Calcium-sensing stromal interaction molecule 2 upregulates nuclear factor of activated T cells 1 and transforming growth factor-β signaling to promote breast cancer metastasis

Abstract

Background Stromal interaction molecule (STIM) 2 is a key calcium-sensing molecule that regulates the stabilization of calcium ions (Ca 2+ ) and therefore regulates downstream Ca 2+ -associated signaling and cellular events. We hypothesized that STIM2 regulates epithelial-mesenchymal transition (EMT) to promote breast cancer metastasis. Methods We determined the effects of gain, loss, and rescue of STIM2 on cellular motility, levels of EMT-related proteins, and secretion of transforming growth factor-β (TGF-β). We also conducted bioinformatics analyses and in vivo assessments of breast cancer growth and metastasis using xenograft models. Results We found a significant association between STIM2 overexpression and metastatic breast cancer. STIM2 overexpression activated the nuclear factor of activated T cells 1 (NFAT1) and TGF-β signaling. Knockdown of STIM2 inhibited the motility of breast cancer cells by inhibiting EMT via specific suppression of NFAT1 and inhibited mammary tumor metastasis in mice. In contrast, STIM2 overexpression promoted metastasis. These findings were validated in human tissue arrays of 340 breast cancer samples for STIM2. Conclusion Taken together, our results demonstrated that STIM2 specifically regulates NFAT1, which in turn regulates the expression and secretion of TGF-β1 to promote EMT in vitro and in vivo, leading to metastasis of breast cancer. Electronic supplementary material The online version of this article (10.1186/s13058-019-1185-1) contains supplementary material, which is available to authorized users.

Calcium signaling: breast cancer’s approach to manipulation of cellular circuitry

“…It is useful to organize the data in this way as an attempt to more comprehensively understand the detailed mechanisms underlying cancer-mediated changes in calcium signaling (from receptor/channel to calcium signaling to cell function), the advantages these alterations confer to cancer, and the best approaches for designing new calcium-focused therapies. High expression levels of calcium channels, calcium pumps, and GPCRs (Table 1; protein or mRNA measurements) have been reported in patient breast cancer tissues over normal tissue (STIM1/2 (Miao et al 2019), ORAI3 (Faouzi et al 2011), SPCA2 pumps (Feng et al 2010), P2X7 channels (Tan et al 2015), TRPA1/TRPC1/ TRPC3/TRPC6/TRPC7/TRPV6/TRPM7/TRPM8 channels (Aydar et al 2009;Bolanz et al 2008;Chodon et al 2010;Dhennin-Duthille et al 2011;Guilbert et al 2008Guilbert et al , 2009Liu et al 2014;Meng et al 2013;Takahashi et al 2018;Tsavaler et al 2001), IP 3 Rs2/3 (Singh et al 2017b), S100 proteins (Cross et al 2005)) and high expression is correlated with breast tumor grade (RYRs (Abdul et al 2008), TRPV6 channels (Dhennin-Duthille et al 2011;Peters et al 2012), TRPM8 channels (Yapa et al 2018), TRPV4 channels (Peters et al 2017), SPCA1 pumps (Grice et al 2010), ORAI1 (McAndrew et al 2011, P 2 Y6 GPCRs , PMCA2 pumps (Peters et al 2016;VanHouten et al 2010), mitochondrial calcium uniporter (MCU) (Curry et al 2013), S100 proteins (McKiernan et al 2011)). Cancer patient samples over normal samples have also shown increased expression of CREB1/2 (Chhabra et al 2007;Fan et al 2012;Sofi et al 2003), PKCζ (Paul et al 2015;Smalley et al 2019)...…”

“…High expression levels of calcium channels, calcium pumps, and GPCRs (Table 1 ; protein or mRNA measurements) have been reported in patient breast cancer tissues over normal tissue (STIM1/2 (Miao et al 2019 ), ORAI3 (Faouzi et al 2011 ), SPCA2 pumps (Feng et al 2010 ), P2X7 channels (Tan et al 2015 ), TRPA1/TRPC1/TRPC3/TRPC6/TRPC7/TRPV6/TRPM7/TRPM8 channels (Aydar et al 2009 ; Bolanz et al 2008 ; Chodon et al 2010 ; Dhennin-Duthille et al 2011 ; Guilbert et al 2008 , 2009 ; Liu et al 2014 ; Meng et al 2013 ; Takahashi et al 2018 ; Tsavaler et al 2001 ), IP 3 Rs2/3 (Singh et al 2017b ), S100 proteins (Cross et al 2005 )) and high expression is correlated with breast tumor grade (RYRs (Abdul et al 2008 ), TRPV6 channels (Dhennin-Duthille et al 2011 ; Peters et al 2012 ), TRPM8 channels (Yapa et al 2018 ), TRPV4 channels (Peters et al 2017 ), SPCA1 pumps (Grice et al 2010 ), ORAI1 (McAndrew et al 2011 ), P 2 Y6 GPCRs (Azimi et al 2016 ), PMCA2 pumps (Peters et al 2016 ; VanHouten et al 2010 ), mitochondrial calcium uniporter (MCU) (Curry et al 2013 ), S100 proteins (McKiernan et al 2011 )). Cancer patient samples over normal samples have also shown increased expression of CREB1/2 (Chhabra et al 2007 ; Fan et al 2012 ; Sofi et al 2003 ), PKCζ (Paul et al 2015 ; Smalley et al 2019 ), and CAMKII (Chi et al 2016 ), increased nuclear localization of NFAT2 (Quang et al 2015 ), and increased phosphorylation of PKCζ (Paul et al 2015 ), CAMKII (Chi et al 2016 ), and CREB2 (Fan et al 2012 ).…”

“…A list of differentially expressed calcium-related proteins in breast cancer patient tissue at the protein or transcript level is shown. Comparisons are made with respect to malignant vs. non-malignant tissue, tumor grade, and survival outcomes Protein Comparison Method Reference Calcium Channels STIM1/2 STIM1 Malignant > adjacent High expression predicts poor survival IHC mRNA Miao et al 2019 McAndrew et al 2011 ORAI1 ORAI3 Correlated with basal subtype Malignant > Normal mRNA mRNA McAndrew et al 2011 Faouzi et al 2011 RyR Correlated with tumor grade IHC Abdul et al 2008 IP3R2/3 Malignant > normal IHC/mRNA Singh et al 2017b MCU Correlated with basal subtype mRNA Curry et al 2013 P2X7 Malignant > normal Western blot Tan et al 2015 TRPA1 Malignant > normal IHC/mRNA Takahashi et al 2018 TRPC1/C6 TRPC3/C6 TRPC6 TRPC7 Malignant > normal Malignant > normal Malignant > normal Malignant > adjacent IHC/mRNA mRNA IHC/mRNA mRNA Dhennin-Duthille et al 2011 Aydar et al 2009 Guilbert et al 2008 Tsavaler et al 2001 TRPM7 Malignant > normal Malignant > normal High expression predicts poor survival Malignant > normal IHC/mRNA mRNA mRNA IHC/mRNA Guilbert et al 2009 Meng et al 2013 Middelbeek et al 2012 Dhennin-Duthille et al 2011 ...…”

“…A list of calcium-related proteins used in studies to test in parallel the effects of knockdown on both cellular function and calcium signaling is shown. The rationale is based on the overexpression of calcium channels, GPCR, calcium pumps, and calcium effectors and enzymes seen in patient tissue and aims to link cellular functional outcomes with changes in calcium signaling Protein knockdown Functional outcome Associated calcium signaling alteration Reference Calcium Channels ORAI3 ORAI3 ORAI1 Inhibited in vitro proliferation and viability in MCF-7 Inhibited in vivo tumor growth in MCF-7 Reduced in vitro proliferation and viability in MCF-7 and MDA-MB-231 Reduced SOCE – Reduced SOCE Faouzi et al 2011 Motiani et al 2013 McAndrew et al 2011 ORAI1 or STIM1 Reduced in vitro migration and invasion, and in vivo metastasis in MDA-MB-231 Reduced SOCE Yang et al 2009 STIM1 Inhibited in vivo tumor growth and metastasis in MDA-MB-231 – Miao et al 2019 IP3R3 Inhibited in vitro migration in MDA-MB-231 Switched from ATP-stimulated global intracellular calcium signal to an oscillating one Mound et al 2017 IP3R2/3 Reduced in vitro viability in MCF-7 – Singh et al 2017a TRPM7 Reduced in vitro migration and in vivo metastasis in MDA-MB-231 Reduced in vitro viability in MCF-7 – Reduced resting calcium concentrations Middelbeek et al 2012 Guilbert et al 2009 TRPM8 Reduced in vitro migration in MDA-MB-231 – Liu et al 2014 TRPV6 Reduced in vitro viability in T47D Reduced in vitro migration and invasion in MDA-MB-231 Reduced in vitro viability in T47D – – Reduced TRPV6 calcium flux Bolanz et al 2008 Dhennin-Duthille et al 2011 Peters et al 2012 MCU Reduced in vivo tumor growth and metastasis in MDA-MB-2...…”

“…Experimental reductions in many other different calcium signaling related proteins can inhibit the tumorigenic and invasive capacity for breast cancer cells (Table 2 ). The knockdown of P 2 Y2 GPCRs was able to reduce in vivo primary tumor growth and metastatic lesions (Jin et al 2014 ; Zhang et al 2017 ) and in vitro invasion and migration (Jin et al 2014 ), STIM1 could inhibit tumor growth and metastasis (Miao et al 2019 ), PKCζ reduced in vitro migration and invasion and in vivo metastasis (Paul et al 2015 ; Smalley et al 2019 ), TRPM7 channels led to decreased in vitro migration and in vivo metastasis (Middelbeek et al 2012 ), NFAT was able to reduce in vitro invasion (Kim et al 2018 ) and in vivo tumor growth (Quang et al 2015 ), TRPV6 (Bolanz et al 2008 ) or TRPM7 (Guilbert et al 2009 ) reduced cell viability, VGCCs reduced cell invasion (Jacquemet et al 2016 ), TRPV6 reduced migration/invasion (Dhennin-Duthille et al 2011 ), ORAI3 inhibited in vivo tumor growth (Motiani et al 2013 ), MCU decreased in vivo tumor growth and metastasis (Tosatto et al 2016 ), and proliferation of breast cancer cells was reduced when VGCCs (Taylor et al 2008 ) or PMCA2 (Peters et al 2016 ) were targeted for knockdown. These data show that many calcium channels, pumps, GPCRs, and effectors are necessary for the migratory, invasive, proliferative, tumorigenic, or metastatic capacity of cancer cells.…”