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. 2021 Nov 25;13(23):5937.
doi: 10.3390/cancers13235937.

Orai3 Regulates Pancreatic Cancer Metastasis by Encoding a Functional Store Operated Calcium Entry Channel

Samriddhi Arora  1 , Jyoti Tanwar  2   3 , Nutan Sharma  1 , Suman Saurav  1 , Rajender K Motiani  1
Affiliations

Orai3 Regulates Pancreatic Cancer Metastasis by Encoding a Functional Store Operated Calcium Entry Channel

Samriddhi Arora et al. Cancers (Basel). .

Abstract

Store operated Ca2+ entry (SOCE) mediated by Orai1/2/3 channels is a highly regulated and ubiquitous Ca2+ influx pathway. Although the role of Orai1 channels is well studied, the significance of Orai2/3 channels is still emerging in nature. In this study, we performed extensive bioinformatic analysis of publicly available datasets and observed that Orai3 expression is inversely associated with the mean survival time of PC patients. Orai3 expression analysis in a battery of PC cell lines corroborated its differential expression profile. We then carried out thorough Ca2+ imaging experiments in six PC cell lines and found that Orai3 forms a functional SOCE channel in PC cells. Our in vitro functional assays show that Orai3 regulates PC cell cycle progression, apoptosis and migration. Most importantly, our in vivo xenograft studies demonstrate a critical role of Orai3 in PC tumor growth and secondary metastasis. Mechanistically, Orai3 controls G1 phase progression, matrix metalloproteinase expression and epithelial-mesenchymal transition in PC cells. Taken together, this study for the first-time reports that Orai3 drives aggressive phenotypes of PC cells, i.e., migration in vitro and metastasis in vivo. Considering that Orai3 overexpression leads to poor prognosis in PC patients, it appears to be a highly attractive therapeutic target.

Keywords: Orai3; metastasis; pancreatic cancer; store operated calcium entry.

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Conflict of interest statement

Authors report no conflict of interest.

Figures

Figure 1
Figure 1
Orai3 expression is inversely associated with the mean survival time of pancreatic cancer patients. (A) Orai3 expression analysis in GEPIA (Gene Expression Profiling Interactive Analysis) database having 179 pancreatic tumor samples (T) and 171 normal pancreas samples (N). Here “*” indicates that Orai3 expression is significantly higher in pancreatic tumor samples in comparison to normal pancreas samples. (B) Pancreatic cancer patients’ survival analysis in GEPIA wherein blue trace corresponds to low Orai3 expression (n = 89 patients) and red trace represents high Orai3 expression (n = 89 patients) clearly suggesting that higher Orai3 expression is associated with less patient survival time. (C) Pancreatic cancer patients’ survival analysis in “The Human Protein Atlas” database where blue trace corresponds to low Orai3 expression (n = 124 patients) and red trace represents high Orai3 expression (n = 52 patients) demonstrates that higher Orai3 levels are associated with poor prognosis and leads to decrease in patients’ survival time. (D) Western blot analysis for examining Orai3 protein levels in well-established pancreatic cancer cell lines and transformed “normal” pancreatic cells. (E) ImageJ based densitometric analysis of Orai3 expression in pancreatic cancer cell lines suggest differential Orai3 expression profile (n = 4).
Figure 2
Figure 2
Characterization of SOCE in a battery of Pancreatic cancer cell lines. (A–F) Representative Ca2+ imaging traces of HPNE, BxPC3, MiaPaCa2, Capan1, Panc1 and CFPAC1 cells where “n” denotes the number of cells in that particular trace. (G) Quantitation of basal Ca2+ levels across different cell lines. (H) The amplitude of ER Ca2+ release was calculated from a number of experiments and data are presented in dot plot graphs. (I) The extent of SOCE was calculated from several experiments and data are presented in dot plot graphs. (J) 2-APB induced potentiation of Orai3 mediated SOCE was calculated from several experiments and data are presented in dot plot graphs. The total number of cells imaged in (G–J) are reported as “n  =  x, y” where “x” denotes total number of cells imaged and “y” denotes number of traces recorded.
Figure 3
Figure 3
Orai3 encodes a functional SOCE channel in Panc1 cells. (A,B) Lentiviral transduced Panc1 cells stably expressing either GFP tagged shNT control or GFP tagged shRNA targeting Orai3 (fluorescence and merged images of bright field plus fluorescence are presented). (C) Representative Western blot showing knockdown of Orai3 in shOrai3 Panc1 stables in comparison to shNT control Panc1 stable cells. (D) Densitometric analysis of Orai3 silencing in 3 independent shNT and shOrai3 Panc1 stable clones. (E) Representative Ca2+ imaging trace of shNT Panc1 stables where “n = 40” denotes the number of cells in that particular trace. (F) Representative Ca2+ imaging trace of shOrai3 Panc1 stables where “n = 42” denotes the number of cells imaged in that particular trace. (G) The extent of SOCE was calculated from 281 shNT and 247 shOrai3 cells, which were imaged from several autonomous experiments/traces (11 traces of shNT and 9 traces of shOrai3 originating from 3 independent clones of shNT and shOrai3 Panc1 stables) and data from autonomous traces are presented in dot plot graphs. (G’) Data for extent of SOCE from all individual cells from 3 independent clones are presented as clone N°1 yellow rounds, clone N°2 blue squares, clone N°3 green diamond. (H) 2-APB induced potentiation of Orai3 mediated SOCE was calculated from around 250 cells/condition. These cells were imaged during several autonomous experiments/traces (10 traces of shNT and 9 traces of shOrai3 originating from 3 independent clones of shNT and shOrai3 Panc1 stables) and data are presented in dot plot graphs. (H’) Data for 2-APB induced SOCE potentiation from all individual cells from 3 independent clones are presented as clone N°1 yellow rounds, clone N°2 blue squares, clone N°3 green diamond. The total number of cells imaged are reported in (G,H) as “n  =  x, y” where “x” denotes total number of cells imaged and “y” denotes number of traces recorded. Data presented are mean ± S.E.M. Unpaired Student’s t-test was performed for statistical analysis. p-value < 0.05 was considered as significant and is presented as “*”; p-value < 0.01 is presented as “**”and p-value < 0.0001 is presented as “****”.
Figure 4
Figure 4
Orai3 regulates Panc1 but not HPNE cell viability. (A) MTT assay-based cell viability analysis (24–96 h time points) in Panc1 shNT and Panc1 shOrai3 stable cells (n = 5). (B,C) Lentiviral transduced HPNE cells stably expressing either GFP tagged shNT control or GFP tagged shRNA targeting Orai3 (fluorescence and merged images of bright field plus fluorescence are shown). (D) Representative Western blot showing knockdown of Orai3 in shOrai3 HPNE stables in comparison to shNT control HPNE stable cells. (E) Densitometric analysis of Orai3 silencing in 4 independent shNT and shOrai3 HPNE stable clones. (F) MTT assay-based cell viability analysis (24–96 h time points) in HPNE shNT and HPNE shOrai3 stable cells (n = 4). Data presented as mean  ±  S.E.M. Unpaired Student’s t-test was performed for statistical analysis. p-value < 0.05 was considered as significant and is presented as “*”; p-value < 0.01 is presented as “**”and p-value < 0.001 is presented as “***”.
Figure 5
Figure 5
Orai3 contributes to Panc1 cell-cycle progression. (A,B) Representative data showing FACS-based cell-cycle analysis of Panc1 shNT and Panc1 shOrai3 stable cells. Cell-cycle analysis was performed with three independent biological experiments using propidium iodide. (C) Quantitative analysis of % cells in different phases of cell cycle in the case of Panc1 shNT and Panc1 shOrai3 stable cells (n = 4). (D) Western blot analysis for cyclin D1 expression in shNT and shOrai3 Panc1 stable cell lines. (E) Densitometric analysis of cyclin D1 levels in shNT and shOrai3 Panc1 stable cells. (F) Western blot analysis for Cdk4 expression in shNT and shOrai3 Panc1 stable cell lines. (G) Densitometric analysis of Cdk4 levels in shNT and shOrai3 Panc1 stable cells. Data presented as mean ± S.E.M. Unpaired Student’s t-test was performed for statistical analysis. p-value < 0.05 was considered as significant and is presented as “*”.
Figure 6
Figure 6
Orai3 regulates basal apoptosis of Panc1 cells. (A–D) Representative FACS-based analysis of apoptosis using propidium iodide and TRITC conjugated annexin V for unstained, i.e., negative control Panc1 cells (A); positive control Panc1 cells (B); shNT Panc1 stable cells (C) and shOrai3 Panc1 stable cells (D). (E) Quantitative analysis of % early apoptotic cells in shNT and shOrai3 Panc1 stable cell lines from 4 independent experiments. Data presented as mean ± S.E.M. Paired t-test was performed for statistical analysis. p-value < 0.05 was considered as significant and is presented as “*”.
Figure 7
Figure 7
Orai3 controls migration of Panc1 cells. Scratch wound healing (A–C) and transwell migration assays (D–F) were performed for evaluating the role of Orai3 in regulating Panc1 migration. (A) Representative wound healing images at 0, 6, 12 and 24 h time points in the case of shNT Panc1 stable cells. (B) Representative wound healing images at 0, 6, 12 and 24 h time point in case of shOrai3 Panc1 stable cells. (C) Quantitative analysis of %wound healing over the period of 24 h in shNT and shOrai3 Panc1 stable cells from 4 independent experiments. (D) Representative micrographs of crystal violet stained transwell migrated shNT Panc1 stable cell line. (E) Representative micrographs of crystal violet stained transwell migrated shOrai3 Panc1 stable cells. (F) Quantitative analysis of number of migrated cells in the transwell migration assay from three independent experiments. Data presented as mean ± S.E.M. Unpaired Student’s t-test was performed for statistical analysis. p-value < 0.05 was considered as significant and p-value < 0.01 is denoted as “**”.
Figure 8
Figure 8
Orai3 regulates pancreatic cancer progression in vivo. (A) Weekly tumor volume measurements in NOD SCID mice injected with either shNT Panc1 cells or shOrai3 Panc1 cells (n = 5 mice/condition). (B). Tumor weight measurements after sacrificing mice at 12-week post-injections (either shNT Panc1 or shOrai3 Panc1 injections) timepoint. (C). Pictures of shNT and shOrai3 Panc1 tumors harvested after 12 weeks of injections. (D). Western blot analysis for Orai3 expression in shNT Panc1 and shOrai3 Panc1 tumors. (E). Densitometric analysis of validating Orai3 knockdown in shOrai3 Panc1 tumors in comparison to shNT Panc1 tumors. (F). Western blot analysis for cyclin D1 levels in shNT Panc1 and shOrai3 Panc1 tumors. (G). Densitometric analysis of cyclin D1 levels in shNT Panc1 and shOrai3 Panc1 tumors. (H). Western blot analysis for Cdk4 expression in shNT Panc1 and shOrai3 Panc1 tumors. (I) Densitometric analysis of Cdk4 levels in shNT Panc1 and shOrai3 Panc1 tumors. Data presented as mean ± S.E.M. Unpaired Student’s t-test was performed for statistical analysis except from tumor volume wherein two-way ANOVA was performed. p-value < 0.01 is denoted as “**”; p-value < 0.001 is presented as “***” and p-value < 0.0001 is denoted as “****”.
Figure 9
Figure 9
Orai3 regulates pancreatic cancer metastasis. (A) Whole-body bio-fluorescence imaging signals at 12-week post-injection timepoint in the case of shNT Panc1 stables injected mice. (A’) Whole-body bio-fluorescence imaging signals in shOrai3 Panc1 stable cells injected mice at 12-week post-injection timepoint. The initial xenografts at the site of injections are marked in red circles while metastatic spread is highlighted with yellow arrows. (B) Thoracoabdominal region bio-fluorescence imaging signals at 12-week post-injection time-point in case of shNT Panc1 stables injected mice. (B’) Thoracoabdominal region bio-fluorescence imaging signals in shOrai3 Panc1 stable cells injected mice at 12-week post-injection timepoint. The thoracoabdominal bio-fluorescence imaging signals are identified in red circles. The time interval between whole body and thoracoabdominal bio-fluorescence imaging was 3 to 5 min. (C) Western blot analysis for MMP2 protein expression in shNT Panc1 and shOrai3 Panc1 tumors. (D) Densitometric analysis of MMP2 levels in shNT Panc1 and shOrai3 Panc1 tumors. (E) Western blot analysis for E-cadherin protein expression in shNT Panc1 and shOrai3 Panc1 tumors. (F) Densitometric analysis of E-cadherin levels in shNT Panc1 and shOrai3 Panc1 tumors. Data presented as mean ± S.E.M. Un-paired t-test was performed for statistical analysis. p-value < 0.05 was considered as significant and is presented as “*”.
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