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Despite the established benefits of long‐term endocrine therapy, women with hormone receptor‐positive breast cancer remain at risk for late relapse. The basis of this is multi‐factorial including genetic, epigenetic, and host factors. In this study we have explored the epigenetic regulation of estrogen receptor (ER)‐dependent molecular and cellular phenotype by hsa‐miR‐18a‐5p using well‐established human ER‐positive (ER+) breast cancer cell lines. miR‐18a was overexpressed in MCF7 and ZR‐75‐1 and this led to an increase in the proliferative ability of the cells and concurrently resulted in decreased expression of luminal markers and higher expression of the basal marker, cytokeratin 14. The cells became more migratory with a significant repression of E‐cadherin and activation of the Wnt noncanonical pathway. We observed an activation of the planar cell polarity (PCP) pathway with increased activation of JNK pathway and eventually change in actin dynamics. There was increased F‐actin polymerization in cells with higher expression of miR‐18a. Examination of miR‐18a expression in a set of human ER+ breast cancer specimens showed a negative correlation between miR‐18a and ESR1 transcripts as well as ER protein. Kaplan‐Meier survival analysis of the cohort stratified by tumor hsa‐miR‐18a‐5p levels produced significant differences in disease‐free survival (log rank P, Through the manuscript, we have tried to explore the epigenetic regulation of estrogen receptor (ER) dependent molecular and cellular phenotype by hsa‐miR‐18a‐5p. High levels of miR‐18a activates the epigenetic pathway of repression of the luminal phenotype through activation of Wnt pathway.

PTPN11/SHP2, a non-receptor protein tyrosine phosphatase is a prominent target of the receptor tyrosine kinase that participates in positive feedback signalling of the human epidermal growth factor receptors and helps in growth and migration. PTPN11/SHP2 is widely believed to be an oncoprotein, although it’s possible tumor-suppressor role is also reported. Our analysis of breast cancer metadata shows, PTPN11/SHP2 copy number loss in luminal A subtype is correlated to poor disease-specific survival and late-stage cancer at diagnosis. Analysis of the level 4 Reverse Phase Protein Array (RPPA) data available on the TCGA database resulted in positive correlations between the lower expression levels of constitutively active variant, the phospho-SHP2-Y542, of PTPN11/SHP2 and larger tumor size and lymph node positivity. We experimentally examined possible negative regulation of growth by PTPN11/SHP2 using MCF10A, a normal breast epithelial cell line. Knock-down of PTPN11/SHP2 resulted in increased cell migration, cell shape changes to mesenchymal morphology, and increased survival in cells treated with epirubicin, a DNA-damaging drug. However, it did not alter the rate of cell proliferation. It is possible that PTPN11/SHP2 might function as a tumor suppressor by potentiating proliferating cells with increased cell migration and resistance to apoptosis. Statement of Significance Molecules like PTPN11/SHP2, among many others that show dual specificity in tumorigenesis in the same tissue depending on the upstream signaling cues, present challenges in the field of targeted drug therapy. This study puts forth the importance of understanding the mechanism of one of the two outcomes and thereby helps better clinical management of a subgroup of cancer.

Background: MicroRNA mediated molecular alterations are involved in the initiation and progression of cancer. Altered expression of multiple microRNAs is associated with endocrine resistance in hormone receptor positive HER2 negative (HR+/HER2-ve) cancer. The role of miR-221 in inducing epithelial to mesenchymal transition (EMT) is well documented especially in cell line model systems. However, the detailed mechanism of specific microRNAs in intrinsic and acquired resistance to endocrine therapy needs to be worked out. In addition, more needs to be done in the documentation of these mechanisms in human breast cancer specimens with complete clinical documentation and long-term follow-up. In this study, we have evaluated the clinical significance of miR-221 and its mechanistic role in EMT using human specimens and cell line models. Materials and Methods: Formalin fixed paraffin embedded tumor from 129 HR+/HER2-ve breast cancer patients with a median follow up of 63 months were used for estimation of miR-221 by quantitative real time PCR. Expression levels of genes which are direct targets of miR-221 and related genes in EMT were analysed from these tumors. Survival between miR-221 high and low groups was compared by Kaplan Meier survival curves and prognostic relevance was estimated by Cox proportional hazard model. Cell line experiments to investigate the role of miR-221 in inducing EMT through integrin β6 are underway in both wild type and tamoxifen resistant MCF-7 cell lines (A gift from Prof Ben Ho Park, Johns Hopkins University School of Medicine). Results: A significant elevated level of miR-221 was observed in small proportion (14%) of HR+/HER2-ve tumors. miR-221 expression had an inverse correlation with both ER protein and ESR1 mRNA levels within HR+/HER2-ve tumors. Tumors with high levels of miR-221 showed significantly higher expression of integrin β6 which is a robust marker of EMT. Patients with high expression of miR-221 had a poorer survival in Kaplan Meier analysis. Results of interrogation of EMT mediated through integrin related pathways involving miR-221 in cell line models will be presented. Discussion: The association between miR-221 and integrin β6 in HR+/HER2-ve breast cancer with endocrine resistance suggests a potential link between an epigenetic regulator and a mediator of tumor-stromal interaction. The other mediators involved in this pathway are being investigated. miR-221 could be potentially used as a marker for identification of a poor prognostic subtype within HR+/HER2-ve breast cancers. Citation Format: Prabhu JS, Kaul R, Korlimarla A, Desai K, Gangadharan C, Rajarajan S, Nair MG, Alexander A, Kaluve R, Manjunath S, Correa M, Prasad MSN, Patil S, Srinath BS, Sridhar TS. Epithelial mesenchymal transition associated with high miR-221 and integrin β6 leads to poor prognosis in hormone receptor positive HER2 negative breast cancers [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-07-10.

MicroRNA mediated aberrant gene regulation has been implicated in several diseases including cancer. Recent research has highlighted the role of epigenetic modulation of the complex process of breast cancer metastasis by miRNAs. miRNAs play a crucial role in the process of metastatic evolution by facilitating alterations in the phenotype of tumor cells and the tumor microenvironment that promote this process. They act as critical determinants of the multi-step progression starting from carcinogenesis all the way to organotropism. In this review, we focus on the current understanding of the compelling role of miRNAs in breast cancer metastasis.

Integrin αvβ6 is involved in the transition from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) of the breast. In addition, integrin β6 (ITGB6) is of prognostic value in invasive breast cancers, particularly in HER2+ subtype. However, pathways mediating the activity of integrin αvβ6 in clinical progression of invasive breast cancers need further elucidation. We have examined human breast cancer specimens (N = 460) for the expression of integrin β6 (ITGB6) mRNA by qPCR. In addition, we have examined a subset (N = 147) for the expression of αvβ6 integrin by immunohistochemistry (IHC). The expression levels of members of Rho–Rac pathway including downstream genes (ACTR2,ACTR3) and effector proteinases (MMP9,MMP15) were estimated by qPCR in the HER2+ subset (N = 59). There is a significant increase in the mean expression of ITGB6 in HER2+ tumors compared to HR+HER2‐ and triple negative (TNBC) subtypes (P = 0.00). HER2+ tumors with the highest levels (top quartile) of ITGB6 have significantly elevated levels of all the genes of the Rho–Rac pathway (P‐values from 0.01 to 0.0001). Patients in this group have a significantly shorter disease‐free survival compared to the group with lower ITGB6 levels (HR = 2.9 (0.9–8.9), P = 0.05). The mean level of ITGB6 expression is increased further in lymph node‐positive tumors. The increased regional and distant metastasis observed in HER2+ tumors with high levels of ITGB6 might be mediated by the canonical Rho–Rac pathway through increased expression of MMP9 and MMP15.

Background: Generation of BT-474 and MDA-MB-231 xenografts in immunocompromised mice provides one means of dissecting the molecular events involved in local invasion versus distant metastasis. Recently, Glunde et al have shown an interdependence of genes involved in cell-cell and cell-matrix adhesion (E-cadherin and integrin β1) and extra-cellular matrix degradation (MMP-2 and 9) in allowing cells to metastasize. Further, Fite el al have identified a set of microRNAs (miRs) up-regulated by E-cadherin (a pre-EMT marker) and down-regulated by Vimentin (post-EMT marker) in acquiring an invasive phenotype. We have performed a detailed analysis of integrins, matrix metallo-proteases and key miRs to better understand the molecular events underlying these disparate behaviours. Methods: We injected BT-474 (N=5) and MDA-MB-231 (N=5) cells orthotopically into SCID mice. Xenografts were assessed for local growth rate and monitored for distant metastasis. The implanted tumors as well as the distant metastatic foci were harvested. Markers involved in local invasion, distant metastasis and tumor-stroma interactions including miRs were compared between BT-474 and MDA-MB-231 cell lines and their xenografts by q-RT-PCR, immunofluorescence and immunohistochemistry. Results: As expected BT-474 xenografts showed a higher rate of tumor growth when compared to MDA-MB-231. Histological examination of BT-474 tumors confirmed only locally invasive tumor growth with infiltrated blood capillaries and vessels; no macro and microscopic metastases were observed in the organs collected. On the contrary, MDA-MB-231 xenografts showed highly undifferentiated tumor growth and frank lung metastasis and extra-pulmonary tumor growth in one of the five mice injected despite slow rate of local growth. Expression of matrix metalloproteases – MMP-2 & 9 was more than 30 fold upregulated in MDA-MB-231 xenografts as compared to BT-474. Elevated level of E-cadherin was observed in BT-474 but was absent in MDA-MB-231. The most interesting differences were seen in the levels of miRs and cell-surface integrins. High levels of miR-18a, miR-93 and miR-182 were observed in BT-474 implants when compared to MDA-MB-231 which had a much lower level of these miRs. On the contrary, higher levels of integrin β3, and β1 were observed in MDA-MB-231 tumors when compared to BT-474. Integrin β6 was absent in both. The reciprocal relationship between these markers is being examined and compared between locally invasive tumors and metastatic triple negative breast cancers from our case series of human specimens (N=250). Conclusion: miRs and integrins known to be involved in invasion are differentially regulated in tumors that are locally invasive compared to ones with distant metastasis. The level of the key targets of these miRs as well as additional integrins is being examined. Understanding the epigenetic regulations leading to metastasis via tumor-stroma interaction might help in discerning differential tumor behaviour. Citation Format: Lawrence PV, Desai K, Prabhu JS, Korlimarla A, Nair MG, Sridhar TS. Differential regulation of microRNAs and integrins influences metastatic potential: Comparison between locally invasive BT-474 and metastatic MDA-MB-231 xenografts [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-06-11.

An in vitro cell line model was established to exemplify tumor stem cell concept in oral cancer. We were able to identify CD147 expressing fractions in SCC172 OSCC cell line with differing Hoechst dye efflux activity and DNA content. In vivo tumorigenic assay revealed three fractions enriched with stem-like cells capable of undergoing mesenchymal transition and a non-tumorigenic fraction. The regeneration potential and transition of one fraction to other imitated the phenotypic switch and functional disparities evidenced during oral tumor progression. Knowledge of these additional stem-like subsets will improve understanding of stem cell based oral epithelial tumor progression from normal to malignant lesions.

Resistance to anthracycline based chemotherapy is a major limitation in the treatment of breast cancer, particularly of the triple negative sub-type that lacks targeted therapies. Resistance that arises from tumor-stromal interaction facilitated by integrins provides the possibility of targeted disruption. In the present study, we demonstrate that integrin β3 signaling inhibits apoptosis induced by a DNA-damaging chemotherapeutic agent, epirubicin, in MDA-MB-231 breast cancer cells. Drug efflux based mechanisms do not contribute to this effect. We show that integrin β3 employs the PI3K-Akt and the MAPK pathway for enabling cell survival and proliferation. Further, our results indicate that integrin β3 helps inhibit epirubicin induced cytotoxicity by repression of the pro-apoptotic protein BAD, thus promoting an anti-apoptotic response. Myristoylated RGT peptide and a monoclonal antibody against integrin β3 brought about a reversal of this effect and chemosensitized the cells. These results identify β3 integrin signaling via repression of BAD as an important survival pathway used by breast cancer cells to evade chemotherapy induced stress.

Flowcytometry is a reliable method for identification and purification of live cells from a heterogeneous population. Since permeabilized cells cannot be sorted live in a FACS sorter, its application in isolation of functional cells largely depends on antibodies for surface markers. In various fields of biology we find intracellular markers that reveal subpopulations of biological significance. Cell cycle stage specific molecules, metastatic signature molecules, stemness associated proteins etc. are examples of potential markers that could improve the research and therapy enormously. Currently their use is restricted by lack of techniques that allow live detection. Even though a few methods like aptamers, droplet-based microfluidics and smartflares are reported, their application is limited. Here, for the first time we report a simple, cost-effective and efficient method of live sorting of cells based on the expression of an intracellular marker using a fluorophore-tagged binding peptide. The target molecule selected was a histone chaperone, HIRA, the expression of which can predict the fate of differentiating myoblast. Our results confirm that the peptide shows specific interaction with its target; and it can be used to separate cells with differential expression of HIRA. Further, this method offers high purity and viability for the isolated cells.

Classically, isolation of CSCs from tumors exploits the detection of cell surface markers associated with normal stem cells. Invariable expression of these cell surface markers in almost all proliferating tumor cells that albeit impart specific functionality, the universality, and clinical credibility of CSC phenotype based on markers is still dubious. Side Population (SP) cells, as defined by Hoechst dye exclusion in flow cytometry, have been identified in many solid tumors and cell lines and the SP phenotype can be considered as an enriched source of stem cells as well as an alternative source for the isolation of cancer stem cells especially when molecular markers for stem cells are unknown. SP cells may be responsible for the maintenance and propagation of tumors and the proportion of SP cells may be a predictor of patient outcome. Several of these markers used in cell sorting have emerged as prognostic markers of disease progression though it is seen that the development of new CSC-targeted strategies is often hindered by poor understanding of their regulatory networks and functions. This review intends to appraise the experimental progress towards enhanced isolation and drug screening based on property of acquired chemoresistance of cancer stem cells.