A Reciprocal Shift in Transient Receptor Potential Channel 1 (TRPC1) and Stromal Interaction Molecule 2 (STIM2) Contributes to Ca2+ Remodeling and Cancer Hallmarks in Colorectal Carcinoma Cells*

Background: Changes in Ca2+ handling in tumor cells might provide novel targets for cancer. Results: Colon carcinoma cells show enhanced store-operated Ca2+ entry and currents and depleted Ca2+ stores associated with changes in STIM1/STIM2 ratio and TRPC1. Conclusion: Ca2+ remodeling in colon cancer is driven by a reciprocal shift in TRPC1 and STIM2. Significance: STIM1/STIM2 and TRPC1 should be investigated further as novel targets for colon cancer. We have investigated the molecular basis of intracellular Ca2+ handling in human colon carcinoma cells (HT29) versus normal human mucosa cells (NCM460) and its contribution to cancer features. We found that Ca2+ stores in colon carcinoma cells are partially depleted relative to normal cells. However, resting Ca2+ levels, agonist-induced Ca2+ increases, store-operated Ca2+ entry (SOCE), and store-operated currents (ISOC) are largely enhanced in tumor cells. Enhanced SOCE and depleted Ca2+ stores correlate with increased cell proliferation, invasion, and survival characteristic of tumor cells. Normal mucosa cells displayed small, inward Ca2+ release-activated Ca2+ currents (ICRAC) mediated by ORAI1. In contrast, colon carcinoma cells showed mixed currents composed of enhanced ICRAC plus a nonselective ISOC mediated by TRPC1. Tumor cells display increased expression of TRPC1, ORAI1, ORAI2, ORAI3, and STIM1. In contrast, STIM2 protein was nearly depleted in tumor cells. Silencing data suggest that enhanced ORAI1 and TRPC1 contribute to enhanced SOCE and differential store-operated currents in tumor cells, whereas ORAI2 and -3 are seemingly less important. In addition, STIM2 knockdown decreases SOCE and Ca2+ store content in normal cells while promoting apoptosis resistance. These data suggest that loss of STIM2 may underlie Ca2+ store depletion and apoptosis resistance in tumor cells. We conclude that a reciprocal shift in TRPC1 and STIM2 contributes to Ca2+ remodeling and tumor features in colon cancer.


We have investigated the molecular basis of intracellular Ca 2؉ handling in human colon carcinoma cells (HT29) versus normal human mucosa cells (NCM460) and its contribution to cancer features. We found that Ca 2؉ stores in colon carcinoma cells are partially depleted relative to normal cells. However, resting Ca 2؉ levels, agonist-induced Ca 2؉ increases, store-operated Ca 2؉ entry (SOCE), and store-operated currents (I SOC ) are largely enhanced in tumor cells. Enhanced SOCE and depleted
Ca 2؉ stores correlate with increased cell proliferation, invasion, and survival characteristic of tumor cells. Normal mucosa cells displayed small, inward Ca 2؉ release-activated Ca 2؉ currents (I CRAC ) mediated by ORAI1. In contrast, colon carcinoma cells showed mixed currents composed of enhanced I CRAC plus a nonselective I SOC mediated by TRPC1. Tumor cells display increased expression of TRPC1, ORAI1, ORAI2, ORAI3, and STIM1. In contrast, STIM2 protein was nearly depleted in tumor cells. Silencing data suggest that enhanced ORAI1 and TRPC1 contribute to enhanced SOCE and differential store-operated currents in tumor cells, whereas ORAI2 and -3 are seemingly less important. In addition, STIM2 knockdown decreases SOCE and Ca 2؉ store content in normal cells while promoting apoptosis resistance. These data suggest that loss of STIM2 may underlie Ca 2؉ store depletion and apoptosis resistance in tumor cells. We conclude that a reciprocal shift in TRPC1 and STIM2 contributes to Ca 2؉ remodeling and tumor features in colon cancer.
Critical cancer hallmarks include enhanced cell proliferation, apoptosis resistance, and acquired ability to migrate and invade foreign tissues (1), cell functions that are regulated by intracellular Ca 2ϩ signals. Increasing evidence suggests that tumor cells may undergo a deep remodeling of their Ca 2ϩ homeostasis (2,3), likely contributing to cancer features. However, mechanisms and contribution of Ca 2ϩ deregulation are largely unknown (4), and no data are available in many types of cancer, including colon cancer. Store-operated Ca 2ϩ entry (SOCE), 4 a ubiquitous Ca 2ϩ entry pathway involved in many physiological functions, particularly in nonexcitable cells, has been proposed to be remodeled in some cancers (5). This pathway is triggered by the release of Ca 2ϩ from intracellular stores induced by phospholipase C activation after receptor stimulation. SOCE is believed to be mediated by the interaction of Stim1 (6), a Ca 2ϩ sensor at the endoplasmic reticulum (ER), and Orai1, a pore-forming protein of store-operated channels (SOCs) at the plasma membrane that enables Ca 2ϩ influx (7,8). It is also widely accepted that STIM1/ORAI1 interactions are responsible for I CRAC activation underlying SOCE in some cell types (8). However, other store-operated currents (I SOC ) less selective for Ca 2ϩ might be mediated by canonical transient receptor potential (TRPC) channels, particularly TRPC1 and TRPC4 (2,9). Some of the above proteins have been reported to be up-regulated in cancer. For instance, TRP channels, including several TRPCs, TRPV6, and TRPM8, are overexpressed in several tumor cells, thus suggesting they may have oncogenic potential (10 -13). SOCE and TRPC6 have been reported to control human hepatoma cell proliferation, and their blockade inhibits * This work was supported in part by Grants BFU2009-08967 and BFU2012-external medium at 37°C. Cells were epi-illuminated alternately at 340 and 380 nm using bandpass filters, and light emitted above 520 nm at both excitation lights was filtered by the dichroic mirror, collected every 5-10 s with a ϫ40, 1.4 NA, oil objective. For estimation of Ca 2ϩ store content, we assessed the effects of cyclopiazonic acid or ionomycin on [Ca 2ϩ ] cyt in the absence of extracellular Ca 2ϩ . In the case of ionomycin, the increases in [Ca 2ϩ ] cyt tended to saturate Fura2 signals. Accordingly, experiments with ionomycin were performed using the low affinity probe Fura4F/AM.
Cell Proliferation-Cells were seeded in 6-well plates at about 10 ϫ 10 5 cells and incubated with supplemented DMEM or containing test solutions. Wells were counted by triplicate at time 0 and after 72 or 96 h. Cell viability was estimated using trypan blue staining.
Flash Photolysis of Caged-IP 3 and Confocal Microscopy-Cells were plated in glass bottom culture dishes and loaded with Fluo4/AM (2 M) and caged-IP 3 (0.5 M) for 1 h. Images were taken using a Leica TCS SP5 confocal microscope (Leica Microsystems, Mannheim, Germany) using a ϫ40 objective. Fluo4 was excited at 488 nm, and emissions between 503 and 571 nm were collected every 3 s. Photolysis of caged-IP 3 /acetoxymethyl ester was performed at 405 nm. The images were analyzed in LAS AF Lite software (Leica Microsystems, Mannheim, Germany). Background was subtracted from all images, and fluorescence intensity (F) was normalized to the resting fluorescence intensity (F 0 ).
Invasion Assay-Cell invasion assay was performed using BD Biocoat TM Matrigel TM invasion chambers (BD Biosciences) containing a membrane with 8-m pores. HT29 cells (1 ϫ 10 6 cells) in DMEM were seeded to the upper chamber. DMEM containing 20% FBS was added in the lower chamber as chemoattractant. After 48 h, noninvading cells were removed with a cotton swab from the upper chamber. Cells invading the outer side of the insert were fixed in methanol and stained with toluidine blue solution and 1% chloride double salt (Panreac, Barcelona, Spain). Cells per field were counted randomly at ϫ200 magnification.
Annexin V Staining Assay-Cell survival assay was performed by flow cytometry using FITC annexin V (BD Biosciences) and propidium iodide (Sigma). Cells were treated with 1 or 2 mM H 2 O 2 for 30 or 150 min, respectively, depending on the experiment and then detached with trypsin/EDTA, centrifuged at 290 ϫ g, and washed with cold PBS. The cells were then suspended in binding buffer (0.1 M Hepes, pH 7.4, 1.4 M NaCl, and 25 mM CaCl 2 ) at a density of 1 ϫ 10 6 cells/ml. After that, 1 ϫ 10 5 cells were incubated with 5 l of annexin V and 10 l of propidium iodide (50 g/ml) for 15 min at room temperature in the dark. Cells were analyzed using Gallios Flow Cytometer (Beckman Coulter, Brea, CA), and the results were processed with Kaluza Analysis Software (Beckman Coulter, Brea, CA).
Electrophysiological Recordings-I SOC in colonic cells was recorded using a Port-a-Patch planar patch clamp system (Nanion Technologies, Munich, Germany) in the whole-cell, voltage clamp configuration at room temperature (20 Ϯ 2°C). Cultured cells (3-5 days after plating) were detached with Detachin and suspended at a cell density of 1-5 ϫ 10 6 cells/ml in external recording solution contained (in mM) the following: 145 NaCl, 2.8 KCl, 2 MgCl 2 , 10 CaCl 2 , 10 Hepes, 10 D-glucose, pH 7.4. For siRNA assays, recordings were performed 48 h after silencing. Suspended cells were placed on the NPC©1 chip surface, and the whole-cell configuration was achieved. Internal recording solution containing (in mM) 50 CsCl, 60 CsF, 10 NaCl, 20 EGTA, 10 Hepes, 2 Na ϩ -ATP, pH 7.2 (adjusted with CsOH), was deposited in recording chips, having resistances of 3-5 megohms. The high concentration of EGTA was used to deplete stores and to activate I SOC in intact and in silenced cells.
In some experiments in which I SOC was activated with thapsigargin or ATP, internal EGTA was diminished from 20 to 0.2 mM and supplemented with a mitochondrial mixture (in mM) of 2 pyruvic acid, 2 malic acid, and 1 NaH 2 PO 4 . I SOC was assessed using voltage ramps (Ϫ100 to ϩ 100 mV in 200 ms) applied every 5 s, from a holding potential of 0 mV and acquired with an EPC-10 patch clamp amplifier (HEKA). Immediately after the whole-cell configuration was established, the cell capacitance and the series resistances (Ͻ10 megohms) were measured. During recordings, these two parameters were measured, and if they exceeded Ն10% with respect to the initial value, the experiment was discontinued. Resting membrane potentials were estimated by reading the potential of the recorded cell immediately after rupturing the membrane in the current-clamp configuration. Leak currents were eliminated by subtracting the average of the first five ramp currents (obtained just after whole-cell configuration was reached) to all subsequent currents. Inward and outward current amplitudes were measured at Ϫ80 and ϩ80 mV, respectively. Data were normalized with respect to cell capacitance. Liquid junction potential and capacitive currents were cancelled using the automatic compensation of the EPC-10. Data were filtered at 10 kHz and sampled at 5 kHz.
Conventional and Quantitative PCR-Total cellular RNA was isolated from cells using TRIzol reagent (Invitrogen). Extracted RNA integrity was tested by electrophoresis on agarose gels, and the purity and concentration were determined by spectrophotometry. RNA was reverse-transcribed using a high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA) and the cDNA diluted prior to PCR amplification. Nucleotide sequences of the STIM1, ORAI1, ORAI2, and ORAI3 primers used were taken from Ref. 26 and ␤-actin from Ref. 27. The remaining primers were designed with Primer-BLAST (28). Table 1 shows all primer sequences used. Qualitative PCR was performed on a TGradient system (Biometra, Goettingen, Germany) using a Taq polymerase (Fermentas). The reaction protocol consisted of 3 min at 94°C, 35 cycles of 1 min at 94°C, 1 min at 57°C, and 30 s at 72°C and finished at 72°C for 10 min. Real time quantitative-PCR was performed using a SYBR Green I detection in a LightCycler rapid thermal cycler (Roche Applied Science). The PCR protocol started with 5 min at 95°C followed by 45 cycles of 15 s at 95°C, 20 s at 57 or 60°C, and 5 s at 72°C. ␤-Actin was used as housekeeping gene. The data were normalized by PCR analysis of ␤-actin. Melting curves were used to determine the specificity of PCR products (data not shown).
Gene Silencing-siRNA sequences of human TRPC1, ORAI1, ORAI2, ORAI3, and STIM2 were obtained from Santa Cruz Biotechnology, as well as control siRNA. NCM460 and HT29 cells (1 ϫ 10 6 ) were transfected transiently with 50 pmol of siRNA using Nucleofector II (Amaxa Biosystems, Cologne, Germany) and the W-017 program according to the manufacturer's instructions. After transfection, cells were grown in culture for 48 h, and then imaging, electrophysiology, and cell survival experiments were performed. The effectiveness of silencing was tested by real time qRT-PCR.
Statistics-When only two means were compared, Student's t test was used. For more than two groups, statistical significance of the data was assessed by analysis of variance and compared using Bonferroni's multiple comparison tests. Differences were considered significant at p Ͻ 0.05.

Store-operated Ca 2ϩ Entry and Cell Proliferation in Colon
Carcinoma Cells-Cell proliferation and SOCE were tested in a series of human colon mucosa (NCM460 and NCM356) and human colon carcinoma cell lines (HT29, SW480-ADH, and  (26) and ␤-actin primers from Wang et al. (27). The remaining primers were designed using BLAST primer software (28). F indicates forward, and R indicates reverse.

Name Primers (5 to 3) Predicted size
SW480-R cells). SOCE was monitored by imaging the increase in cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] cyt ) induced by Ca 2ϩ re-addition to cells previously treated with thapsigargin (1 M, 10 min) in Ca 2ϩ -free medium. Under these conditions, Ca 2ϩ stores are empty (data not shown). Therefore, this procedure enables monitoring maximally activated SOCE when Ca 2ϩ stores are fully depleted. We found that SOCE is small in normal colon mucosa cell lines, and it is largely up-regulated in all three human colon carcinoma cell lines tested (Fig. 1, A and B). Cell proliferation is low in normal mucosa cell lines and increases in carcinoma cells as expected (Fig. 1C). We found that there is an excellent correlation between SOCE and cell proliferation in all five cell lines tested (Fig. 1D) suggesting that increased SOCE contributes to enhanced proliferation in carcinoma cells. These data are consistent with our previous report showing the correlation between SOCE inhibition and prevention of HT29 cell proliferation (25,29). Therefore, increased SOCE may contribute to enhance cell proliferation of colon carcinoma cells.  We tested whether Ca 2ϩ fluxes induced by physiological agonists were also remodeled in colon cancer. For this and subsequent studies, we selected NCM460 and HT29 cells as models of normal and colon carcinoma cells, respectively. The physio-logical agonist ATP increases [Ca 2ϩ ] cyt in both normal and colon carcinoma cells. However, ATP-induced increases in [Ca 2ϩ ] cyt in normal cells are small and transient, whereas in tumor cells [Ca 2ϩ ] cyt increases are much larger and sustained ( Fig. 2A). Fig. 2B shows that resting levels of [Ca 2ϩ ] cyt are also significantly larger in tumor cells. ATP induces both Ca 2ϩ release and (store-operated) Ca 2ϩ entry. We tested both independently in normal and tumor cells. Fig. 2, C and E, shows that ATP-induced Ca 2ϩ release is significantly larger in tumor cells. Similar results are obtained with carbachol ( Fig. 2, D and F). Surprisingly, both agonists induce Ca 2ϩ entry in tumor cells but not in normal cells (Fig. 2, C-F) suggesting that SOCE activation in physiological conditions is somehow prevented in normal cells. Therefore, in normal colonic cells, physiological agonists produce a small and transient increase in  solely to Ca 2ϩ release, whereas tumor cells display a much larger increase due to both enhanced Ca 2ϩ release and SOCE. Ca 2ϩ Release and Ca 2ϩ Store Content in Normal and Tumor Cells-Experiments were designed to ascertain whether IP 3 availability causes differential Ca 2ϩ release and as a consequence different amplitudes of Ca 2ϩ increases between normal mucosa and colon carcinoma cells. Flash photolysis of caged-IP 3 induces Ca 2ϩ release in both normal and tumor cells as shown by confocal imaging of Fluo4-loaded cells. However, the Ca 2ϩ release was still significantly larger in tumor cells (Fig. 2, G and H) suggesting that tumor cells store more Ca 2ϩ and/or are more sensitive to IP 3 than normal cells. Ca 2ϩ store content in normal and colon carcinoma cells was estimated by measuring release of Ca 2ϩ induced by the sarcoplasmic and endoplasmic reticulum Ca 2ϩ -ATPase pump blocker cyclopiazonic acid (CPA) and by low concentrations of the Ca 2ϩ ionophore iono-mycin. Unexpectedly, we found that CPA induces larger [Ca 2ϩ ] cyt increases in normal cells than in tumor cells (Fig. 3A) consistently with a larger Ca 2ϩ store content in normal cells. In fact, a few minutes after CPA treatment, normal cells still responded largely to ATP, whereas tumor cell stores did not respond at all (Fig. 3A). Consistently, ionomycin induces also a larger [Ca 2ϩ ] cyt increase in normal cells (Fig. 3B) than in tumor cells. Thus, contrary to expectations, Ca 2ϩ store content is seemingly larger in normal cells than in tumor cells.
The extent of agonist-induced Ca 2ϩ release relative to total stored Ca 2ϩ was estimated next in normal and tumor cells. For this end, the amount of Ca 2ϩ remaining in the store after ATP was tested using ionomycin in Ca 2ϩ -free medium (Fig. 3C). We found that ATP mobilizes about 20% of total stored Ca 2ϩ in normal cells. In contrast, in tumor cells, ATP releases about 60% of total stored Ca 2ϩ (Fig. 3C). Fig. 3D shows the Ca 2ϩ store content estimated before and after stimulation with ATP in normal and tumor cells. Data indicate that Ca 2ϩ stores in normal cells are overloaded relative to tumor cells, and physiological stimulation does not release much Ca 2ϩ , leaving stores nearly intact. In contrast, Ca 2ϩ stores in tumor cells are substantially depleted in resting conditions and release relatively more Ca 2ϩ in response to stimulation, thus likely enabling cells to reach the threshold for SOCE activation.
It has been reported that Ca 2ϩ store content is critical for apoptosis resistance and survival (4). Accordingly, we have assessed apoptosis resistance (survival) of normal mucosa and carcinoma colon cells by flow cytometry after treatment with H 2 O 2 , a well established agent that promotes oxidative stress, apoptosis, and cell death. Consistently, colon carcinoma (HT29) cells are much more resistant to cell death than normal colonic epithelial (NCM460) cells (Fig. 4, A and B). These data suggest that the low Ca 2ϩ store content of tumor cells may contribute to apoptosis resistance characteristic of human colon carcinoma cells.

Store-operated Currents (I SOC ) in Normal and Colon
Carcinoma Cells-Ion currents involved in SOCE and agonist-induced Ca 2ϩ entry were investigated next using planar patch clamp electrophysiology in the whole-cell voltage clamp configuration. The estimated resting membrane potential for HT29 cells was Ϫ64 Ϯ 2 mV (n ϭ 33) and for NCM460 cells was Ϫ45 Ϯ 4 mV (n ϭ 22). For I SOC activation, Ca 2ϩ stores were passively depleted by dialyzing cells with a recording internal solution containing 20 mM EGTA. NCM460 cells displayed I SOC with small inward current amplitude (Ϫ2.3 Ϯ 0.3 pA/pF, at Ϫ80 mV; n ϭ 18) and without apparent outward current. The current-voltage (I-V) relationship of these I SOC , observed among all normal cells recorded (n ϭ 82), displayed strong inward rectification and very positive reversal potential (Fig.  5A). All these characteristics are similar to the previously reported I CRAC (31). Fig. 5B shows the average time course graph, constructed by plotting the amplitude of the I CRAC -like current (at Ϫ80 mV) with respect to recording time, in which the Ca 2ϩ -dependent inactivation was prevented by the presence of a high concentration of EGTA. I SOC currents in tumor cells were quite different. In HT29 cells, I SOC has a small inward current that was significantly greater than in normal cells (Ϫ4.9 Ϯ 0.15 pA/pF, at Ϫ80 mV; n ϭ 31; Student's t test, p Ͻ  Fig. 5F. The electrophysiological data indicate that Ca 2ϩ store depletion in tumor cells activates I CRAC -like currents with higher amplitude than in normal cells, probably contributing to their increased SOCE. Meanwhile, the nonselective I SOC observed in tumor cells could be an additional pathway for more Ca 2ϩ influx. Similar results are obtained when I SOC was induced by passive depletion of intracellular Ca 2ϩ stores with thapsigargin or the physiological agonist ATP (Fig. 6)  Sensitivity to antagonists was tested next to identify further the channels involved in I SOC in normal and tumor cells. Fig. 7 shows the effects of La 3ϩ , a classic SOCE antagonist of SOCs in normal and tumor cells. La 3ϩ inhibited almost totally I CRAClike currents in normal cells and both inward and outward currents in tumor cells. Low concentrations (30 M) of 2APB largely inhibit the I CRAC -like current of normal cells (Fig. 8A) and the I CRAC -like component of tumor cells (Fig. 8B), but they have no effect on the outward I SOC . At 100 M 2APB, the outward component is now inhibited (Fig. 8C). Average results are shown in Fig. 8D. 2APB is also more efficient in preventing SOCE in normal cells than in tumor cells (data not shown). Results suggest that normal and tumor cells express I SOC , similar to I CRAC , which are sensitive to low concentrations of 2APB. Yet the additional nonselective I SOC observed only in tumor cells is less sensitive to this SOCE antagonist. It has been reported that 2APB may enhance I SOC carried out by ORAI3 (32). In our hands 2APB did not potentiate I SOC in normal or tumor cells (Fig. 8). It has been reported that SOCE is involved in cell migration and invasion in tumor cells (30). Accordingly, colon carcinoma cell invasion was tested in vitro by transwell assay. HT29 cells displayed invasive characteristics. In addition, HT29 cell invasion was inhibited significantly by classic SOCE antagonist 2APB (Fig. 8E).
Ca 2ϩ selectivity and Ba 2ϩ permeability of I SOC was characterized further. The inward component of I SOC was very selective for Ca 2ϩ because removal of Na ϩ (substituted by NMDG ϩ ) did not affect current amplitude (from Ϫ4.9 Ϯ 1.5 pA/pF for Na ϩ medium to Ϫ4.2 Ϯ 2.5 for Na ϩ -free medium; n ϭ 14 -31). These data were consistent with the involvement of the high Ca 2ϩ -selective ORAI1 channel. In contrast, the outward component was nearly abolished in Na ϩ -free medium (from 5.3 Ϯ 0.2 pA/pF for Na ϩ medium to 0.2 Ϯ 0.7 for Na ϩ -free medium; n ϭ 14 -31) suggesting that this cation is the main current carrier of this component. However, it has been reported that Ba 2ϩ decreases currents mediated by ORAI1 but potentiate those mediated by ORAI2 and ORAI3. We found that the inward I SOC carried Ba 2ϩ ions, but the current amplitude was lower than that transported by Ca 2ϩ (from Ϫ4.9 Ϯ 15 pA/pF for Ca 2ϩ to Ϫ2.6 Ϯ 1.4 for Ba 2ϩ ; n ϭ 12-31) (data not shown). Data suggest that ORAI1 channels, rather than ORAI2 and -3, contribute to I CRAC -like currents in tumor cells. In addition, a nonselective channel also contributes to I SOC in tumor cells but not in normal cells.

Expression of SOCE Molecular Players in Normal and Tumor Carcinoma Cells-Expression of molecular candidates involved in I SOC and SOCE in normal and tumor cells was investigated
next. PCR analysis shows expression of probable TRP channels involved in SOCE in normal and tumor cells. We found that only TRPC1 is expressed in both normal and tumor cells (Fig.  9A). TRPV6 and TRPM8 were expressed in normal but not in tumor cells (Fig. 9A). Consistently, TRPM8 agonist menthol had no effect on I SOC in tumor cells (data not shown). Other candidates tested, including TRPC6 and TRPV4, were missing in both cell lines (Fig. 9A). Regarding candidates involved in I CRAC , we found that all members of the ORAI (ORAI1, -2, and -3) and STIM (STIM1 and -2) protein families are expressed in both normal and tumor cells (Fig. 9B). Quantitative, real time RT-PCR studies were carried out on those candidates expressed in both normal and tumor cells (Fig. 9C). Expression values were normalized relative to expression of ␤-actin. The expression profile of these candidates in normal NCM460 cells was STIM2 ϭ ORAI2 Ͼ STIM1 ϭ ORAI1 Ͼ ORAI3 Ͼ Ͼ TRPC1. In HT29 colon carcinoma cells, the expression profile was roughly similar except that STIM1 now doubled the ORAI1 expression (Fig. 9C). More importantly, we found that several transcripts were increased significantly relative to normal cells, including STIM2, ORAI2, STIM1, and TRPC1 (Fig. 9C). ORAI1 and ORAI3 transcripts were similar in normal and tumor cells (Fig. 9C).
Western blotting analysis was carried out to test expression of molecular candidates at the protein level. Fig. 10 shows that expression of almost all tested proteins was increased in tumor cells, including ORAI1, ORAI2, ORAI3, TRPC1, and STIM1 (Fig. 10, A-E  mRNA level. Relative changes were not similar. TRPC1 and STIM1 increased 5.2 and 3.7 times in tumor cells, respectively. ORAI1, ORAI2, and ORAI3 increased 2.3-, 2.9-, and 1.5-fold in tumor cells, respectively (Table 2). Surprisingly, we found that STIM2 protein is nearly lost in colon carcinoma HT29 cells relative to normal colon NCM460 cells (Fig. 10F) despite that stim2 was the most increased transcript in tumor cells (Fig. 9C). An emerging concept in Ca 2ϩ signaling is that stoichiometry of molecular components may influence SOCE and I SOC critically (33). Accordingly, we have estimated the fold change of each component in tumor cells relative to the changes of the remaining proteins. Table 3 shows the fold change ratios of each protein relative to the changes of the rest of the proteins. TRPC1/ORAI1, TRPC1/ORAI2, and TRPC1/ORAI3 ratios increased 2.2-, 1.8-, and 3.5-fold, respectively, in tumor cells, suggesting that SOCs in tumor cells are enriched in TRPC1. In addition, fold change ratios for TRPC1/STIM2, ORAI1/ STIM2, ORAI2/STIM2, ORAI3/STIM2, and STIM1/STIM2 increased by 35-, 13-, 19-, 20-, and 25-fold, respectively. These values suggest that, in tumor cells, STIM2 protein is essentially removed from any possible interaction with other SOCE molecular players. Interestingly, it has been reported that STIM2 may inhibit STIM1-mediated SOCE (34) and may regulate Ca 2ϩ store content (35). Accordingly, loss of STIM2 may impact on both SOCE and Ca 2ϩ store content. Knockdown experiments were carried out next to ascertain the role of the above-mentioned proteins on SOCE, I SOC , and Ca 2ϩ store content.
Effects of TRPC1 and ORAI1 Silencing on SOCE and I SOC in Normal and Tumor Cells-Results suggest that ORAI1 and TRPC1 are likely involved in SOCE and I SOC in colon carcinoma cells. To test for this possibility, ORAI1 and TRPC1 were silenced in HT29 cells using small interference RNA (siRNA) technology. siRNA probes against ORAI1 and TRPC1 decreased significantly the amount of corresponding mRNA (Fig. 11, A and B). ORAI1 silencing decreases significantly SOCE in HT29 cells almost as much as it decreases ORAI1 transcript (Fig. 11A). In contrast, TRPC1 knockdown fails to reduce SOCE in tumor cells (Fig. 11B). The effects of silencing

TABLE 2 Changes (fold increase) in proteins involved in SOCE in tumor cells relative to normal cells
Values correspond to the fold increase for each protein in tumor cells relative to normal cells. Thus, for instance, TRPC1 is 5.2 times more abundant in colon carcinoma cells than in normal colonic cells. Data are taken from the bars shown in Fig.  7. All proteins are more abundant (fold change Ͼ1) in tumor cells except for Stim2, which is decreased by 85% in tumor cells. The most important changes are those observed in TRPC1, Stim1, and Stim2 (shown in boldface). ORAI1 and TRPC1 on I SOC and I CRAC were tested next. In tumor cells, scramble siRNA had no effect on the inward or the outward I SOC (Fig. 11, C and F). As expected, ORAI1 silencing decreases largely the inward I SOC (from Ϫ4.9 Ϯ 0.6 pA/pF for scramble siRNA to Ϫ2,2 Ϯ 0.4 for ORAI1 siRNA; n ϭ 13-19) but also reduces significantly the outward component (from 4.5 Ϯ 1 pA/pF for scramble siRNA to 2.4 Ϯ 1 for ORAI1 siRNA; n ϭ 13-19) (Fig. 11, D and F). TRPC1 silencing nearly abolishes the outward component of the I SOC (from 4.5 Ϯ 1 pA/pF for scramble siRNA to 0.7 Ϯ 0.4 for TRPC1 siRNA; n ϭ 13-17) and also reduces significantly the inward component (from Ϫ4.9 Ϯ 0.6 pA/pF for scramble siRNA to Ϫ1.7 Ϯ 0.5 for TRPC1 siRNA; n ϭ 13-17) (Fig. 11, E and F). In normal cells, the results are quite different (Fig. 12). TRPC1 silencing or scramble siRNA has no effect on I CRAC (Fig. 12, A and B). However, silencing of ORAI1 inhibits I CRAC in normal cells (Fig. 12C). Average data are shown in Fig. 12D. Likewise, silencing of ORAI1 but not TRPC1 inhibits SOCE in normal cells (data not shown). These results indicate that both ORAI1 and TRPC1 contribute to I SOC in colon carcinoma cells, although in normal cells I CRAC is mediated only by ORAI1. We have also tested the contribution of ORAI2 and ORAI3 on SOCE in tumor cells. Fig. 13 shows that, paradoxically, silencing of either ORAI2 or ORAI3 in HT29 cells tends to increase SOCE. However, differences were not statistically significant (Fig. 13). Finally, we have investigated the molecular basis and functional significance of Ca 2ϩ store depletion in tumor cells. For this end, we tested the effects of STIM2 silencing in normal NCM460 cells on Ca 2ϩ store content, SOCE, and apoptosis resistance. STIM2 silencing decreases STIM2 mRNA by 64 Ϯ 6% (data not shown).
We found that STIM2 silencing decreased the rise in [Ca 2ϩ ] cyt induced by ionomycin in Ca 2ϩ -free medium consistently with decreased Ca 2ϩ store content in STIM2-silenced cells (Fig. 14A). In addition, re-addition of external Ca 2ϩ to ionomycin treated is decreased in STIM2-silenced cells suggesting that STIM2 knockdown inhibits SOCE in normal cells. Consistently, SOCE in cyclopiazonic acid-treated cells was reduced in silenced cells (Fig. 14, A and B) relative to control cells. Therefore, these data indicate that STIM2 contributes to SOCE and Ca 2ϩ store content in normal cells, and its silencing leads to decreased SOCE and Ca 2ϩ store content. As Ca 2ϩ store content may be relevant for apoptosis resistance, we next tested the effects of STIM2 silencing on apoptosis resistance. We found that after strong oxidative damage (2 mM H 2 O 2 , 150 min), resistance to apoptosis was similar in control and silenced cells (data not shown). However, when damage was less severe (1 mM H 2 O 2 , 30 min), STIM2-silenced cells proved to be more resistant to cell death than control cells (Fig. 14C). These data indicate that STIM2 participates in SOCE in normal colon epithelial cells, and the inhibition of its expression during tumorigenesis may contribute to Ca 2ϩ store depletion and apoptosis resistance, which are characteristic of tumor cells.

DISCUSSION
We have investigated the remodeling of intracellular Ca 2ϩ handling in colon cancer, its molecular basis, and its contribution to cancer hallmarks. To this end, functional parameters and molecular players involved in Ca 2ϩ homeostasis were studied in normal human mucosa and colon carcinoma cells. All colon carcinoma cell lines tested displayed a much larger SOCE than normal cell lines, which correlated with increased cell proliferation in tumor cells, thus suggesting that enhanced SOCE contributes to increased tumor cell proliferation in colon cancer. Consistently, up-regulation of SOCE has been recently correlated with cancer features in a number of cancers (10,12,13,16,20,21,36). We have shown previously that SOCE antagonists inhibit colon carcinoma cell proliferation (25,29). Now, TABLE 3

Ratio of change in proteins involved in SOCE relative to the change observed in the remaining proteins
Each value corresponds to the fold change of a particular protein in tumor cells relative to the fold change of the remaining proteins. For instance, TRPC1 increases 5.2 fold in tumor cells whereas Orai1 increases only 2.3 times. Thus, TRPC1 protein expression increases 2.2 times more than Orai1. These ratios are very large for any combination of proteins with Stim2 in the denominator as this protein actually decreases in tumor cells.
we show that SOCE antagonist 2APB also inhibits colon carcinoma cell invasion suggesting contribution of SOCE to enhanced proliferation and invasion in these cells. Accordingly, we have investigated the mechanisms for increased SOCE in human colon carcinoma cells. Importantly, NCM460 normal and HT29 carcinoma colon cells have been recently validated as normal and tumor cell models, respectively (37).
Increased SOCE in colon carcinoma cells was associated with enhanced resting [Ca 2ϩ ] cyt , more negative membrane potential, increased I SOC , and enhanced agonist-induced Ca 2ϩ release and entry. As a matter of fact, physiological agonists that induce Ca 2ϩ release (ATP and carbachol) promoted Ca 2ϩ entry only in tumor cells. This differential response could be due to the fact that Ca 2ϩ stores in normal cells are overloaded relative to tumor cells, and the agonist-induced Ca 2ϩ store emptying is rather limited. In this scenario, the threshold for SOCE activation could be beyond reach, and SOCE is not permitted unless Ca 2ϩ stores are fully depleted by, for instance, thapsigargin. In contrast, Ca 2ϩ stores in tumor cells are substantially depleted, and Ca 2ϩ release is enhanced, thus putting SOCE threshold at reach and favoring SOCE activation in physiological conditions. This partial Ca 2ϩ store depletion in colon carcinoma cells could contribute also to cancer features. First, it favors SOCE activation and therefore cell proliferation and invasion as stated above. Second, it may also contribute to apoptosis resistance, another hallmark of cancer cells. Interestingly, it has been reported recently that Ca 2ϩ store content may be critical for survival. Specifically, large Ca 2ϩ stores favor enhanced transfer to mitochondria and mitochondrial Ca 2ϩ overload, whereas reduced Ca 2ϩ store content prevents mitochondrial Ca 2ϩ overload and apoptosis (38). Consistently, we show that colon carcinoma cells display substantially depleted stores and enhanced resistance to cell death. Taken together, data suggest that the "Ca 2ϩ signature" of colon carcinoma cells shown here and made of enhanced SOCE and depleted Ca 2ϩ stores may contribute to enhanced proliferation, invasion, and survival characteristics of cancer cells. What mechanisms underlie enhanced SOCE and depleted Ca 2ϩ stores in human colon carcinoma cells? Regarding SOCE, our combined functional and molecular analysis reveals that SOCE enhancement in tumor cells is mediated by the following: 1) up-regulation of ORAI1 and STIM1 proteins, which likely  mediate enhanced I CRAC and SOCE in tumor cells; 2) overexpression of TRPC1 protein that correlated with the emergence of a nonselective I SOC ; and 3) the switch of the levels of expression between STIM1 and STIM2 Ca 2ϩ sensors proteins (Fig.  15). More specifically, differences in ion channel expression and ER Ca 2ϩ sensors may contribute to enhance SOCE in tumor cells. This possibility was addressed directly by measuring SOCs in normal and tumor cells. Interestingly, I SOC was strikingly different. Normal cells display a small I CRAC current, whereas tumor cells showed a mix of currents, including enhanced I CRAC plus and additional nonselective I SOC . It has been reported that SOCE can be supported by different I SOC expressed in the same cell (9, 39 -41). To our knowledge, this is the first report showing that I SOC currents in normal cells are strikingly different compared with their tumor cell counterparts.
I CRAC in normal and colon carcinoma cells is likely mediated by ORAI1 and STIM1/STIM2 proteins because all of them are expressed in both cell lines, and the biophysical and pharmacological characteristics of recorded currents match those described for canonical I CRAC (31,42,43). In our hands, I CRAC in both normal and tumor colon cells displayed voltage-independent activation, strong inward rectification, and reversal potential in very positive voltages. Also, I CRAC was inhibited by 2APB at a low concentration (30 M). In addition, the well known potentiating effect of low concentrations of 2APB on ORAI3-containing SOCs (32,44) was not observed. Moreover, the extent of I CRAC was unaffected by the absence of extracellular Na ϩ ions and reduced when Ba 2ϩ was used instead Ca 2ϩ , thus indicating a high Ca 2ϩ selectivity and the involvement of ORAI1 channels (42,45,46). However, the emergent I SOC restricted to tumor cells was nonselective showing a reversal potential near 0 mV. Unlike I CRAC , the emergent I SOC was not sensitive to low concentrations of 2APB (30 M), and the current amplitude of the outward component was significantly decreased by removal of extracellular Na ϩ ions, thus suggesting involvement of a TRPC member (47,48). At the molecular level, several candidates were excluded because they are not expressed in tumor cells, including TRPV6, TRPM8, TRPC6, and TRPV4. In contrast, TRPC1 was expressed in normal and tumor cells, and its abundance increased quite significantly in colon cancer cells, thus suggesting contribution of TRPC1 to the nonselective, emergent I SOC of tumor cells. Knockdown experiments corroborated the molecular identity of SOCs underlying SOCE. ORAI1-containing channels mediate I CRAC in normal and tumor cells. Overexpression of ORAI1 and STIM1 is involved in increased SOCE and I SOC in tumor cells. Consistently, ORAI1 silencing prevented SOCE in tumor cells. However silencing of either ORAI2 or ORAI3 had no significant effect on SOCE in colon carcinoma (HT29) cells. Meanwhile, TRPC1 channels are involved in the nonselective I SOC but do not contribute to SOCE. Moreover, the low Ca 2ϩ store content may, in turn, modulate the expression of the TRPC1 channel. For example, it has been shown that prolonged depletion of Ca 2ϩ stores enhances TRPC1 expression and increases [Ca 2ϩ ] cyt responses to agonists without affecting SOCE (49). Silencing data are also consistent with the possibility of functional interactions between ORAI1 and TRPC1 channels. ORAI1 knockdown prevents mainly I CRAC but also reduces significantly the outward component mediated by TRPC1. Conversely, TRPC1 silencing nearly abolishes outward I SOC , but it also reduces significantly the extent of the inward component. Consistently, it has been shown that STIM1 may drive interactions between ORAI1 and TRPC1 (39,50).
Our results pose the question regarding what is the role played by TRPC1 up-regulation in colon cancer. TRPC channel has been the subject of a long term controversy about its role as a SOC channel (33). In our experimental conditions, the nonselective I SOC is likely mediated by TRPC1-containing channels. The most interesting matter is that, in human carcinoma colon cells, TRPC1 protein showed the largest change (up-regulation) together with STIM2 (down-regulation). Accordingly, these changes could represent the most critical events underlying Ca 2ϩ remodeling and acquisition of cancer features. In support of this view, it has been reported that TRPC channels are overexpressed and regulate cell proliferation in human nonsmall cell lung, breast, liver, stomach, and glioma cancer (13,(51)(52)(53)(54). The nonselective channel TRPC1 permeates Na ϩ and Ca 2ϩ , and consequently, it may have a role as a Ca 2ϩ influx pathway or as a modulator of membrane potential. For instance, TRPC1 may control the driving force for Ca 2ϩ influx during SOCE (55). Moreover, TRPC1 could support cell prolif-  eration of tumor cells because one of its physiological roles is the modulation of the cell cycle progression through the regulation of cell volume (56). In addition, it has been reported recently that the interaction between STIM1 and TRPC1 is essential for cell migration after wounding in rat intestinal, epithelial cells (57). Moreover, it has been shown that the rise in STIM2 relative to STIM1 favors STIM1/STIM2 heteromers that suppress STIM1 translocation to the plasma membrane and its interaction with TRPC1 (57). Therefore, our finding that TRPC1/STIM2, STIM1/STIM2, and ORAI1/STIM2 ratios increase by 35-, 25-, and 13-fold, respectively, in colon cancer cells suggests that STIM2 depletion may enable STIM1 translocation to the plasma membrane and STIM1 interaction with TRPC1, providing an explanation for both enhanced SOCE and functional expression of an emergent, nonselective I SOC in tumor cells. Further research is needed to ascertain more precisely the role of TRPC1 in colonic tumorigenesis. What mechanisms are involved in the low level of Ca 2ϩ store content in colon carcinoma cells? Ca 2ϩ store content at the ER depends on the balance between Ca 2ϩ uptake mediated by sarcoplasmic and ER Ca 2ϩ -ATPase pumps and Ca 2ϩ exit through unknown leak channels (4  contrast, the STIM2 EF hand displays a low apparent affinity for Ca 2ϩ (K d ϳ500 M) and senses rather small decreases in [Ca 2ϩ ] within the ER with an EC 50 of 406 M (35). Accordingly, in normal mucosa cells expressing both STIM1 and STIM2, it is likely that Ca 2ϩ levels inside the ER Ca 2ϩ store are set by STIM2 that activates first when the Ca 2ϩ store content falls below 500 M. This view is consistent with the large Ca 2ϩ store content found in normal cells where Stim2 is relatively more abundant. However, in tumor cells, STIM2 depletion may render STIM1 as the only Ca 2ϩ sensor available. In this scenario, STIM1 could set Ca 2ϩ levels within the ER close to 200 M. Our finding that the STIM1/STIM2 ratio increases by 25-fold in tumor cells where Ca 2ϩ stores remain substantially depleted is entirely consistent with this possibility. Our knockdown experiments also support this view. STIM2 knockdown in normal cells decreased Ca 2ϩ store content in a significant manner. More importantly, STIM2 silencing induced apoptosis resistance to normal cells, thus confirming the important role of STIM2 loss in Ca 2ϩ store emptying and enhanced cell survival. Interestingly, down-regulation of STIM2 and Ca 2ϩ store depletion may contribute to increase TRPC1 in tumor cells in another way. It has been reported that depletion of Ca 2ϩ stores with thapsigargin increases TRPC1 protein levels without affecting SOCE (49). Thus, TRPC1 functional expression depends on the filling state of Ca 2ϩ stores. This view is supported by a report showing that in Darier disease, a disorder of skin epithelia, a rare mutation that prevents operation of SERCA2 depletes Ca 2ϩ stores, and this condition promotes a compensatory up-regulation of TRPC1 (58). Therefore, increased expression of TRPC1 and perhaps other SOCE com-  FIGURE 15. Hypothesis of molecular basis of Ca 2؉ remodeling in colon cancer. The "Ca 2ϩ signature" of colon carcinoma cells is enhanced SOCE, differential SOCs, and depleted Ca 2ϩ stores. This remodeling is associated with increased protein expression of TRPC1, STIM1, ORAI1, ORAI2, and ORAI3 in tumor cells along with loss of STIM2 protein. Normal cells show small SOCE mediated by canonical I CRAC carried by ORAI1. STIM2 protein in normal cells may limit STIM1/ORAI1 interaction and may signal for large Ca 2ϩ store content, thus preventing SOCE activation and TRPC1 functional expression. In this scenario, cell proliferation and migration are limited, and cells are prone to die as Ca 2ϩ stores are loaded. Loss of STIM2 renders cells under control of STIM1 that set Ca 2ϩ store content to a lower level. Like in Darier disease, depletion of Ca 2ϩ stores likely promotes TRPC1 functional expression. In addition, loss of STIM2 may also favor interaction of STIM1 with ORAI1 and TRPC1 resulting in enhanced I CRAC and the appearance of a nonselective current. ponents in colon cancer could be secondary to Ca 2ϩ store depletion associated with the loss of STIM2.
Taken together, the above data suggest that the critical event in Ca 2ϩ remodeling in colon cancer could be STIM2 protein down-regulation. As a cautionary note, we must acknowledge that our results are derived from comparison of a few normal and colon carcinoma cell lines that may not reflect entirely human colorectal carcinogenesis. Further research is required to test whether our results apply to other tumor cell lines and real tumor cells. However, in support of the potential relevance of STIM2 loss in colon cancer, recent data suggest that STIM2 is a tumor suppressor but the action mechanism is unknown. The STIM2 gene located at 4p15 has been recently identified as a candidate gene for tumorigenesis in glioblastoma multiforme (23) and colon cancer (22). Paradoxically, STIM2 transcript is actually overexpressed in 64% of all human colon cancers tested (22). However, these results are controversial because, as stated by the own authors, it is intriguing that a gene with a suppressor phenotype is so frequently overexpressed in colon cancer (22). It is worth noting, however, that STIM2 was tested only at the transcript level. Interestingly, STIM2 transcript, which is upregulated also in prostate cancer, has been recently shown to be down-regulated during the transition from moderate to high Gleason grade (59). Thus, up-regulation of the mRNA level of STIM2 is not necessarily reflected as overexpression of the protein. In agreement, we show that STIM2 transcript is overexpressed in colon carcinoma (HT29) cells, although STIM2 protein is nearly lost in the same cells. Finally, it has also been reported recently that increases in STIM1/STIM2 ratios are associated with a poor prognosis in breast cancer (60).
In summary, we show here that human colon carcinoma cells show increased store-operated Ca 2ϩ entry, enhanced and modified store-operated currents, and partially depleted Ca 2ϩ stores relative to their normal counterparts. These changes correlate with increased cell proliferation, invasion, and survival characteristic of tumor cells. Finally, most changes can be explained by changes in molecular players involved in SOCE, particularly a reciprocal shift in TRPC1 and STIM2 expression, thus suggesting TRPC1 and STIM2 as novel targets for colorectal cancer. Further research is required to ascertain more precisely the role of these molecular players in colon carcinogenesis.