Abstract LB-082: The Kindlin2-p53-SerpinB2 signaling axis is required for the regulation of cellular senescence in breast cancer

Abstract

Abstract Physiological cellular senescence is defined as the irreversible arrest of cell proliferation. However, in cancer, pathological aberrations of signaling pathways that regulate specific oncogenes and tumor suppressor genes, can also lead to senescence in cancer cells. In both physiological and pathological settings, senescence is regulated by at least two well-defined pathways; i.e. the p53/p21 and p16INK4a/pRB pathways. In cancer, it is now well established that senescence is a driver of hyperplastic pathology. Kindlin-2 (FERMT2) belongs to the FERM domain-containing family of intracellular proteins that have been established as playing a key role in the activation of integrins. Several recent studies, including those from our group, have associated Kindlin-2 with the pathology of cancers originating from different organs, including breast cancer (BC). For example, we have shown that Kindlin-2 is involved in the regulation of the tumor microenvironment in BC by recruiting macrophages to the tumor site and their polarization to a pro-tumorigenic state. We have also established Kindlin-2 as a major regulator of the EMT process in BC. The role of Kindlin-2 in senescence in BC is, however, not well understood. Our RNA-Seq analyses determined that loss of Kindlin-2 expression in several BC cell lines resulted in a significant increase (at least 30-fold) in SerpinB2 and p21 expression levels, both known associated with cancer cell senescence, compared to the control cells, using RT-PCR, western blot analyses, and by immunostaining of mouse tumors xenografts derived from Kindlin-2-deficient MDA-MB-231 and 4T1 BC cells. Concomitant with the increase of SerpinB2 and p21 expression, we observed a significant increase of β-galactosidase staining, a marker for senescence, in both the Kindlin-2-defficient cells as well as senescent cells, clearly implicating Kindlin-2 in cancer cell senescence via the regulation of expression of senescence-specific genes. Further investigations revealed that Kindlin-2 can be found in nuclear immunocomplexes that also contain p53 in non-senescent cells; and to a much lesser extent in senescent cells. This latest observation led us to hypothesize that binding of Kindlin-2 to p53 may have an inhibitory effect on the function of p53 as a senescence-inducer gene by inhibiting the binding of p53 to the promoter of SerpinB2 and p21. This hypothesis was confirmed by chromatin immunoprecipitation analyses which showed that Kindlin-2, like p53, can bind to the promoters of both SerpinB2 and p21. The binding of Kindlin-2 to SerpinB2 and p21 promoters was, however, weakened in senescent cells. More importantly, in the Kindlin-2-deficient cancer cells, the binding of p53 to the promoters of both SerpinB2 and p21 was significantly much stronger, further supporting our hypothesis that Kindlin-2, by being present in the same immunocomplexes as p53, inhibits the binding of p53 to SerpinB2 and promoters to induce their expression and, therefore, cancer cell senescence. Collectively, our data provide the underpinnings of a new signaling axis (Kindlin-2/p53/SerpinB2-p21), in which Kindlin-2 regulates cancer cell senescence through its binding to p53 and modulating the p53-mediated regulation of expression of senescence-specific genes, thereby identifying a novel role of Kindlin-2 in the regulation of BC progression and metastasis. Citation Format: Khalid Sossey-Alaoui, Elzbieta Pluskota, Dorota Szpak, Edward F. Plow. The Kindlin2-p53-SerpinB2 signaling axis is required for the regulation of cellular senescence in breast 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 LB-082.