Dysregulated transient receptor potential channel 1 expression and its correlation with clinical features and survival profile in surgical non‐small‐cell lung cancer patients

Abstract Background Transient receptor potential channel 1 (TRPC1) facilitates the tumor growth, metastasis, and chemoresistance in a series of neoplasms, while its correlation with clinical features and survival profile in NSCLC patients remains elusive. Hence, this study aimed to explore this topic. Methods Totally, 192 NSCLC patients were enrolled. Protein and mRNA expression of TRPC1 in carcinoma tissue and para‐carcinoma tissue were evaluated by immunohistochemistry (IHC) assay and reverse transcription quantitative polymerase chain reaction (RT‐qPCR) assay, respectively. Results Immunohistochemistry score and mRNA expression of TRPC1 were higher in carcinoma tissue compared with para‐carcinoma tissue (both p < 0.001). Besides, increased TRPC1 IHC score (p = 0.004) and elevated TRPC1 mRNA overexpression (p = 0.016) were linked with occurrence of LYN metastasis; meanwhile, increased TRPC1 IHC score (p = 0.015) and raised TRPC1 mRNA expression (p = 0.009) were also linked with advanced TNM stage, whereas TRPC1 IHC score and TRPC1 mRNA expression were not correlated with other clinical features (all p > 0.05). Additionally, TRPC1 protein high (p = 0.007) and TRPC1 mRNA high (p = 0.015) were correlated with poor disease‐free survival (DFS) but not correlated with overall survival (OS). Moreover, multivariate Cox's proportional hazards regression analysis showed that high TRPC1 protein expression (p = 0.046) and advanced TNM stage (p < 0.001) were independently correlated with poor DFS. However, TRPC1 protein and mRNA expression were not linked with OS (both p > 0.05), while poor differentiation (p = 0.003) and advanced TNM stage (p < 0.001) were independently associated with worse OS. Conclusions TRPC1 is unregulated in NSCLC tissue with its overexpression relating to the occurrence of LYN metastasis and worse DFS in NSCLC patients.


| INTRODUC TI ON
Lung cancer is one of the main causes of cancer-related death worldwide with an estimated 2.2 million new cancer cases and 1.8 million deaths in 2020, among which 85% cases are diagnosed as non-smallcell lung cancer (NSCLC). 1,2 With the development of novel drug and the realization of precise medicine, the outcome of NSCLC is improved to some extent. 3,4 Unfortunately, a nonnegligible proportion of NSCLC patients still cannot respond to the current therapy with an unfavorable five-year survival, which ranges from 4% to 17%. 5,6 Hopefully, accumulative studies have demonstrated the prognostic potential of new biomarkers for NSCLC. To be specific, accumulating researches disclose that a series of hallmarks such as miR-155, CDK4, and MMP-14 are selected for monitoring disease progression and prognostication, [7][8][9] whereas this issue still needs further investigation; thus, the significance of detecting novel hallmarks for NSCLC should be emphasized.
Transient receptor potential channel 1 (TRPC1), a family member of non-selective cation channel proteins, mediates store-operated calcium entry. 10,11 Besides, accumulating evidences illustrate that TRPC1 may have an oncogenic potential, which facilitates the proliferation, invasion, and dedifferentiation in numerous carcinomas. 10,11 More importantly, several studies illuminate that TRPC1 is highly expressed and shows prognostic value in some carcinomas. 12,13 Detailly, TRPC1 is upregulated in breast cancer tissue and is correlated with advanced clinicopathological features as well as poor prognosis in breast cancer patients. 14,15 Likewise, TRPC1 is overexpressed and links with unsatisfying clinicopathological characteristics or poor prognosis in patients with colorectal cancer and gastric cancer. [16][17][18] However, its clinical involvement in NSCLC remains elusive. Hence, the current study aimed to investigate the aberrant expression of TRPC1 and its relation to clinical features and survival profile in NSCLC patients.

| Participants
With the approval of the Institutional Review Board of Huangshi

| Data collection
Clinical data were abstracted from the medical records, which included demographics, comorbidities, tumor markers, surgical information, and adjuvant chemotherapy regimens. Surgical resection was operated for the corresponding patients according to the disease status: for TNM stage I-II patients, pulmonary lobectomy plus lymph node dissection or pneumonectomy plus lymph node dissection was performed; for TNM stage III patients, pulmonary lobectomy, pneumonectomy, or lymph node dissection were performed.

| Sample collection
All 192 patients' FFPE samples of carcinoma tissue and paracarcinoma tissue were collected to evaluate the expression of TRPC1 protein by IHC assay. Meanwhile, 110 of 192 patients also had fresh-frozen carcinoma tissue and para-carcinoma tissue samples, which were obtained to determine the expression of TRPC1 mRNA by reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay.

| IHC assay
The procedure of IHC assay was in accord with a previous study. 19 In brief, the goat polyclonal anti-TRPC1 antibody (1:150; Abcam) was applied as primary antibody, and the rabbit anti-goat IgG (H&L) (1:2000; Abcam) was applied as secondary antibody. The experiment was carried out in strict accordance with the instructions.

| RT-qPCR assay
RT-qPCR was performed to evaluate the TRPC1 mRNA expression.
The total RNA was extracted by TRIzol™ Reagent (Thermo Fisher Scientific). RNA was reversely transcribed to cDNA using iScript™ cDNA Synthesis Kit (with random primer; Bio-Rad). Moreover, qPCR was performed using QuantiNova SYBR Green PCR Kit (Qiagen).
Primers were designed in accordance with a previous study. 20 Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal references; meanwhile, TRPC1 expression was calculated using the 2 -ΔΔCt method.

| Definition
According to the total IHC score, the expression of TRPC1 protein was categorized as high expression (IHC score >3) and low expression (IHC score ≤3). According to the result of RT-qPCR assay, the expression of TRPC1 mRNA was classified as high expression (>2.268) and low expression (≤2.268) by its median value (2.268) in carcinoma tissue. and adjuvant chemotherapy (yes vs. no)) were incorporated in multivariable analysis using forward stepwise (conditional LR) method to screen out independent prognostic factors. All tests were twosided, and p < 0.05 indicated a statistical significance.

| Clinical characteristics
Totally, 192 NSCLC patients were analyzed with a mean age of 61.9 ± 11.6 years (

| Protein and mRNA expression of TRPC1
TRPC1 protein expression in carcinoma tissue as well as paracarcinoma tissue was detected by IHC assay ( Figure 1A). To be specific, TRPC1 IHC score was higher in carcinoma tissue compared with para-carcinoma tissue (t = 13.822, p < 0.001, Figure 1B); meanwhile, ROC analysis disclosed that TRPC1 IHC score exhibits a good capability in distinguishing carcinoma tissue from para-carcinoma tissue (AUC: 0.775, (95% confidence interval (CI): 0.729-0.821), Figure 1C).
Additionally, subgroup analysis was performed on account of whether patients underwent adjuvant therapy, which illuminated that TRPC1 protein high (χ 2 = 3.950, p = 0.047, Figure S1A) and TRPC1 mRNA high (χ 2 = 4.674, p = 0.031, Figure S1b) were linked with poor accumulating DFS in patients undergoing adjuvant chemotherapy, whereas other subgroup analysis revealed that there was no more correlation between TRPC1 expression and survival profiles ( Figure S1C-H). independently associated with worse OS ( Figure 5B).

| DISCUSS ION
TRPC1 serves as an iron channel facilitating Ca 2+ entry in the store- Two previous studies reveal that TRPC1 is highly expressed in NSCLC tissue. 32,33 In the present study, it was illustrated that TRPC1 was not only overexpressed in NSCLC tissue but also distinguished NSCLC tissue from normal tissue. Possible explanation could be that: the overexpression of TRPC1 might reflect the faster speed of cell proliferation; meanwhile, the malignant proliferation speed of NSCLC cells is faster than that of normal cells. Therefore, TRPC1 is overexpressed in NSCLC tissue.
It is illustrated that TRPC1 overexpression is linked with larger tumor size, the occurrence of lymph node metastasis and elevated TNM stage in breast cancer patients. 14 Collectively, TRPC1 is unregulated in NSCLC tissue with its overexpression relating to the occurrence of LYN metastasis and worse DFS in NSCLC patients, which suggests that it could serve as a potential index for prognostication in NSCLC patients.

ACK N OWLED G M ENTS
None.

CO N FLI C T O F I NTE R E S T
The authors of this work have nothing to disclose.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.