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Mitotic kinases such as polo-like kinase 1 (PLK1) and aurora kinase B (AURKB) are known to phosphorylate survivin, an IAP family member, to mediate cell survival and proliferation. Interestingly, the triad is significantly overexpressed in multiple tumors including triple-negative breast cancer (TNBC), an aggressive breast cancer subtype, associated with high proliferative capacity, propensity for distant metastasis and therapeutic resistance. Not only TNBCs disproportionately afflict African-American (AA) women compared to European-American (EA) women, AAs demonstrate a much more aggressive disease course compared to their EA counterparts. Largely, most efforts on examining molecular underpinnings of breast cancer-related racial disparity have so far focused on differences in gene expression, epigenetic aspects, and single-nucleotide polymorphisms (SNPs) in tumor tissues. Herein, employing racially distinct TNBC cells, we illuminate the role of survivin phosphorylation, an important post-translational modification, in dictating enhanced cell proliferation and survival in AA TNBC cells compared to EA TNBC cells. Using the publically-available TCGA dataset, our comprehensive in silico analysis of various mitotic and cyclin-dependent kinases yielded a significantly higher gene expression of PLK1 (p=0.061) and AURKB (p=0.003) in AA (n=41) compared to EA TNBC (n=86) patients. Surprisingly, we found no race-related differences in survivin gene expression in TCGA breast dataset (n=127, p=0.343). In addition, there was no statistical difference in survivin protein levels as shown by immunohistochemical staining of survivin in TNBC samples (n=142, p=0.45) from Emory Hospital, Atlanta. Based on these intriguing findings, we hypothesized that the differential functional activity of survivin in AA vs EA TNBC cells augments tumor proliferation in AA TNBC cells. Literature suggests that phosphorylation of survivin at Ser-20 and Thr-117 plays a role in the activity of the chromosome passenger complex (CPC) facilitating mitosis and cell proliferation. We observed an upregulation of phosphorylated substrates of PLK1 and AURKB, namely, p-survivin [Ser-20] and p-survivin [Thr-117], respectively, in AA compared to EA TNBC cell lines. Since most proteins are regulated post-translationally to perform diverse activities, we next knocked down survivin expression in AA, and EA TNBC cell lines, using a siRNA approach. Survivin ablation significantly attenuated cell proliferation, cell cycle progression, but not invasion, and migration in AA TNBC cells compared to EA TNBC cells. These data strongly support our premise that higher functional activity of survivin confers enhanced proliferative capacity on AA TNBC cells compared to EA TNBC cells, suggesting that survivin may be a viable therapeutic target for AA TNBC patients. Further studies intended to tease out the role of survivin phosphorylation in TNBC-related racial disparity are currently under way in our lab. Citation Format: Chakravarthy Garlapati, Shriya Joshi, Rida Padmashree, Ritu Aneja. Polo-like kinase 1 (PLK1) and aurora kinase B (AURKB) collude with survivin to augment tumor cell proliferation in African-American TNBC: Implications for racial disparity [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B065.

The tumor microenvironment is a crucial player in tumorigenesis, proliferation, and survival. Communication between its various components through microvesicular trafficking is central to the interaction between tumor cells and the surrounding milieu. Exosomes, nanovesicles released by all cell types, carry various proteins and nucleic acids. Once released into the extracellular environment, exosomes are taken up by surrounding cells or the exosomes travel in various bodily fluids to reach distant parts of the body. Those exosomes derived from tumor cells contain proteins and nucleic acids essential to the development and maintenance of tumors. One such protein, Survivin, a member of the Inhibitor of Apoptosis (IAP) protein family, has been identified in virtually all cancer cells. Survivin function is closely linked with subcellular location. Nuclear Survivin plays a key role in cell division and cell cycle regulation as a member of the chromosome passenger complex. Cytosolic and mitochondrial Survivin primarily facilitate the inhibition of apoptosis. Recent research has identified a unique pool of Survivin in the extracellular environment. Functions tied to this location are as yet undefined, however, evidence suggests that this population may contribute to increased proliferation, invasiveness and therapeutic resistance when taken into recipient cancer cells. Using tandem affinity purification we have identified a relationship between Survivin and TNF receptor 1 (TNFR1), as well as transferrin receptor 2 (TfR2). Both of these receptors play a role in endocytosis of extracellular proteins. Transferrin receptors are upregulated on cancer cells to facilitate the increased iron demand of proliferating cells. We therefore hypothesize that exosomal Survivin is taken up by adjacent cancer cells via receptor-mediated endocytosis. We have shown that in the presence of antibodies to these receptors, there is a decreased uptake of extracellular Survivin (from conditioned media). Results were similar when conditioned media was introduced to HeLa cells with siRNA knockdowns of each receptor, as well as to CHO cells deficient in these receptors. We conclude that uptake of extracellular Survivin into cancer cells is dependent upon these two endocytosis receptors. The relationship of Survivin and exosomes in the extracellular environment is indicative that exosomes transport Survivin out of tumor cells and into the surroundings. These results suggest that exosomal Survivin enters cancer cells by way of receptor-mediated endocytosis. Understanding this mechanism of signaling between cancer cells shows the potential intercellular trafficking of oncoproteins may play in tumor maintenance and progression. It also provides us with a novel means to combat the resulting enhanced aggressive nature of the disease. Citation Format: Amber Gonda, Salma Khan, Heather R. Ferguson Bennit, Ron B. Moyron, Nathan R. Wall. Extracellular Survivin uptake by cancer cells via receptor-mediated endocytosis. [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 5089.

Faithful transmission of genomic information to the two daughter cells requires highly coordinated cell cycle regulation. Aurora kinases have been implicated in mediating critical events during mitosis and cytokinesis. Aurora B is the enzymatic component of the chromosome passenger complex (CPC) which shows a characteristic localization during various phases of mitosis. Both the expression level and kinase activity of Aurora B is upregulated in several primary tumors including breast cancer and colon cancer and is associated with poor prognosis. Therefore, Aurora B is an attractive anti-cancer target and several chemical inhibitors of Aurora B are already in clinical trials. Inhibition of Aurora B results in cell cycle arrest. However, the signaling pathways responsible for the phenotypes observed after Aurora B inhibition have not yet been fully explored. We have recently shown that the p53 target gene p21 (CDKN1A) is activated both at protein and mRNA levels after pharmacological inhibition of Aurora B and that p21-activation is dependent on p53 and on the p38 MAP kinase. While the stabilization of p53 and its recruitment to the p21 promoter occur independently of p38, transcriptional elongation of p21 depends on active p38 after Aurora B is inhibited. We further show that partial inhibition of Aurora B is sufficient to induce p21. FISH (Fluorescence in-situ hybridization) assays indicate increased aneuploidy without inhibition of cytokinesis when Aurora B is partially inhibited. Increased aneuploidy is correlated with generation of reactive oxygen species (ROS) and proteotoxic stress, but not with DNA damage. In summary, our data suggest that aneuploidy due to partial inhibition of Aurora B results in proteotoxic stress and in the production of ROS, which contributes to the induction of p21 through activation of p38 MAPK. Significantly, Aurora B inhibitors and drugs that act on aneuploid cells (AICAR and 17AAG) synergize in the inhibition of cell proliferation and in inhibition of transformation. Thus, combining Aurora B inhibitors with drugs that act on aneuploid cells may result in a more effective cancer therapy than Aurora B inhibitors alone. Note: This abstract was not presented at the conference. Citation Format: Geeta Kumari, Tanja Ulrich, Stefan Gaubatz. Aneuploidy due to inhibition of Aurora B induces p21 through activation of p38 by proteotoxic stress and ROS. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A33.

Eukaryotic cells rely on a mitotic checkpoint in order to preserve their proper ploidy. This mechanism stops mitosis progression before anaphase until each kinetochore is adequately bound to mitotic microtubules, and tension is generated which splits up chromatids towards opposite poles of the mitotic spindle. The resulting paused state of the cell cycle is known as mitotic arrest, and has been long known to induce cell death. However, the mechanisms driving mitotic arrest-mediated cell death have not yet been elucidated. It has been hypothesized that an anti-apoptotic/pro-apoptotic protein interplay might define the mitotic arrest outcome. We are interested in defining the role of survivin and its splicing variants, survivin Δex3 and survivin 2b, in the mitotic arrest-induced cell death. Survivin is a multifaceted member of the IAP family of proteins with functions in the chromosome passenger complex that has been shown to be over expressed in nearly every human tumor type. Noteworthy, Survivin Δex3 retains IAP activity but Survivin 2b has been shown to act as a pro-apoptotic factor. In this study, we defined the mitotic index (MI) of HCT116 cells in response to different concentrations of taxol and vinblastine. The results show that vinblastine induces a wide MI oscillation in contrast to taxol, which produces a homogeneous behavior in the cell population. This difference in population behavior might be explained by the different mechanisms of action of taxol and vinblastine. From the maximum taxol-induced MI point on, the percentage of sub-2n cells increased at the expense of mitotic cells, as assessed by flow cytometry. These results suggest that, in our model, cells die by exposure to taxol in a mitotic arrest-dependent manner. We also determined, by RT-PCR, that survivin variants are endogenously expressed in HCT116 cells. Furthermore, in immunoblot experiments, we found that survivin protein level decreases throughout taxol treatment in contrast to Bub1 levels, a member of the spindle assembly checkpoint. These results suggest that the decreased survivin level might be related to mitotic arrest-dependent cell death. The analysis of the intracellular localization of survivin variants throughout the mitotic arrest showed that survivin is localized in mitotic chromosomes throughout mitosis (before anaphase) independently of the time of treatment with taxol and vinblastine. Noteworthy, we also found that survivin Δex3 localized to the cytosol, and its level decreases throughout mitosis while survivin 2b protein was diffusely localized in the cytosol and mitotic chromosomes, and its level and localization remained unchanged. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-84. doi:1538-7445.AM2012-LB-84

Survivin is a 16.5 kD protein that is highly expressed in most cancer cells. Although it is also expressed in embryonic tissues and proliferating adult cells, it may be a promising target for cancer therapy if it has distinct functions in normal versus malignant cells. It is well established that survivin has an essential role in dividing cells as a member of the chromosome passenger complex (CPC). We recently showed that survivin is highly expressed in orthochromatic erythroblasts, cells that have exited the cell cycle and undergoing enucleation. Based on this observation, we predicted that survivin would play a novel role in maturation of these late erythroblasts. To discover the function of survivin in these post-mitotic cells, we first analyzed the localization of survivin and other members of the CPC by immunofluorescence. We discovered that survivin is expressed in cytoplasm, but did not colocalize with aurora B kinase or INCENP in primary enucleating erythroblasts. Next, we overexpressed survivin in the murine erythroleukemia (MEL) cell line, which undergoes terminal differentiation but does not enucleate, and quantified the effect on maturation by flow cytometry. In contrast to control MEL cells, which down-regulate survivin and fail to enucleate, nearly 15% of the survivin overexpressing cells showed nuclear extrusion. To obtain insight into this novel effect of survivin, we purified survivin protein complexes from MEL cell lysates and performed mass spectrometry. We found that EPS-15 (EGFR pathway substrate clone 15) and clathrin, two proteins that are involved in endocytic vesicle trafficking, interact with survivin. We confirmed these interactions by immunoprecipitation and immunofluorescence studies in primary human erythroblasts. Next, we individually knocked down survivin and EPS-15 in primary human erythroblast cultures and demonstrated that both proteins are required for optimal enucleation. Moreover, deletion of the survivin gene in fetal liver erythroblasts isolated from survivin floxed embryos, reduced the enucleation efficiency without affecting cell survival or differentiation. In order to determine how survivin and EPS15 contribute to enucleation, we performed light microscopic and ultrastructural examination of primary murine red cells undergoing terminal maturation. We observed that loss of survivin was associated with a decrease in the number of large cellular vacuoles within erythroblasts. Furthermore, by immunogold-EM, we found that survivin co-localized with EPS-15 within LAMP1-positive vacuoles in late erythroblasts. Finally, we observed that vacuolin-1, a small molecule that increases the number and size of vacuoles, rescued the enucleation defect of survivin-/- erythroblasts. Together, these results suggest that survivin plays an important and novel role in vesicle trafficking within maturing red blood cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2984. doi:10.1158/1538-7445.AM2011-2984