Abstract
Store-operated Ca2+ entry (SOCE) provides a major Ca2+ entry route in cancer cells. SOCE is mediated by the assembly of Stim and Orai proteins at endoplasmic reticulum (ER)-plasma membrane junctions upon depletion of the ER Ca2+ store. Additionally, Stim and Orai proteins underpin constitutive Ca2+ entry in a growing number of cancer cell types due to the partial depletion of their ER Ca2+ reservoir. Herein, we investigated for the first time the structure and function of SOCE in primary cultures of colorectal carcinoma (CRC) established from primary tumor (pCRC) and metastatic lesions (mCRC) of human subjects. Stim1-2 and Orai1-3 transcripts were equally expressed in pCRC and mCRC cells, although Stim1 and Orai3 proteins were up-regulated in mCRC cells. The Mn2+-quenching technique revealed that constitutive Ca2+ entry was significantly enhanced in pCRC cells and was inhibited by the pharmacological and genetic blockade of Stim1, Stim2, Orai1 and Orai3. The larger resting Ca2+ influx in pCRC was associated to their lower ER Ca2+ content as compared to mCRC cells. Pharmacological and genetic blockade of Stim1, Stim2, Orai1 and Orai3 prevented ER-dependent Ca2+ release, thereby suggesting that constitutive SOCE maintains ER Ca2+ levels. Nevertheless, pharmacological and genetic blockade of Stim1, Stim2, Orai1 and Orai3 did not affect CRC cell proliferation and migration. These data provide the first evidence that Stim and Orai proteins mediate constitutive Ca2+ entry and replenish ER with Ca2+ in primary cultures of CRC cells. However, SOCE is not a promising target to design alternative therapies for CRC.
Highlights
Colorectal carcinoma (CRC) is the most common gastrointestinal cancer in the world [1] and, at early stage, surgical resection followed by adjuvant chemotherapy in higher risk population to eradicate distant micrometastases is the standard treatment when the disease is diagnosed [2]
The comparison of ΔCt values of the mRNAs obtained by qRT-PCR showed that there was no difference in the transcript levels of Stim1-2 and Orai1-3 between pCRC and mCRC cells (Figure 1)
Immunoblots revealed a major band of ≈100 kDa for Stim1 (Figure 2A) and Stim2 (Figure 2B) and of ≈33 kDa for Orai1 (Figure 2C) and Orai3 (Figure 2D), as expected from their molecular sizes [5]
Summary
Colorectal carcinoma (CRC) is the most common gastrointestinal cancer in the world [1] and, at early stage, surgical resection followed by adjuvant chemotherapy in higher risk population to eradicate distant micrometastases is the standard treatment when the disease is diagnosed [2] In those patients showing disease recurrence or being metastatic at diagnosis, 5-year survival falls dramatically from 80-90% to 10%-20% despite development of effective combinations of chemotherapeutic agents along with the introduction of targeted therapies including monoclonal antibodies against epidermal growth factor receptor or vascular endothelial growth factor (VEGF) [2]. Cancer cell lines, easy to obtain, handle and expand, cannot recapitulate the complex biology that underpins neoplastic transformation This hurdle is further exacerbated by the growing evidence that the molecular and genetic profile of each tumor may vary from one patient to another, which led to the concept of personalized medicine to treat cancer patients [21]. A recent study revealed that SOCE does not control proliferation in primary cultures of human metastatic renal cell carcinoma (RCC) [22] and glioblastoma cells [23]
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