiation, growth and progression. Results: Utilizing extensive data mining and metagenomic analyses, we discovered presence of toxin producing Bacteriodes fragilis in malignant breast. B. fragilis is known for its potential to initiate and/or promote colon cancer. Its pathogenicity has been attributed to its unique toxin BFT. Mice infected with B. fragilis exhibited significant circulating BFT and distinct morphological alterations in mammary gland. While no changes were observed in cell growth and clonogenicity upon BFT treatment, significant increase in migration and invasion potential and decreased adhesion of MCF10A and MCF7 cells were observed. BFT-treated cells displayed acquisition of fibroblast-like appearance and increased formation of pseudopodia/microtentacles emanating from the cell membrane along with molecular markers of epithelial-to-mesenchymal transition. Decreased expression of epithelial marker, E-cadherin along with elevated levels of mesenchymal markers, N-cadherin and vimentin were observed. BFT also increased expression of EMT-related transcription factors, Snail, Slug and Twist. BFT-treated cells attained stem cell-like phenotype exhibiting an increased ability to form secondary and tertiary mammospheres and elevated expression of pluripotency-factors (Oct4, Nanog and Sox2). Mechanistic studies showed BFT induced expression and nuclear translocation of cleaved NOTCH1 and βcatenin resulting in activation of downstream targets. Inhibition of Notch1 and βcatenin using γ-secretase inhibitor and ICG001 successfully inhibited functional effects of BFT. Further, BFT-pretreated MCF7 cells exhibit increased tumor growth and form multifocal tumors in mice. MCF10A-KRas cells, pretreated with BFT, also showed increased tumor progression and multifocal tumors in mice. In vivo limiting dilution assay using breast tumors from BFT-pretreated MCF7 cells exhibited a striking increase in tumor-initiating cells. Follow-up analyses of these tumors demonstrated increased migratory, invasive, and mammospheres-forming behavior confirming that brief BFT exposure elicits long-term molecular changes. Conclusion: Collectively, these findings present the first in vitro and in vivo evidence to show that Bacteriodes fragilis Toxin induces EMT, invasion/migration and stem cell-like phenotype and leads to concomitant activation of Notch and βcatenin axes. Citation Format: Sheetal Parida, Shaoguang Wu, Nethaji Muniraj, Sumit Siddharth, Arumugam Nagaligam, Christina Hum, Panagiotis Mistriotis, Konstantinos Konstantopoulos, Cynthia L. Sears, Dipali Sharma. Bacteroides fragilis: A potential pathogen orchestrating EMT and stemness in breast epithelial cells via concomitant activation of Notch and βcatenin axes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2834.

Abstract 3025: Investigation of AKT3 in the resistance of non-small cell lung cancer to EGFR-TKI

Abstract Lung cancer is the leading cause of cancer deaths worldwide, and EGFR-TKI is the first-line treatment for non-small cell lung cancer (NSCLC) harboring EGFR activation mutation. However, most patients develop acquired resistance around 12 months. Two resistant cells, HCC827/IR (IRESSA resistance) and H1975/AR (AZD9291 resistance) exhibiting EMT phenotypes were generated to investigate the molecular and cellular characteristics of the EGFR-TKI acquired resistance. The expression and activation of EGFR were reduced in both resistant cells. Upregulation of AXL and FGFR1 were found in HCC827/IR but not H1975/AR cells. HCC827/IR (without T790M) and H1975/AR (without C797S) showed “off target resistance”. Activation of AKT (p-AKT) in both parental cells was more sensitive to EGFR-TKI than that in resistant cells. RNA-seq revealed that AKT3 was upregulated in both resistant cells. High expression of AKT3 was predicted to correlate with the poor survival of lung adenocarcinoma patients. Knockdown of AKT3 in HCC827/IR cells inhibited cell migration and increased the protein expression of E-cadherin, as well as inhibited S phase population to reduce cell proliferation. Knockdown of AKT3 in H1975/AR cells enhanced AZD9291-induced inhibition on AKT activation (p-AKT). Immunoprecipitation of AKT3 in both resistant cells demonstrated its involvement in AKT activation, and AKT3 activation (p-AKT3) was not sensitive to inhibition by gefitinib and AZD9291 in resistant cells. We also found that protein but not mRNA of AKT3 was upregulated in gefitinib-treated HCC827/IR and AZD9291-treated H1975/AR cells. Therefore, AKT3 might serve as a predictive biomarker for EGFR-TKIs therapy, and its upregulation might be one of the mechanisms for EGFR-TKIs acquired resistance. In addition, combination of AZD9291 with AKT inhibitors elicited synergistic inhibition on cell viability of H1975/AR cells. This work was supported by the National Health Research Institutes (NHRI)- NHRI-EX107-10707BI. Citation Format: Shih-Hsiang Huang, Jin-Yuan Shih, Ching-Chow Chen. Investigation of AKT3 in the resistance of non-small cell lung cancer to EGFR-TKI [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3025.

Abstract 1332: High Throughput isolation and expansion of circulating tumor cells (CTCs) from Non-small cell lung cancer (NSCLC) patients for personalized treatments

Abstract Background: Circulating tumor cells (CTCs) have emerged as important blood-based surrogate markers of primary tumors. Current methods for isolation of lung CTCs mostly rely on biomarker dependent antibody-based capture, missing populations that may be stem-like in nature. Results: We have applied the microfluidic Labyrinth device for high throughput, label-free, size-based isolation of CTCs from non-small cell lung cancer patients (NSCLC). The Labyrinth device was optimized and tested for inertial separation of cancer cells using the human lung cancer cell line H1650. The recovery and purity were >82% and >78%, respectively, operating at a flow rate of 2.5 mL/min. Using the biomarker-independent Labyrinth separation device, heterogeneous CTC populations were isolated from metastatic NSCLC patients (n=21). Heterogeneous CTC populations were detected, including CTCs (PanCK+ and CD45-), CTCs expressing EpCAM or Vimentin, and CTCs expressing both markers representing an EMT-like population of CTCs. Using Labyrinth, we were able to isolate CTCs from 100% of patients with an average yield of 180±168 CTCs/mL. Among the captured CTCs, EpCAM- CTCs were significantly more common than EpCAM+ CTCs (115.7 vs. 39.1 CTCs/mL respectively). Cell clusters of 2 or more CTCs were also observed in 95% of patients; 79% of these clusters were negative for EpCAM expression, whereas 35% expressed Vimentin, suggestive of an EMT phenotype. Recovered CTCs from patients with RET, ROS1 and ALK rearranged tumors showed aberrations matching with the primary tumor for each gene using FISH analysis. We have successfully expanded the recovered CTCs from 2 patients and screened for therapeutic targeting. We have found that TPX-0005 might be effective in these patients and would direct them to a clinical trial using this compound. Conclusion: The label-free Labyrinth device demonstrated the capability of collecting recovered CTCs from the device using a continuous processing technique while in a suspension state. This advantage opens the opportunities not only for CTC expansion off-chip, but also for ex-vivo drug testing to direct patient-specific therapies. Citation Format: Mina Zeinali, Maggie Lee, Arthi Nadhan, Anvya Mathur, Wei Huang, Eric Lin, Ramdane Harouaka, Max S. Wicha, Nallasivam Palanisamy, Mathias Hafner, Rishindra Reddy, Gregory P. Kalemkerian, Bryan J. Schneider, Khaled A. Hassan, Nithya Ramnath, Sunitha Nagrath. High Throughput isolation and expansion of circulating tumor cells (CTCs) from Non-small cell lung cancer (NSCLC) patients for personalized treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1332.

Abstract 3822: ORIC-101 reverses a GR-driven EMT-like phenotype and sensitizes TNBC cells to chemotherapy

Abstract The Glucocorticoid Receptor (GR) is a member of the superfamily of nuclear hormone receptors that is activated by human cortisol and synthetic glucocorticoids such as dexamethasone (Dex). Upon ligand binding, GR translocates to the nucleus and regulates the expression of a wide spectrum of genes involved in diverse biological processes, including inflammation, immunity, metabolism, cell cycle, and differentiation. Dysregulated cortisol levels are associated with poor prognosis, drug resistance, and increased cancer recurrence. Multiple studies have shown that GR inhibition reverses resistance to chemotherapy in cancers of epithelial origin including prostate, bladder, renal, ovarian, pancreatic, and triple negative breast cancer (TNBC). We have recently reported the discovery of ORIC-101, a potent GR antagonist with a unique cytochrome P450 inhibition profile that makes this compound particularly suitable for combination with taxanes such as paclitaxel (Rew Y et al, 2018). Consistent with previous reports, our data showed that activation of GR promoted growth of TNBC cells in 3D culture conditions and protected TNBC cells from paclitaxel. Treatment with ORIC-101 fully reversed these effects. To understand the molecular basis of the observed GR-mediated chemotherapy resistance, we set out to isolate the pool of TNBC cells that escaped from paclitaxel treatment in the presence of Dex. Molecular profiling of these “chemotherapy escapees” pointed to a number of glucocorticoid-regulated biological pathways, including basal stem cell lineage genes and mesenchymal markers, suggesting the acquisition of an EMT-like phenotype in chemo-resistant cells. In support of this finding, we found using ChIP-seq analysis that GR directly bound within the promoter/enhancer regions of well-established EMT genes such as SNAI2 and FN1, and regulated their expression in response to Dex treatment. Functionally, RNAi-mediated knockdown of SNAI2 partially restored sensitivity to paclitaxel, suggesting that the GR-driven EMT phenotype contributes to paclitaxel resistance in TNBC cells. Consistent with the in vitro observations, immunohistochemical analysis showed that GR activation upregulated the levels of both basal stem cell and mesenchymal markers in “chemotherapy escapees” from paclitaxel-treated TNBC xenografts. Importantly oral administration of ORIC-101 fully blocked these effects. Altogether, we found that activation of GR drove an EMT-like phenotype in TNBC cells in vitro and in vivo. ORIC-101 reversed these effects and sensitized TNBC cells to chemotherapy. Our findings thus provide mechanistic insights into the role of GR as a mediator of therapy resistance in TNBC. Clinical evaluation is being planned to assess the therapeutic potential of ORIC-101 in combination with standard-of-care chemotherapeutic agents. Citation Format: Haiying Zhou, Jessica Sun, Wayne Kong, Yosup Rew, Xiaohui Du, John Eksterowicz, Daqing Sun, Qiuping Ye, Omar Kabbarah, Valeria R. Fantin. ORIC-101 reverses a GR-driven EMT-like phenotype and sensitizes TNBC cells to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3822.

Tumor suppressor role of cytoplasmic polyadenylation element binding protein 2 (CPEB2) in human mammary epithelial cells

BackgroundOver-expression of cyclooxygenase (COX)-2 promotes breast cancer progression by multiple mechanisms, including induction of stem-like cells (SLC). Combined gene expression and microRNA microarray analyses of empty vector vs COX-2- transfected COX-2 low MCF7 breast cancer cell line identified two COX-2-upregulated microRNAs, miR-526b and miR-655, both found to be oncogenic and SLC-promoting. Cytoplasmic Polyadenylation Element-Binding Protein 2 (CPEB2) was the single common target of both microRNAs, the functions of which remain controversial. CPEB2 has multiple isoforms (A-F), and paradoxically, a high B/A ratio was reported to impart anoikis-resistance and metastatic phenotype in triple- negative breast cancer cells. We tested whether CPEB2 is a tumor suppressor in mammary epithelial cells.MethodsWe knocked-out CPEB2 in the non-tumorigenic mammary epithelial cell line MCF10A by CRISPR/Cas9-double nickase approach, and knocked-down CPEB2 with siRNAs in the poorly malignant MCF7 cell line, both lines being high CPEB2-expressing. The resultant phenotypes for oncogenity were tested in vitro for both lines and in vivo for CPEB2KO cells. Finally, CPEB2 expression was compared between human breast cancer and non-tumor breast tissues.ResultsCPEB2 (isoform A) expression was inversely correlated with COX-2 or the above microRNAs in COX-2-divergent breast cancer cell lines. CPEB2KO MCF10A cells exhibited oncogenic properties including increased proliferation, migration, invasion, EMT (decreased E-Cadherin, increased Vimentin, N-Cadherin, SNAI1, and ZEB1) and SLC phenotype (increased tumorsphere formation and SLC marker-expression). Tumor-suppressor p53 protein was shown to be a novel translationally-regulated target of CPEB2, validated with polysome profiling. CPEB2KO, but not wild-type cells produced lung colonies upon intravenous injection and subcutaneous tumors and spontaneous lung metastases upon implantation at mammary sites in NOD/SCID/IL2Rϒ-null mice, identified with HLA immunostaining. Similarly, siRNA-mediated CPEB2 knockdown in MCF7 cells promoted oncogenic properties in vitro. Human breast cancer tissues (n = 105) revealed a lower mRNA expression for CPEB2 isoform A and also a lower A/B isoform ratio than in non-tumour breast tissues (n = 20), suggesting that CPEB2A accounts for the tumor-suppressor functions of CPEB2.ConclusionsCPEB2, presumably the isoform A, plays a key role in suppressing tumorigenesis in mammary epithelial cells by repressing EMT, migration, invasion, proliferation and SLC phenotype, via multiple targets, including a newly-identified translational target p53.

Arsenic-Induced Neoplastic Transformation Involves Epithelial–Mesenchymal Transition and Activation of the β-Catenin/c-Myc Pathway in Human Kidney Epithelial Cells

Arsenic contamination is a serious environmental and public health issue worldwide including the United States. Accumulating evidence suggests that kidney is one of the target organs for arsenic-induced carcinogenesis. However, the mechanism of arsenic-induced renal carcinogenesis is not well understood. Therefore, the objective of this study was to evaluate the carcinogenicity of chronic exposure to an environmentally relevant concentration of arsenic on kidney epithelial cells and identify the molecular mechanism underlying this process. HK-2 kidney epithelial cells were treated with arsenic for acute, long-term, and chronic durations, and cellular responses to arsenic exposure at these time points were evaluated by the changes in growth, morphology, and expression of genes. The results revealed a significant growth increase after long-term and chronic exposure to arsenic in HK-2 cells. The morphological changes of EMT and stem cell sphere formation were also observed in long-term arsenic exposed cells. The anchorage-independent growth assay for colony formation and cell maintenance in cancer stem cell medium further confirmed neoplastic transformation and the induced cancer stem cell properties of arsenic-exposed cells. Additionally, the expression of marker genes confirmed the increased growth, EMT, and stemness during arsenic-induced carcinogenesis. Moreover, the increase expression of β-catenin and c-Myc further suggested the role of these signaling molecules during carcinogenesis in HK-2 cells. In summary, results of this study suggest that chronic exposure to arsenic even at a relatively lower concentration can induce neoplastic transformation through acquisitions of EMT, stemness, and MET phenotypes, which might be related to the β-catenin/c-Myc signaling pathway.

Loss of Disabled-2 Expression in Pancreatic Cancer Progression

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer type characterized by rapid metastasis and resistance to chemotherapy, properties that are shared by cancer stem cells (CSCs). In pancreatic cancer, tumor cells which possess the properties of CSCs also phenotypically resemble cells that have undergone epithelial-to-mesenchymal transition or EMT. Disabled-2 (Dab2) is a multifunctional scaffold protein frequently downregulated in cancer that has been linked to the process of EMT. However, the role of Dab2 in pancreatic cancer development and progression remains unclear. Downregulation of Dab2 expression in pancreatic cancer cell lines was found to trigger induction of genes characteristic of EMT and the CSC phenotype, while overexpression of Dab2 in the Panc1 cell line blocked the process of TGFβ-stimulated EMT. In addition, selective inhibition of the TGFβRI/RII receptors was found to reverse genes altered by Dab2 downregulation. Dab2 mRNA expression was found to be decreased in PDAC tumor samples, as compared to levels observed in normal pancreatic tissue. Methylation of the Dab2 gene promoter was demonstrated in Stage I PDAC tumors and in the MiaPaCa2 cell line, suggesting that promoter methylation may silence Dab2 expression early in pancreatic cancer progression. These results suggest that Dab2 may function as a tumor suppressor in pancreatic cancer by modulation of the TGFβ-stimulated EMT and CSC phenotype.

LINC01354 interacting with hnRNP-D contributes to the proliferation and metastasis in colorectal cancer through activating Wnt/\u03b2-catenin signaling pathway

BackgroundLong non-coding RNAs (lncRNAs) have been identified to play an important role in the development and progression of various tumors, including colorectal cancer (CRC). However, the regulatory molecular mechanism by lncRNA in CRC initiation and progression has not been fully clarified.MethodsTCGA database was used to identify the involvement of LINC01354 in CRC. qRT-PCR and western blot were used to determine RNA and protein expression. The gain- and loss-of-function assays were conducted to explore the function of LINC01354 in the progression of CRC. In order to investigate the LINC01354-mediated mRNA in CRC tumorigenesis, we applied the profiling analysis as well as GO and KEGG analysis. Pulldown and RIP assays were applied to detect the interaction of hnRNP-D with LINC01354 and β-catenin.ResultsThe upregulation of LINC01354 in CRC and its prognostic significance were identified by TCGA database and confirmed in CRC tissues. Functionally, forced expression of LINC01354 promoted, while knockdown of LINC01354 inhibited cell proliferation, migration and EMT phenotype formation of CRC cells. A significant enrichment of the Wnt/β-catenin signaling pathway genes under LINC01354 overexpression. In addition, LINC01354 modulated the mRNA stability of β-catenin through interacting with hnRNP-D, thereby activating Wnt/β-catenin signaling pathway.ConclusionsOur investigations proposed novel regulatory axis of LINC01354/hnRNP-D/Wnt/β-catenin, which might be in favor of exploring novel therapeutic regimens for the clinical treatment of CRC.

138P - PD-L1 confers primary resistance to EGFR-TKI in EGFR mutant non-small cell lung cancer via inducing EMT phenotype

138P - PD-L1 confers primary resistance to EGFR-TKI in EGFR mutant non-small cell lung cancer via inducing EMT phenotype

PTEN inactivation induces epithelial-mesenchymal transition and metastasis by intranuclear translocation of β-catenin and snail/slug in non-small cell lung carcinoma cells

ObjectiveEpithelial-mesenchymal transition (EMT) is the key event in distant metastasis of diverse tumors including lung cancer. Recent evidence suggests the involvement of phosphatase and tensin homolog (PTEN) in EMT phenotypes. However, the molecular mechanism of EMT induced by PTEN inactivation is not clear in lung cancer. We aimed to investigate the role of PTEN inactivation in acquisition of EMT in lung cancer cells. MethodsWe knocked out the PTEN in PTEN proficient lung cancer cells lines (A549 and NCI-H460) using CRISPR/Cas-9 system and observed the growth, EMT phenotypes, and EMT related molecules. We also explored the in vivo effect of PTEN inactivation on tumor cell growth and distant metastasis using nude mouse injection. ResultsPTEN knockout (KO) cells showed faster growth, migration and invasion than PTEN wild-type (WT) cells. When we injected the cells into nude mice, PTEN-KO cells showed faster growth and higher metastatic potential. In PTEN-KO cells, the levels of phosphorylated AKT (Ser-473 and Thr-308) were profoundly elevated and the expressions of phosphorylated GSK-3β (Ser9, inactive form) increased, while that of β-catenin decreased. Regarding the EMT markers, the expression of E-cadherin decreased but those of N-cadherin, vimentin and MMP-2 increased in the PTEN-KO cells. Especially, PTEN-KO cells showed the almost complete intra-nuclear shift of β-catenin and no β-catenin signal was observed in the cell membrane. Accordingly, PTEN-KO cells exhibited morphological changes such as loss of cell-to-cell contact, pseudopodia and the round shape, which are the typical phenotypes of EMT. Snail and Slug were also dominantly accumulated in the nucleus after PTEN inactivation. ConclusionAll these data consistently support that PTEN inactivation contributes to EMT by nuclear translocation of β-catenin and Snail/Slug in lung cancer cells.

Inhibition of LOXL2 Enhances the Radiosensitivity of Castration-Resistant Prostate Cancer Cells Associated with the Reversal of the EMT Process.

Introduction Radiotherapy is the mainstay in the treatment of prostate cancer. However, significant radioresistance of castration-resistant prostate cancer (CRPC) cells constitutes a main obstacle in the treatment of this disease. By using bioinformatic data mining methods, LOXL2 was found to be upregulated in both androgen-independent prostate cancer cell lines and radioresistant tumor samples collected from patients with prostate cancer. We speculate that LOXL2 may play an important role in the radioresistance of CRPC cells. Methods The effect of LOXL2 knockdown on the radiosensitivity of androgen-independent prostate cancer cells lines was measured by the clonogenic assay and xenograft tumor experiments under in vitro and in vivo conditions, respectively. In studies on the mechanism, we focused on the EMT phenotype changes and cell apoptosis changes induced by LOXL2 knockdown in DU145 cells. The protein levels of three EMT biomarkers, namely, E-cadherin, vimentin, and N-cadherin, were measured by western blotting and immunohistochemical staining. Cell apoptosis after irradiation was measured by flow cytometry and caspase-3 activity assay. Salvage experiment was also conducted to confirm the possible role of EMT in the radiosensitization effect of LOXL2 knockdown in CRPC cells. Results LOXL2 knockdown in CRPC cells enhanced cellular radiosensitivity under both in vitro and in vivo conditions. A significant reversal of EMT was observed in LOXL2-silenced DU145 cells. Cell apoptosis after irradiation was significantly enhanced by LOXL2 knockdown in DU145 cells. Results from the salvage experiment confirmed the key role of EMT process reversal in the radiosensitization effect of LOXL2 knockdown in DU145 cells. Conclusions LOXL2 plays an important role in the development of cellular radioresistance in CRPC cells. Targeting LOXL2 may be a rational avenue to overcome radioresistance in CRPC cells. A LOXL2-targeting strategy for CRPC treatment warrants detailed investigation in the future.

The effect of sodium butyrate and cisplatin on expression of EMT markers.

Histone modifications play a key role in the epigenetic regulation of gene transcription in cancer cells. Histone acetylations are regulated by two classes of enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDACs are increased in ovarian carcinomas and they are involved in carcinogenesis and resistance to chemotherapeutic agents. In our study we investigated anticancer effect of HDAC inhibitor sodium butyrate (NaBu) on cisplatin-sensitive and cisplatin-resistant ovarian cell lines A2780 and A2780cis. A2780 and A2780cis were treated with NaBu alone or in combination with cisplatin (CP). NaBu inhibited the growth of both cell lines and enhanced cytotoxic effect of CP. Exposure to NaBu for 24 h induced cell cycle arrest. The expressions of EMT-related genes and proteins were further investigated by qPCR and western blot analysis. Loss of E-cadherin has been shown to be crucial in ovarian cancer development. We found that NaBu dramatically induce expression of E-cadherin gene (CDH1) and protein levels in A2780 and A2780cis. We investigated correlation between transcription and methylation of CDH1gene. Methylation level analysis in 32 CpG sites in CDH1 gene (promoter/exon1 regions) was performed using bisulfite NGS (Next Generation Sequencing). We found that cisplatin-resistant cell line A2780cis cells differ from their cisplatin-sensitive counterparts in the CDH1 methylation. Methylation in A2780cis cells is elevated compared to A2780. However, NaBu-induced expression of CDH1 was not accompanied by CDH1 demethylation. NaBu treatment induced changes in expression of EMT-related genes and proteins. Interestingly E-cadherin zinc finger transcriptional repressor SNAIL1 was upregulated in both cell lines. Mesenchymal marker vimentin was downregulated. Matrix metalloproteases (MMPs) are necessary for pericellular proteolysis and facilitate migration and invasion of tumour cells. NaBu induced mRNA expression of MMPs, mild changes in activities of gelatinases MMP2 and MMP9 were detected. Our data demonstrate that NaBu sensitizes cisplatin-resistant ovarian cancer cells, re-established E-cadherin expression, but it was not able to reverse the EMT phenotype completely.

TLX3 repressed SNAI1-induced epithelial-mesenchymal transition by directly constraining STAT3 phosphorylation and functionally sensitized 5-FU chemotherapy in hepatocellular carcinoma.

TLX3 has an established role as a sequence-specific transcription factor with vital functions in the nervous system. Although several studies have shown that TLX3 is aberrantly up-regulated in leukemia, its expression and function in hepatocellular carcinoma (HCC) remain unknown. We found that TLX3 expression was decreased in 68/100 (68%) HCC cases and negatively correlated with the expression of p-STAT3, SNAI1, and Vimentin, while it was positively associated with E-cadherin expression. ITRAQ proteomic profiling revealed significantly less TLX3 expression in primary HCC tumors than in portal vein tumor thrombi. Comparison of Kaplan-Meier curves showed that down-regulation of TLX3 in HCC was associated with poor post-surgical survival. TLX3 over-expression inhibited HCC cell viability, proliferation, migration, invasion and enhanced 5-FU treatment, whereas silencing TLX3 produced the opposite results. Further experiments showed that TLX3 attenuated the EMT phenotype. In vivo experiments showed that knockdown of TLX3 promoted the growth of HCC xenografts and attenuated the anti-tumor effects of 5-FU treatment. Gene expression microarray analysis revealed that TLX3 inhibited IL-6/STAT3 signaling. In additional mechanistic studies TLX3 reversed the EMT phenotype of HCC cells by binding to STAT3, inhibiting STAT3 phosphorylation, and down-regulating SNAI1 expression. Taken together, loss of expression of TLX3 induces EMT by enhancing IL-6/STAT3/SNAI1 signaling, and accelerates HCC progression while also attenuated the effect of 5-FU on HCCs.