The possible advantages of aberrant NFAT activation

As identified in section 2.4.5, an alternative pathway activated in STIM1 overexpressing cells upon heighted SOCE is the NFAT signaling pathway. The NFAT family of transcription factors is best described in the immunology field, where it regulates T cell activation and differentiation, while also affecting the function of other immune cells (Müller and Rao, 2010). Still, NFATs have been reported to be crucial in other cell systems as well and their dysregulation associated with several diseases, including heart problems and cancer (Dewenter et al., 2017; Mancini and Toker, 2009). In the case of cancer, NFATs have been described to promote angiogenesis, metastasis and tumor progression (Mancini and Toker, 2009). Thus, aberrant NFAT activation in STIM1 overexpressing cells may confer these tumors with novel oncogenic properties.

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Angiogenesis is a hallmark of cancer, in which tumors upon different stimuli, including hypoxia, activate HIF1a, upregulating and secreting VEGF, while also reprogramming their metabolism to adapt to oxygen-deprived environments.

Endothelial cells surrounding the tumor, in turn, can bind VEGF, which stimulates their proliferation and fosters angiogenesis (Majmundar et al., 2010). In many endothelial cells, the response to VEGF and the subsequent upregulation of angiogenic genes, such as COX2, has been proven to be SOCE and NFAT-dependent (Mancini and Toker, 2009; Suehiro et al., 2014). Interestingly, NFATs have also been described to induce HIF1a expression to promote a metabolic rewiring in T cells. T cell proliferation has been shown to be dependent on a metabolic reprogramming elicited by SOCE and subsequent NFAT and Akt/mTOR signaling pathway activation. In this case, NFATs promoted the upregulation of the transcription factors HIF1a and IRF4, while Akt/mTOR signaling lead to c-MYC activation. Together, NFATs, HIF1a, IRF4 and c-MYC induced the expression of various metabolic genes involved in glycolysis, oxidative phosphorylation and nucleotide metabolism (Vaeth et al., 2017). Taken together, there is evidence suggesting that NFATs control several players involved in angiogenesis and the response to hypoxia. Thus, it is possible that in STIM1 overexpressing tumors, NFATs may promote the upregulation of HIF1a and the initiation of angiogenesis upon hypoxia and other SOCE-promoting stimuli. Furthermore, it is plausible, that similar to T cells, NFAT activation upon different stimuli, may rewire the metabolic dependencies of STIM1 overexpressing cells.

Even though, the above speculations have to be tested, an NFAT-dependent upregulation of HIF1a could confer STIM1 amplifying cells with an alternative pathway to respond to stress signals, which is independent from the ER stress response pathway. Furthermore, an NFAT-driven metabolic rewiring upon SOCE stimulation, may help the cells cope with several metabolic stresses. Thus, it is possible that potential metabolic changes, elicited by the upregulation of RRM1 during the establishment of gemcitabine resistance, are further accommodated by a metabolic reprogramming elicited by aberrant NFAT activation due to STIM1 upregulation. This would, in turn, favor the co-amplification of RRM1 and STIM1 during the establishment of gemcitabine resistance.

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Aberrant NFAT activation in STIM1 overexpressing tumors may not only lead to increased angiogenesis and a possible metabolic reprogramming, but also to heightened metastatic rates. In melanoma, increased NFAT signaling in epithelial cells lead to heightened BMP2 secretion, which promoted cancer cell dedifferentiation and metastasis. NFAT activity has also been associated with increased cell migration and invasion in breast cancer, while being characterized as the driver of several metastatic factors in colon cancer (Jauliac et al., 2002; Tripathi et al., 2014; Yiu and Toker, 2006). In PDAC, aberrant NFATc1 activation led to increased expression of several EMT genes (Hendrikx et al., 2019). By dimerizing with SOX2, NFATc1 drove the upregulation of the EMT-promoting transcription factors ZEB1, TWIST and SNAI1 (Singh et al., 2015). Thus, it is highly likely that cells expressing higher STIM1 and therefore SOCE levels, present a more dedifferentiated phenotype and higher metastatic potential.

Interestingly, NFATs have also been extensively characterized as drivers of pancreatic cancer development and growth. Several studies demonstrated that NFAT is key for inflammation-driven pancreatic cancer development (Baumgart et al., 2014, 2016). The promoter of NFAT itself has also been shown to be methylated by EZH2 and consequently silenced in pancreatic acinar cells. This was reversed upon KRAS activation during PDAC development, leading to the de-repression and concomitant activation of NFATc1 (Chen et al., 2017). Moreover, NFATc2 has been shown to promote the silencing of the tumor suppressor CDKN2B further fostering tumorigenesis (Baumgart et al., 2012). NFATs further promote c-MYC expression and cell proliferation in pancreatic cancer (Buchholz et al., 2006; König et al., 2010c). For example, TGFβ has been reported to activate NFAT, which in turn displaces SMAD3 allowing TGFβ responsive genes, such as c-MYC, to be activated, ultimately promoting tumor growth (Singh et al., 2010). Therefore, it is highly likely that STIM1 overexpressing pancreatic cancer cells display a tumorigenic and proliferative advantage elicited by aberrant NFAT activation compared to STIM1 lowly expressing cells.

In order to validate all aforementioned hypotheses, functional assays comparing low and high STIM1 expressing cells under resting conditions as well as under several stresses would have to be conducted. Xenograft experiments could also be performed, in which STIM1 high and low expressing cells are injected into mice and

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tumor progression, volume and metastasis occurrence are monitored. Still, even though the consequences of an aberrant NFAT activation in STIM1 overexpressing cells have to be better characterized, previous studies highly suggest that ectopic NFAT activation may confer these cells with important additional tumorigenic properties.

3.6. The benefits and drawbacks of targeting calcium signaling in