Reversal Of EMT Research Articles

Diallyl disulphide suppresses the cannonical Wnt signaling pathway and reverses the fibronectin-induced epithelial mesenchymal transition of A549 lung cancer cells.

Globally, non-small cell lung cancer is a leading cause of cancer-related mortality and about 40% of these cancers are detected in the metastatic stage. Epithelial mesenchymal transition (EMT) plays a critical role during malignant transformation, and the extracellular matrix component, fibronectin (FN), is a known inducer of invasion and metastasis. Diallyl disulphide (DADS), a bioactive component of garlic, exhibits a wide spectrum of biological activities including the inhibition of cancer cell migration and invasion. The present study was aimed at deciphering the effect of DADS on the regulation of FN-induced EMT in A549 lung cancer cells. DADS suppressed the FN-induced invasion and migration potential of A549 cells which may be attributed to the reduced activity of gelatinases. DADS suppressed the FN-aggravated EMT of A549 cells by the upregulation of the epithelial markers, E-cadherin and cytokeratin-18, and the downregulation of the mesenchymal markers, N-cadherin and vimentin, and the transcription factors, snail, slug and twist. DADS was effective in inhibiting the nuclear translocation of β-catenin and the phosphorylation of glycogen synthase kinase-3β and in suppressing the activity of dishevelled homolog 2 and T-cell-factor/lymphoid enhancer factor in FN-induced A549 cells. Cumulatively, this study indicated that DADS might be able to reverse FN-induced EMT in A549 cells via the suppression of Wnt signaling.

Nerigoside suppresses colorectal cancer cell growth and metastatic potential through inhibition of ERK/GSK3β/β-catenin signaling pathway

BackgroundNerigoside (NG), a cardenolide isolated from a commonfolk medicine, Nerium oleander Linn. (Apocynaceae), has not been explored for its biological effects. To date, cardenolides have received considerable attention in pharmacology studies due to their direct effects of apoptosis-induction or growth-inhibitory against tumor in vitro and in vivo. Whether and how NG exerts anticancer effects against colorectal cancer remains to be elucidated. PurposeThe aim of this study was to investigate the anticancer effect of NG in human colorectal cancer cells. MethodsTo test anticancer effect, we compared potency of NG in two colorectal cancer cell lines, HT29 and SW620 by WST-1 and colony proliferation assays. And we investigated mechanism of anticancer activities by analyzing players in apoptotic and ERK/GSK3β/β-catenin signaling pathways in HT29 and SW620 cells treated with NG. ResultsIn this study, we showed that NG markedly suppressed the cell viability and colony formation of colorectal cancer cells HT29 and SW620, with no significant toxic effect on non-cancer cells NCM460. Annexin V-FITC/PI and CFSE labeling results revealed that NG suppressed cell proliferation in low concentration, along with reducing expression of PCNA, while NG induced apoptosis in high concentration,. Meanwhile, NG significantly arrested cell migration by reversal of EMT and cell cycle on G2/M. Then, we found that the ERK and GSK3β/β-catenin signaling pathway were noticeably blocked in CRC cells after treatment with NG. According to western blot, NG upregulated the expression of p-GSK3β/GSK3β and decreased especially the expression of β-catenin in nuclear. In addition, Wnt signaling and its target genes were suppressed in response to NG. Then, the Ser9 phosphorylation of GSK3β can be reduced / raised by GÖ 6983 / LiCl, respectively. Thus, we further confirmed that the GSK3β/β-catenin axis is involved in NG-prevented cell proliferation. ConclusionNG inhibited the growth of colorectal cancer cells by suppressing ERK/GSK3β/β-catenin signaling pathway. And the GSK3β/β-catenin axis is involved in preventing cell proliferation and migration by NG-treatment. These results suggest that NG may be used to treat colorectal cancer, with better outcome by combining with GSK3β inhibitor to block Wnt pathway.

Daurinoline suppressed the migration and invasion of chemo-resistant human non-small cell lung cancer cells by reversing EMT and Notch-1 and sensitized the cells to Taxol

Non-small cell lung cancer (NSCLC) is one of the most common malignancies, and Taxol is a cornerstone in the treatment. However, taxol-resistance eventually limits the clinical effects and applications. Daurinoline could restore the sensitivity of resistant MCF-7/adr and KBv200 cells. Whereas, the effect of daurinoline on the chemo-resistant NSCLC cells and the mechanism has not been elucidated. In this study, daurinoline was firstly demonstrated that inhibited the proliferation, migration, invasion and EMT phenotype of chemo-resistant NSCLC cells. And these effects were associated with EMT and Notch-1 reversal. Moreover, daurinoline could significantly enhance the anti-tumor effect of Taxol rather than epirubicin, adriamycin and cisplatin. And the reverse fold (RF) value of daurinoline was greater than terfenadine reported before. There are little cytotoxic effects of daurinoline and its derivatives reported by L.W. Fu, et al. (2001). Therefore, daurinoline may be a potential anti-tumor agent or chemosensitizer for chemo-resistant NSCLC patients.

Adiponectin inhibits migration and invasion by reversing epithelial‑mesenchymal transition in non‑small cell lung carcinoma.

Adiponectin is the most abundant adipokine in the tumor microenvironment. The role of this protein in tumor progression, however, remains controversial. In the present study, we aimed to investigate the effects of adiponectin on the abilities of migration and invasion in non-small cell lung carcinoma (NSCLC). Using NSCLC cell lines, we examined the effects of adiponectin on cell migration and invasion using Transwell assays. Expression of epithelial-mesenchymal transition markers was examined via microscopy and western blotting. We also performed a knockdown of Twist, AdipoR1 and AdipoR2 in NSCLC cells with siRNAs. The addition of adiponectin to NSCLC cells inhibited both the migration and invasion abilities. Furthermore, we found that NSCLC cells displayed increased epithelial marker expression and downregulation of mesenchymal marker expression following adiponectin administration. Twist AdipoR1 and AdipoR2 knockdown reversed the inhibitory effects of adiponectin on migration and invasion in NSCLC and epithelial-mesenchymal transition. Exogenous adiponectin significantly impaired the migratory and invasive capacities of NSCLC cells through reversal of EMT, suggesting that adiponectin may be a novel promising therapeutic approach against NSCLC.

Valproic acid sensitizes metformin-resistant human renal cell carcinoma cells by upregulating H3 acetylation and EMT reversal

BackgroundMetformin (Met) is a widely available diabetic drug and shows suppressed effects on renal cell carcinoma (RCC) metabolism and proliferation. Laboratory studies in RCC suggested that metformin has remarkable antitumor activities and seems to be a potential antitumor drug. But the facts that metformin may be not effective in reducing the risk of RCC in cancer clinical trials made it difficult to determine the benefits of metformin in RCC prevention and treatment. The mechanisms underlying the different conclusions between laboratory experiments and clinical analysis remains unclear. The goal of the present study was to determine whether long-term metformin use can induce resistance in RCC, whether metformin resistance could be used to explain the disaccord in laboratory and clinical studies, and whether the drug valproic acid (VPA), which inhibits histone deacetylase, exhibits synergistic cytotoxicity with metformin and can counteract the resistance of metformin in RCC.MethodsWe performed CCK8, transwell, wound healing assay, flow cytometry and western blotting to detect the regulations of proliferation, migration, cell cycle and apoptosis in 786-O, ACHN and metformin resistance 786-O (786-M-R) cells treated with VPA, metformin or a combination of two drugs. We used TGF-β, SC79, LY294002, Rapamycin, protein kinase B (AKT) inhibitor to treat the 786-O or 786-M-R cells and detected the regulations in TGF-β /pSMAD3 and AMPK/AKT pathways.Results786-M-R was refractory to metformin-induced antitumor effects on proliferation, migration, cell cycle and cell apoptosis. AMPK/AKT pathways and TGF-β/SMAD3 pathways showed low sensibilities in 786-M-R. The histone H3 acetylation diminished in the 786-M-R cells. However, the addition of VPA dramatically upregulated histone H3 acetylation, increased the sensibility of AKT and inhibited pSMAD3/SMAD4, letting the combination of VPA and metformin remarkably reappear the anti-tumour effects of metformin in 786-M-R cells.ConclusionsVPA not only exhibits synergistic cytotoxicity with metformin but also counteracts resistance to metformin in renal cell carcinoma cell. The re-sensitization to metformin induced by VPA in metformin-resistant cells may help treat renal cell carcinoma patients.

Novel application of the published kinase inhibitor set to identify therapeutic targets and pathways in triple negative breast cancer subtypes.

Triple negative breast cancers (TNBCs) have high recurrence and metastasis rates. Acquisition of a mesenchymal morphology and phenotype in addition to driving migration is a consequential process that promotes metastasis. Although some kinases are known to regulate a mesenchymal phenotype, the role for a substantial portion of the human kinome remains uncharacterized. Here we evaluated the Published Kinase Inhibitor Set (PKIS) and screened a panel of TNBC cell lines to evaluate the compounds’ effects on a mesenchymal phenotype. Our screen identified 36 hits representative of twelve kinase inhibitor chemotypes based on reversal of the mesenchymal cell morphology, which was then prioritized to twelve compounds based on gene expression and migratory behavior analyses. We selected the most active compound and confirmed mesenchymal reversal on transcript and protein levels with qRT-PCR and Western Blot. Finally, we utilized a kinase array to identify candidate kinases responsible for the EMT reversal. This investigation shows the novel application to identify previously unrecognized kinase pathways and targets in acquisition of a mesenchymal TNBC phenotype that warrant further investigation. Future studies will examine specific roles of the kinases in mechanisms responsible for acquisition of the mesenchymal and/or migratory phenotype.

Abstract 842: Breast cancer: Relationship between GPC3 and EMT process

Abstract Epithelial-mesenchymal transition (EMT) is a process whereby epithelial cells acquire mesenchymal fibroblast-like properties and display reduced intracellular adhesion and increased motility. Glypican-3 (GPC3) is a heparan sulfate proteoglycan associated with cancer. We have established that GPC3 regulates the EMT process underwent by breast cancer cells. However, cellular and molecular events underlying this modulation are not completely described. At the present work we analyzed the effect of GPC3 on cell adhesion and on the expression of molecules associated with this property. In addition, molecular regulators of EMT were also studied. We employed a murine breast cancer cell line (LM3: GPC3-) as well as human lines (MDA-MB231: GPC3- and MCF-7: GPC3+). We have blocked GPC3 expression in MCF-7 cells, while it was overexpressed in MDA-MB231 and LM3 ones. It is know that EMT depends on a reduction in cell adhesion. We determined that GPC3 overexpression induced an increase in the homotypic adhesion of MDA-MB231 and LM3 cells, although GPC3 silencing did not lead significant changes in the organization of MCF-7 spheroids. GPC3 was also able to modulate the adhesion to extracellular matrix components. GPC3 overexpressing cells were more adherent to fibronectin than controls, but no changes were observed in their adhesion to laminin. E-Cadherin, a molecule involved in homotypic adhesion, is considered a suppressor of invasion and growth of many epithelial cancers. Here, we have corroborated by WB that GPC3 overexpression induces the E-Cadherin upregulation in LM3 and MDA-MB231 cells, whereas GPC3 silencing inhibits the expression of this epithelial marker in MCF-7 cells. We also confirmed the called "cadherin switch", since N-Cadherin was downregulated in GPC3 overexpressing cells and upregulated in GPC3 silenced ones. β-Catenin is a key downstream effector in the Wnt pathway. We confirmed that GPC3 expressing breast cancer cells exhibit a canonical Wnt signaling inhibition. β-Catenin also activates Slug. Similar to Snail, Slug binds to the E-Cadherin promoter to repress transcription. By WB and qPCR we demonstrated that GPC3 induces a decrease in Slug and Snail levels. Surprisingly, Twist expression was higher in GPC3 expressing cells. It was reported that Slug is able to represses integrins expression. However, β1-Integrin levels were decreased in GPC3 overexpressing MDA-MB231 cells, while they were increased in GPC3 blocked MCF-7 sublines. Our results suggest the key role of GPC3 in the EMT regulation. GPC3 would modulate cell adhesion and expression of E-Cadherin, N-Cadherin and β1-Integrin. In addition, GPC3 would regulate the expression of different EMT molecules (as Slug, Snail and Twist) as well as signaling pathways involved in that process (like canonical Wnt). The set and balance of these regulations would result in the reversal of EMT, reinforcing the function of GPC3 as an inhibitor of mammary tumor progression. Citation Format: Lilian F. Castillo, Gisela V. Novack, Elisa Bal de Kier Joffé, María G. Peters. Breast cancer: Relationship between GPC3 and EMT process [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 842. doi:10.1158/1538-7445.AM2017-842

Abstract 1820: Synergistic lethality of mAbs with an EMT reversal agent, Nintedanib, in epithelial ovarian cancer

Abstract Epithelial-mesenchymal transition (EMT) has long been associated with cancer progression and metastasis. As a reversible process, it has the potential to be a target for cancer therapy. However, this concept of reversing EMT has not yet been widely explored in current treatment strategies. With the identification of Nintedanib as an EMT reversal agent, we aim to broaden the utility of this triple angiokinase inhibitor by identifying agents that show synergistic lethality with it, using monoclonal antibodies (mAbs) as a combinatory therapeutic. Nintedanib alone is not significantly cytotoxic to cells, but it causes cell cycle arrest and reverses EMT. By selecting for this sub-population of cells that are sensitive to Nintedanib, their subsequent eradication can then be more intricately directed. Since the anti-cancer application of EMT reversal agents are not fully developed, it is vital to investigate mechanisms that sensitize cancer cells to cytotoxic agents following EMT reversal.Our group has generated panels of mAbs that are able to bind differentially to various breast and ovarian cancer cell lines along the EMT spectrum. Some mAbs preferentially bind to Epithelial lines, while some have stronger affinity to Mesenchymal lines. This implies that mAbs have the ability to differentiate between epithelial and mesenchymal phenotypes, uncovering a new dimension to the capability of mAbs which have yet to be explored.Using a mesenchymal ovarian cancer cell line as a starting model, Nintedanib-treated cells were screened with the mAbs and those showing a >2-fold increase in binding were shortlisted for further validation. Based on this criteria, 26 mAbs were identified and further characterized in functional Antibody Drug Conjugate (ADC) assays with mAbs conjugated to a toxic drug, Saporin (or Zap). Mabs successfully internalizing and killing the cancer cells will then be further chosen for antigen characterization and in vivo functional studies. Ultimately, there is huge potential for mAbs to be discovered that shows synergy with Nintedanib to enhance lethality to various types of cancer cells. A novel clinical strategy, the application of EMT reversal in terms of utilising a synthetic lethality-like approach allows better design of combinatory therapeutics, increasing treatment efficacy that might revolutionalise cancer treatment. Citation Format: Jocelyn Teo, Heng Liang Tan, Ruby Yun-Ju Huang, Andre Choo. Synergistic lethality of mAbs with an EMT reversal agent, Nintedanib, in epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1820. doi:10.1158/1538-7445.AM2017-1820

EMT reversal in human cancer cells after IR knockdown in hyperinsulinemic mice.

Type 2 diabetes (T2D) is associated with increased cancer risk and cancer-related mortality. Data herein show that we generated an immunodeficient hyperinsulinemic mouse by crossing the Rag1(-/-) mice, which have no mature B or T lymphocytes, with the MKR mouse model of T2D to generate the Rag1(-/-) (Rag/WT) and Rag1(-/-)/MKR(+/+) (Rag/MKR) mice. The female Rag/MKR mice are insulin resistant and have significantly higher nonfasting plasma insulin levels compared with the Rag/WT controls. Therefore, we used these Rag/MKR mice to investigate the role of endogenous hyperinsulinemia on human cancer progression. In this study, we show that hyperinsulinemia in the Rag/MKR mice increases the expression of mesenchymal transcription factors, TWIST1 and ZEB1, and increases the expression of the angiogenesis marker, vascular endothelial growth factor A (VEGFA). We also show that silencing the insulin receptor (IR) in the human LCC6 cancer cells leads to decreased tumor growth and metastases, suppression of mesenchymal markers vimentin, SLUG, TWIST1 and ZEB1, suppression of angiogenesis markers, VEGFA and VEGFD, and re-expression of the epithelial marker, E-cadherin. The data in this paper demonstrate that IR knockdown in primary tumors partially reverses the growth-promoting effects of hyperinsulinemia as well as highlighting the importance of the insulin receptor signaling pathway in cancer progression, and more specifically in epithelial-mesenchymal transition.

Abstract 1873: The role of p21-activated kinase 4 (PAK4) in cancer stemness and epithelial-to-mesenchymal transition

Abstract The p21-activated kinase 4 (PAK4) is a key downstream effector of the Rho family GTPases. PAK4 is found to be over-expressed in many oncogenic Ras-driven cancers and cell lines but not in their normal counterparts. Mesenchymal pancreatic ductal adenocarcinoma (PDAC) stem cells (CSCs) that are triple positive for stemness markers (CD133+, CD44+, and EpCam+) as well as Ras and snail transduced human mammary epithelial cells (HMLER-snail) showed enhanced expression of PAK4 along with activation of Rho, Rac1 and CDC42. PAK4 RNA interference resulted in disruption of PDAC CSC spheroids, reduction of mesenchymal markers (i.e. vimentin and snail) and suppression of CSCs potential to form subcutaneous tumors in mice. These findings point to a novel role of PAK4 in promoting oncogenic Ras-driven EMT and stemness. Using PDAC CSCs (MiaPaCa-2 and L3.6pl) and human mammary epithelial cells (HMECs) that are transduced with Ras and the EMT promoter snail (HMLE-Snail and HMLER-Snail), we investigated the impact of PAK4 allosteric modulators [PAMs (KPT-7189, KPT-7523, KPT-9274 and KPT-9307)] on the reversal of EMT and stemness. The toxicity and efficacy of PAMs were evaluated in vitro and in multiple sub-cutaneous xenograft models. PAMs show anti-proliferative activity in vitro against different PDAC CSCs and HMECs while sparing normal cells. Cell growth inhibition was concurrent with apoptosis induction, suppression of colony formation and reversal of mesenchymal morphology to epithelial (MET). PAMs not only reduced PAK4 RNA and protein steady state levels but also inhibited proliferative and anti-apoptotic signals downstream of PAK4. PAMs caused suppression of EMT markers (EpCAM, vimentin and snail) and re-expression of an epithelial promoter E-cadherin. PAM treatment inhibited RhoA, Rac1 and CDC42 activation in GLISA assays. KPT-9274 showed remarkable anti-tumor activity in sub-cutaneous xenograft models of CSCs and residual tumors showed suppression of EMT markers that was concurrent with inhibition of PAK4 signaling. These studies open a novel therapeutic opportunity against EMT and cancer stem cells through PAK4 inhibition. Citation Format: Asfar S. Azmi, Irfana Muqbil, Amro Aboukameel, William Senapedis, Erkan Baloglu, Yosef Landesman, Sharon Shacham, Michael Kauffman, Philip A. Philip, Ramzi M. Mohammad. The role of p21-activated kinase 4 (PAK4) in cancer stemness and epithelial-to-mesenchymal transition. [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 1873.

An epithelial marker promoter induction screen identifies histone deacetylase inhibitors to restore epithelial differentiation and abolishes anchorage independence growth in cancers.

Epithelial–mesenchymal transition (EMT), a crucial mechanism in development, mediates aggressiveness during carcinoma progression and therapeutic refractoriness. The reversibility of EMT makes it an attractive strategy in designing novel therapeutic approaches. Therefore, drug discovery pipelines for EMT reversal are in need to discover emerging classes of compounds. Here, we outline a pre-clinical drug screening platform for EMT reversal that consists of three phases of drug discovery and validation. From the Phase 1 epithelial marker promoter induction (EpI) screen on a library consisting of compounds being approved by Food and Drug Administration (FDA), Vorinostat (SAHA), a histone deacetylase inhibitor (HDACi), is identified to exert EMT reversal effects by restoring the expression of an epithelial marker, E-cadherin. An expanded screen on 41 HDACi further identifies 28 compounds, such as class I-specific HDACi Mocetinosat, Entinostat and CI994, to restore E-cadherin and ErbB3 expressions in ovarian, pancreatic and bladder carcinoma cells. Mocetinostat is the most potent HDACi to restore epithelial differentiation with the lowest concentration required for 50% induction of epithelial promoter activity (EpIC-50).The HDACi exerts paradoxical effects on EMT transcriptional factors such as SNAI and ZEB family and the effects are context-dependent in epithelial- and mesenchymal-like cells. In vitro functional studies further show that HDACi induced significant increase in anoikis and decrease in spheroid formation in ovarian and bladder carcinoma cells with mesenchymal features. This study demonstrates a robust drug screening pipeline for the discovery of compounds capable of restoring epithelial differentiation that lead to significant functional lethality.

Blocking the PAH2 domain of Sin3A inhibits tumorigenesis and confers retinoid sensitivity in triple negative breast cancer.

Triple negative breast cancer (TNBC) frequently relapses locally, regionally or as systemic metastases. Development of targeted therapy that offers significant survival benefit in TNBC is an unmet clinical need. We have previously reported that blocking interactions between PAH2 domain of chromatin regulator Sin3A and the Sin3 interaction domain (SID) containing proteins by SID decoys result in EMT reversal, and re-expression of genes associated with differentiation. Here we report a novel and therapeutically relevant combinatorial use of SID decoys. SID decoys activate RARα/β pathways that are enhanced in combination with RARα-selective agonist AM80 to induce morphogenesis and inhibit tumorsphere formation. These findings correlate with inhibition of mammary hyperplasia and a significant increase in tumor-free survival in MMTV-Myc oncomice treated with a small molecule mimetic of SID (C16). Further, in two well-established mouse TNBC models we show that treatment with C16-AM80 combination has marked anti-tumor effects, prevents lung metastases and seeding of tumor cells to bone marrow. This correlated to a remarkable 100% increase in disease-free survival with a possibility of “cure” in mice bearing a TNBC-like tumor. Targeting Sin3A by C16 alone or in combination with AM80 may thus be a promising adjuvant therapy for treating or preventing metastatic TNBC.

Berberine induces neuronal differentiation through inhibition of cancer stemness and epithelial-mesenchymal transition in neuroblastoma cells

BackgroundBerberine, a plant alkaloid, has been used since many years for treatment of gastrointestinal disorders. It also shows promising medicinal use against metabolic disorders, neurodegenerative disorders and cancer; however its efficacy in neuroblastoma (NB) is poorly explored. HypothesisEMT is important in cancer stemness and metastasis resulting in failure to differentiate; thus targeting EMT and related pathways can have clinical benefits. Study designPotential of berberine was investigated for (i) neuronal differentiation and cancer stemness inhibition, (ii) underlying molecular mechanisms regulating cancer-stemness and (iii) EMT reversal. MethodsUsing neuro2a (N2a) neuroblastoma cells (NB); we investigated effect of berberine on neuronal differentiation, cancer-stemness, EMT and underlying signalling by immunofluorescence, RT-PCR, Western blot. High glucose-induced TGF-β mediated EMT model was used to test EMT reversal potential by Western blot and RT-PCR. STRING analysis was done to determine and validate functional protein-interaction networks. ResultsWe demonstrate berberine induces neuronal differentiation accompanying increased neuronal differentiation markers like MAP2, β-III tubulin and NCAM; generated neurons were viable. Berberine attenuated cancer stemness markers CD133, β-catenin, n-myc, sox2, notch2 and nestin. Berberine potentiated G0/G1 cell cycle arrest by inhibiting proliferation, cyclin dependent kinases and cyclins resulting in apoptosis through increased bax/bcl-2 ratio. Restoration of tumor suppressor proteins, p27 and p53, indicate promising anti-cancer property. The induction of NCAM and reduction in its polysialylation indicates anti-migratory potential which is supported by down regulation of MMP-2/9. It increased epithelial marker laminin and smad and increased Hsp70 levels also suggest its protective role. Molecular insights revealed that berberine regulates EMT via downregulation of PI3/Akt and Ras-Raf-ERK signalling and subsequent upregulation of p38-MAPK. TGF-β secretion from N2a cells was potentiated by high glucose and negatively regulated by berberine through modulation of TGF-β receptors II and III. Berberine reverted mesenchymal markers, vimentin and fibronectin, with restoration of epithelial marker E-cadherin, highlighting the role of berberine in reversal of EMT. ConclusionCollectively, the study demonstrates prospective use of berberine against neuroblastoma as elucidated through inhibition of fundamental characteristics of cancer stem cells: tumorigenicity and failure to differentiation and instigates reversal in the EMT.

Abstract P5-08-38: Low lactate dehydrogenase B expression correlates with decreased distant-metastasis free- and recurrence-free survival post-chemotherapy in basal-like breast cancer

Abstract Background: Metabolism is an important differentiating feature of cancer cells. Lactate dehydrogenases (LDH A/B) are metabolically important proteins involved in the critical inter-conversion of pyruvate to lactate and vice versa. Several reports suggest that LDHB levels are elevated in TNBC, compared to other breast cancer subtypes. However, we recently published that LDHB levels are low in TNBC cell lines and restoring LDHB results in decreased cell proliferation, oxidative phosphorylation, and reversal of EMT. Furthermore, in a small patient cohort, we have shown that although LDHB levels are higher in TNBC patients compared to non-TNBC patients, LDHB levels where consistently lower when compared to LDHA levels. Thus, we set out to determine if either "Hi" or "Low" LDHA and LDHB levels effect patient survival. Methods: Utilizing the publically available datasets contained within kmplot,which contains gene expression data and relapse free and overall survivall, we determined mean levels of LDHA and LDHB in breast cancer patients. To analyze the prognostic value, patient samples were split into two groups based upon expression above the mean (considered high expressors, "Hi") or below the mean (considered low expressors, "Low"). The two patient cohorts were compared by a Kaplan-Meier survival plot, and the hazard ratio with 95% confidence intervals and logrank P value calculated. Groups were further stratified based upon LDH levels prior to- and post-chemotherapy. Results: We found that in patients with luminal A and luminal B breast cancer, there were no significant changes in either LDHA (p=0.1) or LDHB (p=0.21) on distant metastasis-free (DMFS) or recurrence–free (RFS) survival. However, in the basal subtype (i.e. patients with ER negative and PR negative breast cancer), low levels of LDHB was significantly associated with poorer DMFS (p=0.025) (n=240) prior to chemotherapy and both DMSF (p=0.048) (n=176) and RFS (p=0.0082) (n=388) post-chemotherapy. Examining the mean expression values for each of these patient populations, we did not observe any significant changes in DMSF or RFS pre or post-chemotherapy, suggesting an intrinsic feature of basal-like patients with low LDHB expression to have a more aggressive phenotype. Interestingly, we did observe significance in RFS (n=581, p=0.0043) in patient with "Hi" LDHA expression pre-chemotherapy, however there was no significant associations of LDHA with RFS (p=0.19) (n=388) or DMSF (n=176, p=0.75) post-chemotherapy. Conclusion: These findings, coupled with our cell line data, showing overexpressing LDHB in TNBC cell lines results in decreased proliferation with increased mitochondrial damage and apoptosis, suggests that lower levels of LDHB expression is indeed associated with an aggressive breast cancer phenotype that undergoes EMT and the Warburg effect. This could contribute to the lack of pathological response after chemotherapy and thus increased risk for later metastasis. Additionally, given the very large number of patients examined within these independent datasets, these findings further suggest that low LDHB expression is a robust prognostic biomarker of clinical outcome in patients with a basal-like phenotype. Citation Format: Dean-Colomb W, Tan M, Tang W, Ambs S, Yates C. Low lactate dehydrogenase B expression correlates with decreased distant-metastasis free- and recurrence-free survival post-chemotherapy in basal-like breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-08-38.

Abstract P1-05-06: Essential role of notch-4/STAT3 signaling in epithelial-mesenchymal transition of tamoxifen-resistant human breast cancer

Abstract Epithelial-mesenchymal transition (EMT) is process in which epithelial cells undergo unique morphologic changes characterized by a transition from epithelial cobblestone phenotype to elongated fibroblastic phenotype (mesenchymal phenotype) leading to increased motility and invasion. Our previous study demonstrated that tamoxifen (TAM)-resistant human breast cancer (TAMR-MCF-7) cells showed the increased expression of mesenchymal marker proteins compared to the parent MCF-7 cells. Notch plays a crucial role in the promotion of EMT during both development and tumor progression. Especially, Notch-1 and Notch-4 were reported as prognostic markers in human breast cancer. Here, we found that the basal expression and activity of Notch-4 were significantly increased in TAMR-MCF-7 cells compared to control MCF-7 cells. Suppression of Notch-4 by either Notch inhibitors or Notch-4 siRNA significantly attenuated the EMT signaling. Interestingly, long-term treatment with DAPT, a Notch inhibitor, eventually led to partial reversal of EMT by up-regulating E-cadherin expression. Activated or tyrosine-phosphorylated STAT3 (pYSTAT3) protein is supposed as a critical signaling molecule in the regulation of tumorigenesis and metastasis of cancer cells. We further found that TAMR-MCF-7 cells exhibited constitutive STAT3 phosphorylation. Suppression of Notch-4 activation attenuated the activated STAT3 elevation in TAMR-MCF-7 cells. We hypothesized that Notch-4 regulated EMT in TAMR-MCF-7 cells via STAT3 signaling. Intrasplenic injection model of liver metastases was performed using TAMR-MCF-7 cells. Mice were received subcutaneous injections daily with DAPT (10 mg/kg) formed smaller tumor size in spleens and showed less micrometastatic tumor burden in livers compared to group treated with vehicle. In conclusion, inhibition of Notch signaling may have efficacy in the treatment of breast cancer metastasis. Citation Format: Bui QT, Kang KW. Essential role of notch-4/STAT3 signaling in epithelial-mesenchymal transition of tamoxifen-resistant human breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-05-06.

Abstract P5-03-08: Eribulin impairs the transport of TGF-β type I receptor leading to inhibition of downstream non-canonical TGF-β signaling necessary for cancer metastasis and survival

Abstract Microtubule targeting agents (MTAs) continue to be some of the most valuable drugs used in the treatment of breast cancers. While decades of research have shown that these drugs cause mitotic arrest in cells by suppressing the dynamic instability of microtubules, recent evidence demonstrates that the ability of MTAs to disrupt microtubule-dependent transport of key signaling components, including proteins and microRNAs, in interphase cells likely contributes to their anticancer actions. TGF-β receptors are known to undergo constant cycling from the plasma membrane to intracellular portions of the cell, a process which is microtubule dependent. This microtubule-dependent trafficking has been shown to regulate the nuclear translocation of the TGF-β type I receptor, TGFβR1. Nuclear translocation of TGFβR1 activates the expression of genes including Snail and MMP2, which facilitate the invasiveness, motility and metastasis of cancer cells. We tested the hypothesis that a 2 h treatment of breast cancer cells with eribulin or 4 other clinically relevant MTAs, would differentially disrupt interphase microtubules and alter the internalization and trafficking of TGFβR1 to the nucleus; thereby impacting downstream signaling events. Cells were serum starved for 12 hours and then treated for 2 h with concentrations of MTAs that caused comparable disruption of the interphase microtubule network; 100 nM was used for the destabilizers, eribulin and vinorelbine and 1 μM was used for the stabilizers, paclitaxel, docetaxel and ixabepilone. Following the 2 h treatment, cells were stimulated with 10 ng/mL TGF-β1 for 30 min. The results show that there are distinct differences between the effects of microtubule stabilizers and destabilizers on TGFβR1 trafficking. Eribulin and vinorelbine decreased the nuclear localization of TGFβR1 in a panel of breast cancer cell lines with initial studies suggesting that eribulin impairs this trafficking to a greater extent. In contrast, the microtubule stabilizers, particularly ixabepilone, increased TGFβR1 localization in the nucleus. Additionally, TGFβR1 was extensively localized along stabilizer-induced microtubule bundles. Overall, our work suggests that eribulin is the most effective MTA at inhibiting TGFβR1 nuclear accumulation and subsequent phosphorylation of Smads 2 and 3. The downstream signaling effects of these MTAs on TGFβR1 induced transcription of Snail and MMP2 are also being investigated. Eribulin induced inhibition of non-canonical TGF-β signaling is consistent with previous studies that show that a 7 day treatment with eribulin reversed TGF-β-mediated EMT in breast cancer cells.1,2 Our data suggest that inhibition of the nuclear transport of TGFβR1 could be a potential mechanism for the eribulin-mediated EMT reversal. These studies begin to shed light into the diverse mechanisms of MTAs and lay the groundwork to identify patient populations that might respond optimally to different MTAs. 1. Yoshida T, et al., Brit J Cancer 110(6): 1497-505, 2014. 2. Dezso Z, et al., PloS One, 9(8): e106131, 2014. This work is funded by Eisai Inc. Citation Format: Rohena CC, Kaul R, Risinger AL, Mooberry SL. Eribulin impairs the transport of TGF-β type I receptor leading to inhibition of downstream non-canonical TGF-β signaling necessary for cancer metastasis and survival. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-03-08.

Abstract B01: B cell lymphoma 9 mediates a cross talk between the canonical Wnt and EGFR signaling in breast cancer

Abstract Human ductal carcinoma in situ (DCIS) are the most common type of non-invasive breast cancers. The five-year survival rate for women diagnosed with non-invasive DCIS is 98% while the five-year survival plummets to 83-27% for breast cancers that have become invasive and have spread to distant sites [also referred to as invasive ductal carcinoma (IDC)]. To study DCIS pathobiology and factors that promote their transition to IDC, we have developed a novel in vivo DCIS model, MIND (mouse-intraductal), that involves intraductal injection of epithelial cells derived from primary human DCIS biopsy and surgical samples thus mimicking the entire process of DCIS to IDC transition. As a complementary approach, we have utilized human DCIS/IDC tandem lesions, which are patient DCIS that show a transition to IDC within the same breast. Analysis of RNA and protein at distinct stages of in situ to IDC using both models showed B cell lymphoma-9 (BCL9) up-regulation to be associated with DCIS transition to IDC. BCL9 is a recently identified co-activator of Wnt-stimulated beta-catenin-mediated transcription. Our studies showed that in vivo silencing of BCL9 led to inhibition of DCIS invasion and reversal of EMT. We have also demonstrated a direct binding interaction between BCL9 and beta-catenin and showed suppression of beta-catenin-mediated transcription by BCL9 knockdown. Analysis of patient DCIS samples revealed a significant correlation between high nuclear BCL9 expression and pathologic characteristics associated with DCIS recurrence: Estrogen receptor (ER) negative and Ki67. Furthermore, analysis of the TCGA data showed BCL9 gene to be upregulated in 26% of breast cancers. This is a significant gene alteration when compared to HER2 (ERBB2) gene (19%) and estrogen receptor (ESR1) gene (8%) alterations in breast cancers. Interestingly, a significantly higher proportion of basal like invasive breast cancers compared to luminal breast cancers showed BCL9 amplification suggesting that BCL9 may predispose to the development of basal breast cancers. We have performed an RPPA analysis on our DCIS cell lines KD BCL9 vs. control. This analysis indicated that BCL9 KD showed down-regulation in a number of genes in the EGFR signaling pathway including p-EGFR, p-HER2, p-STAT3, and p-Src. Conclusion: BCL9 is a molecular driver of DCIS invasive progression. The molecular mechanism for BCL9's role in breast cancer progression is through the enhancement in the canonical Wnt and EGFR signaling. Citation Format: Hanan Elsarraj, Hong Yan, Jennifer Knapp, Anna Tsimelzon, Shixia Huang, Andrew Godwin, Sue Hilsenbeck, Dean Edwards, Fariba Behbod. B cell lymphoma 9 mediates a cross talk between the canonical Wnt and EGFR signaling in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B01.

SphK1 promotes tumor cell migration and invasion in colorectal cancer.

Colorectal cancer (CRC) is one of the most common cancers worldwide. Sphingosine kinase 1 (SphK1), which phosphorylates sphingosine to sphingosine-1-phosphate (S1P), is overexpressed in various types of cancers and may act as an oncogene in tumorigenesis. However, little is known about the role of SphK1 in CRC patients. We studied the expression of SphK1 in 85 cases of CRC tissues by immunohistochemistry, qRT-PCR, and western blot. We also evaluated the effect of SphK1 on cell proliferation and invasion by MTT and transwell invasion assay. SphK1 is overexpressed in CRC tissues and cell lines, and upregulation of SphK1 correlated significantly with the following parameters: lymph node metastasis, liver metastasis, and advanced TNM stage. SphK1 knockdown results in inhibition of cancer cell proliferation. Inhibition of CRC cell migration and invasion is also evident through reversal of EMT by increases in E-cadherin expression and decreases in vimentin expression. In conclusion, SphK1 is associated with the proliferation and invasiveness of CRC cells and the SphK1 gene may contribute to a novel therapeutic approach against CRC.

Abstract LB-B21: BI-69A11 inhibits Epithelial to Mesenchymal Transition exploiting β-catenin pathway in colon carcinoma

Abstract EMT is a potential biological phenomena required for metastatic dissemination. Although targeting EMT for therapeutic implication has been gaining interest to the scientific community, only few molecules have succeeded to employ their anti-metastatic effect so far. BI-69A11 has been earlier proved for its anti-tumor activity in prostate and colon carcinoma. This study aims to elucidate the biological consequence of BI-69A11 on EMT in colon carcinoma in vitro and in vivo. Our findings showed that BI-69A11 increased epithelial signature marker E-cadherin and reciprocally decreased the mesenchymal markers snail, slug and vimentin. Moreover, cellular migration, invasion and adhesion were potentially hampered by BI-69A11 through β-catenin dependent pathway. The underlying molecular mechanism behind BI-69A11 induced inhibition of EMT includes suppression of nuclear transport of β-catenin, diminished phosphorylation of β-catenin accompanied with enhanced E-cadherin- β-catenin complex formation at the membrane. Moreover, BI-69A11 controlled the transcriptional activity and reversed nuclear translocation by decrease in the phosphorylation at ser552 and ser45/Thr41 residue. The growing interaction between E-cadherin and β-catenin complex also promoted the E-cadherin transcription leading to the reversal of EMT. BI-69A11 caused increasing accumulation of vinculin in the membrane that in turn strengthened the focal adhesion junction leading to the inhibition of metastasis. Overall, these evidences present a thoughtful insight towards underlying molecular mechanism of BI-69A11 induced reversal of EMT in colorecteal carcinoma in vitro and in vivo. Citation Format: Ipsita Pal, Mahitosh Mandal. BI-69A11 inhibits Epithelial to Mesenchymal Transition exploiting β-catenin pathway in colon carcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B21.

Regulator of G-protein signaling 4: A novel tumor suppressor with prognostic significance in non-small cell lung cancer

Regulator of G-protein signaling (RGS) family members are regulatory molecules which act as GTPase activating proteins (GAPs) for G alpha subunits of heterotrimeric G proteins. Emerging data indicated that RGS members were involved with tumorigenesis and metastasis. In the current study, we identified RGS4 as a novel tumor suppressor with prognostic significance in non-small cell lung cancer (NSCLC). To be specific, we found that RGS4 expression was higher in normal lung tissues than NSCLC specimens (P = 0.003). Further studies demonstrated that RGS4 was generally down-regulated in NSCLC specimens compared with the matched normal lung tissues, both at mRNA and protein levels. In addition, correlational analysis indicated that RGS4 expression levels negatively correlated with lymph node metastasis (P = 0.009) and TNM stage (P = 0.008). Survival analysis demonstrated that patients with lower RGS4 protein expression exhibited a much worse 5-year overall survival and 5-year disease-free survival than those with high expression. More importantly, we proved that over-expression of RGS4 in NSCLC cells decreased invasion and migration due to inhibition of MMP2/9 and reversal of EMT while down-regulation of RGS4 in normal lung cell lines promoted invasion and migration. At last, nude mice metastatic model proved that over-expression of RGS4 suppressed tumor metastasis in vivo. All of these results confirmed the critical role of RGS4 in NSCLC progression.