Calcium store sensor stromal-interaction molecule 1-dependent signaling plays an important role in cervical cancer growth, migration, and angiogenesis

Abstract

Store-operated Ca(2+) entry (SOCE) is the principal Ca(2+) entry mechanism in nonexcitable cells. Stromal-interaction molecule 1 (STIM1) is an endoplasmic reticulum Ca(2+) sensor that triggers SOCE activation. However, the role of STIM1 in regulating cancer progression remains controversial and its clinical relevance is unclear. Here we show that STIM1-dependent signaling is important for cervical cancer cell proliferation, migration, and angiogenesis. STIM1 overexpression in tumor tissue is noted in 71% cases of early-stage cervical cancer. In tumor tissues, the level of STIM1 expression is significantly associated with the risk of metastasis and survival. EGF-stimulated cancer cell migration requires STIM1 expression and EGF increases the interaction between STIM1 and Orai1 in juxta-membrane areas, and thus induces Ca(2+) influx. STIM1 involves the activation of Ca(2+)-regulated protease calpain, as well as Ca(2+)-regulated cytoplasmic kinase Pyk2, which regulate the focal-adhesion dynamics of migratory cervical cancer cells. Because of an increase of p21 protein levels and a decrease of Cdc25C protein levels, STIM1-silencing in cervical cancer cells significantly inhibits cell proliferation by arresting the cell cycle at the S and G2/M phases. STIM1 also regulates the production of VEGF in cervical cancer cells. Interference with STIM1 expression or blockade of SOCE activity inhibits tumor angiogenesis and growth in animal models, confirming the crucial role of STIM1-mediated Ca(2+) influx in aggravating tumor development in vivo. These results make STIM1-dependent signaling an attractive target for therapeutic intervention.

Fig. 1.

STIM1 expression is associated with cancer metastasis and clinical outcome. (A) Expression pattern of STIM1 in early-stage cervical cancer. Cervical cancer (n = 24) with the pair tissues of carcinoma and adjacent nonneoplastic epithelia were analyzed by immunoblotting. N, nonneoplastic epithelia. T, tumor tissues. (B) Quantitative analyses of STIM1 immunoblotting. STIM1 expression level in normal squamous epithelia was used as control and those in tumor tissues were expressed as the relative of control. (C) Immunofluorescent stainings of STIM1 and E-cadherin in normal cervical epithelia and adjacent cervical cancer tissues. E-cadherin, normal epithelial marker. Nuclei were stained with Hoechst 33258 (blue). Representative images of six different cases. (Scale bars, 20 μm.) (D) The association between STIM1 expression level and tumor size in the same surgical specimen of cervical cancer tissues (n = 24). (E) The tumor expression level of STIM1 was significantly higher in the groups of local pelvic lymph node metastasis. Dashed lines, mean ± SEM.

Fig. 2.

STIM1 is involved in tumor growth and angiogenesis. (A) STIM1 knockdown by shRNA in cervical cancer SiHa and CaSki cell lines. (B) Establishment of STIM1-overexpressed cervical cancer cell lines. Endogenous STIM1 and exogenous STIM1 were differentiated by immunoblotting with STIM1 (Left) and EGFP (Right), respectively. (C and D) STIM1 overexpression enhances tumor angiogenesis and growth. Bilateral dorsal sites of SCID mice were subcutaneously inoculated with mock-transfected (Control) or STIM1-overexpressed cervical cancer cells. Representative tumor xenografts (C), the mean tumor vessel numbers and mean tumor weight (D) 21 d after inoculation. Arrowhead, local spread of tumor mass. (Scale bars, 0.5 cm.) Columns, mean ± SEM (n = 6), *P < 0.01. (E) STIM1 knockdown attenuates tumor growth and angiogenesis. Representative tumor xenografts (Left), mean tumor vessel numbers (Top Right), and mean tumor weight (Bottom Right) 15 d after inoculation of control shRNA- or shSTIM1-transfected cervical cancer cells. Columns, mean ± SEM (n = 6), *P < 0.01. (Scale bar, 1 cm.) (F) STIM1 regulates VEGF-A production. The VEGF-A secretion in various stable pools of cervical cancer cells were measured by ELISA. *P < 0.01, compared with wild type. Columns, mean ± SEM (n = 5).

Fig. 3.

STIM1 modulates the focal-adhesion complex. (A and B) STIM1 is involved in cervical cancer cell migration. Columns, mean ± SEM from at least four different experiments. EGF, 100 ng/mL epidermal growth factor. (C) EGF-induced Ca²⁺ influx is a STIM1-dependent process. Gray lines, [Ca²⁺]_i oscillations of individual cervical cancer SiHa cells. Black lines, the mean trace of [Ca²⁺]_i oscillations. (D and E) STIM1 is necessary for EGF-stimulated calpain activation. (D) Quantitative fluorescent analyses of intracellular calpain activities, measured with a fluorogenic membrane-permeable calpain substrate t-Boc-LM-CMAC. PD151746 (50 μM), a specific inhibitor targeting Ca²⁺-binding site of calpain; PD145305 (50 μM), a negative control for PD151746. Columns, mean ± SEM from at least 100 cells. (E) STIM1 siRNA abolishes EGF-induced cleavage of α-spectrin. Arrow and arrowhead, the full-length (280 kDa) and calpain-digested cleaved form (150/145 kDa) of α-spectrin, respectively. (F) STIM1 affects EGF-stimulated Pyk2 activation. (Left) Representative immunoblots from at least three different experiments. (Right) Densitometric quantification of Pyk2 phosphorylation (Tyr402) levels. Points, mean ± SEM; *P < 0.01, compared with control group. (G) STIM1 modulates the focal adhesion turnover. (Left) Representative images showing focal adhesions. FAK (green), focal-adhesion marker. (Right) Quantitative analyses of focal adhesion size. Focal-adhesion sizes were quantified by measuring the area of FAK staining. Columns, mean ± SEM from at least 20 individual cells of three different experiments. N.S., nonsignificant. (Scale bars, 10 μm.)

Fig. 4.

EGF enhances the interaction between STIM1 and Orai1. (A) STIM1 specifically interacts with Orai1, but not with Orai2, Orai3, TRPC1, or TRPC6 in cervical cancer SiHa cells upon EGF stimulation. Representative immunoblots from at least three different experiments. IP, immunoprecipitation; WCL, whole-cell lysates. (B and C) Time-lapse confocal images of SiHa cells co-overexpressed with EGFP-STIM1 (green) and mOrange-Orai1 (red) in response to EGF stimulation. Arrows, the colocalization between EGFP-STIM1 and mOrange-Orai1 at the cell periphery. The quantitative results with pixel-by-pixel analyses were shown in C. Columns, mean ± SEM from at least 30 cells of three different experiments. (Scale bars, 10 μm.) (D and E) EGF does not enhance the interaction between STIM1 and TRPC1 or TRPC6. (Right) Representative images of STIM1 and TRPC family, such as TRPC6 (D) and TRPC1 (E) in the absence or presence of EGF stimulation. (Left) Colocalization ratio between STIM1 and TRPC6 (D) or TRPC1 (E) at cell periphery by pixel-by-pixel analyses. Columns, mean ± SEM from at least 30 cells of three different experiments. (Scale bar, 10 μm.)

Fig. 5.

STIM1 influences cell-cycle progression. (A) STIM1 knockdown significantly inhibits the proliferation of cervical cancer SiHa cells. Points, mean ± SEM (n = 5); ^#P < 0.05, *P < 0.01, compared with control groups. (B) STIM1-silencing in cervical cancer cells inhibits cell proliferation by arresting cell cycle at S and G2/M phases. Representative FACS measurements to determine cell-cycle stages at the third day posttransfection. (C) STIM1 knockdown induces p21 up-regulation and Cdc25C down-regulation. Representative immunoblots from five different experiments. (D) STIM1 knockdown modestly increases mRNA levels of p21, demonstrated by semiquantitative RT-PCR with β-actin as the internal control. Columns, mean ± SEM (n = 4). (E) STIM1 knockdown slows down p21 protein degradation. The protein stability of p21 was examined in the presence of translational inhibitor cycloheximide (CHX, 20 μg/mL). (Upper) Representative immunoblots from four different experiments. (Lower) Densitometric quantification of p21 protein levels. Points, mean ± SEM (n = 4). (F) STIM1 knockdown inhibits the proteasome-mediated degradation of p21. Cells were preincubated with the protein translation inhibitor cycloheximide (20 μg/mL) for 30 min before the treatment of the proteasome inhibitor MG132 (40 μM), lysosome inhibitor NH₄Cl (40 μM), or protease inhibitor mixture (PIC, 1 unit) for 8 h. (Left) Representative immunoblots from three different experiments. (Right) Densitometric quantification of p21 protein levels. Columns, mean ± SEM (n = 3).

Fig. 6.

Store-operated Ca²⁺ entry affects tumor growth in vivo. (A) STIM1 siRNA, 2-APB (20 μM), or SKF96365 (50 μM) inhibits SOCE of cervical cancer SiHa cells. To measure Ca²⁺ entry, SiHa cells loaded with Fura-2/AM (2 μM) were preincubated in Ca²⁺-free media plus 2 μM thapsigargin for 30 min to deplete the internal Ca²⁺ store. Each trace was averaged from at least 30 single cells. (B–E) Blockade of SOCE retards tumor growth and angiogenesis. (B) Female SCID mice bearing tumor xenograft of SiHa cells were intraperitoneally injected every 3 d (arrows) with control vehicle (n = 6), SKF96365 (2.5 mg/kg; n = 6), or 2-APB (50 μg/kg; n = 6) from the sixth day postinoculation. Representative tumor xenografts (C), mean tumor vessel numbers (D), and mean tumor weight (E) at the 15th day postinoculation. Arrows, rapid tumor growth with obvious angiogenesis. Black arrowheads, the obliteration of blood supply. Blue arrowheads, the extravasation of tumor feeding vessels. Columns, mean ± SEM (n = 6); *P < 0.01, compared with control group. (Scale bars, 1 cm.)