Abstract P4-06-11: Differential regulation of microRNAs and integrins influences metastatic potential: Comparison between locally invasive BT-474 and metastatic MDA-MB-231 xenografts

Abstract

Abstract 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.