**2.4. STIM and Orai proteins in breast cancer**

In nonexcitable cells, such is the case of breast cells, both store-operated calcium entry (SOCE) and store-independent calcium pathways have been described, which involve the activation of stromal interaction molecules (STIM) and Orai proteins.

STIM/Orai proteins play an important role in the breast physiology. To date, in lactation, Orai1 is upregulated, while Stim1, but not Stim2, is downregulated [91]. Moreover, calciuminflux through Orai1 was described to be necessary for mammary epithelial cells for concentrating milk with Ca2+ and for milk expulsion through alveolar unit contraction [91].

In cancer metastasis, STIM/Orai proteins have been described to be involved by two major mechanisms: (i) upregulation or increased functional activation and (ii) molecular switching [92]. In estrogen-receptor-positive breast cancer cells (e.g., MCF7, BT474, ZR751, T47D, and HCC1500 cancerous cell lines) but not in estrogen-receptor-negative breast cancer cells (e.g., MCF10A and 184A1 normal breast epithelial cells, and MDA-MB231, BT20, and HCC1937 cancerous cell lines), Orai 3 mediates the STIM1/2 and Orai3 pathway and the Ca2+-selective Ca2+ release-activated Ca2+ current (I(CRAC)), while Orai1 does not mediate the STIM1/Orai1 pathway [42]. Further on, estrogen-receptor-α knockdown downregulates Orai3 without any effect on Orai1, decreases Orai3-mediated SOCE, and diminishes the I(CRAC) current in estrogen-receptor-positive MCF7 cells [93].

The expression of Orai3 was identified to be higher in MCF-7 breast cancer cells versus normal MCF-10A mammary epithelial cells, while its silencing inhibits the MCF-7 cell proliferation, arrests the cell cycle in the G1 phase, downregulates cyclin-dependent kinases 4/2, cyclins E and D1, and determines the accumulation of p21(Waf1/Cip1) (a cyclin-dependent kinase inhibitor) and p53 (a tumor-suppressing protein) [94].

In STIM1 siRNA- or Orai1 siRNA-treated MDA-MB-231 human breast tumor cells, a reduction in the migration process was identified [95]. Additionally, overexpression of STIM1 and Orai1 in MCF-10A epithelial cells increased their invasiveness [95]. Experiments on immunodeficient NOD/SCID mice injected with MDA-MB-231 human breast tumor cells were stably transfected with STIM1 siRNA, or Orai1 siRNA, but not with control siRNA, which demonstrated the inhibition of metastasis [95].

Interestingly, a signaling pathway independent of endoplasmic reticulum calcium stores or STIM1 and STIM2 protein activation was identified in MCF-7 breast cancer cells (the secretory pathway calcium ATPase 2 (SPCA2)/Orai1 signaling) [96], where SPCA2 is located in the Golgi apparatus.

#### **2.5. Ether à go-go (hEag1) K+ channels in breast cancer**

role in the inhibition of proliferation and apoptosis in MCF-7 human breast cancer cells [81]. Patch-clamp recordings demonstrated the presence of T-type voltage-gated calcium currents

A meta-analysis of public microarray datasets in clinical cancer tissue samples identified the upregulation of several VGCCs transcripts (e.g., *CACNA1C*, *CACNA1D*, *CACNA1B*, *CACNA1G*, and *CACNA1I*) in breast cancer and their involvement in the development and cancer progression [82]. Oppositely, another meta-analysis indicated the downregulation of

Transient receptor potential (TRP) channels have been documented to play an important role in the development and progression of cancer. TRPC1, TRPC6, TRPM7, TRPM8, and TRPV6 channels were described to be upregulated in human breast ductal adenocarcinoma in comparison with the adjacent nontumoral tissue, and correlations with the proliferative parameters or the invasiveness cell capacity have been evidenced [84]. Moreover, several studies documented the role of TRP channels in different breast cancer cell lines. In detail, TRPM7 was demonstrated by silencing experiments to contribute to the migration and invasiveness of MDA-MB-435 breast cancer cells by signaling through the MAPK, but not through Akt, pathway [85]. TRPM8 was detected in BT-474 and MDA-MB-231, but not in MCF7, breast carcinoma cells, while mentholevoked Ca2+-oscillations were recorded in all three breast cancer cell lines [86]. TRPV6 had a high level of expression in breast adenocarcinoma tissue [87]. TRPV1 is functionally expressed in SUM149PT breast cancer cells, a model for a very aggressive form of breast cancer (i.e., triple-

In nonexcitable cells, such is the case of breast cells, both store-operated calcium entry (SOCE) and store-independent calcium pathways have been described, which involve the activation

STIM/Orai proteins play an important role in the breast physiology. To date, in lactation, Orai1 is upregulated, while Stim1, but not Stim2, is downregulated [91]. Moreover, calciuminflux through Orai1 was described to be necessary for mammary epithelial cells for concen-

In cancer metastasis, STIM/Orai proteins have been described to be involved by two major mechanisms: (i) upregulation or increased functional activation and (ii) molecular switching [92]. In estrogen-receptor-positive breast cancer cells (e.g., MCF7, BT474, ZR751, T47D, and HCC1500 cancerous cell lines) but not in estrogen-receptor-negative breast cancer cells (e.g., MCF10A and 184A1 normal breast epithelial cells, and MDA-MB231, BT20, and HCC1937 cancerous cell lines), Orai 3 mediates the STIM1/2 and Orai3 pathway and the Ca2+-selective Ca2+ release-activated Ca2+ current (I(CRAC)), while Orai1 does not mediate the STIM1/Orai1 pathway [42]. Further on, estrogen-receptor-α knockdown downregulates Orai3 without any effect on Orai1, decreases Orai3-mediated SOCE, and diminishes the I(CRAC) current in

trating milk with Ca2+ and for milk expulsion through alveolar unit contraction [91].

(ICaT) in MCF7 breast cancer cells [79].

174 Calcium and Signal Transduction

the same VGCCs transcripts in breast cancer [83].

**2.3. Transient receptor potential channels in breast cancer**

negative breast cancer) [88], and in MCF7 breast cancer cells [89, 90].

of stromal interaction molecules (STIM) and Orai proteins.

**2.4. STIM and Orai proteins in breast cancer**

estrogen-receptor-positive MCF7 cells [93].

Several pieces of evidence indicate the role played by Ether à go-go (hEag1) K<sup>+</sup> channels in breast cancer cells invasiveness. To date, blocking or silencing hEag1 channels in MDA-MB-231 breast cancer cells induces membrane depolarization and subsequent diminishment of Ca2+ influx through Orai1, which affects cell migration and proliferation [97].
