Aberrant TRPC1 expression reflects stromal cervical invasion, lymphovascular invasion, elevated FIGO stage, and poor survival in resectable endometrial carcinoma patients

Abstract Background Transient receptor potential channel 1 (TRPC1) promotes tumor growth and metastasis in endometrial carcinoma (EC) cell lines, whereas its clinical role in EC management remains unclear. Therefore, this study aimed to investigate the association of TRPC1 protein expression with the clinical features and survival of EC patients, then was further validated by TRPC1 mRNA measurement and data from The Human Protein Atlas. Methods TRPC1 protein expression in tumor tissues and normal endometria of 176 resectable EC patients was determined using immunohistochemistry. Besides, TRPC1 mRNA expression of partial patients (n = 80) was detected using RT‐qPCR. Additionally, survival data from The Human Protein Atlas (derived from The Cancer Genome Atlas [TCGA]) was analyzed. Results TRPC1 protein expression was up‐regulated in tumor tissue compared with normal endometrium (p < 0.001). Up‐regulated TRPC1 protein expression was associated with stromal cervical invasion (p = 0.044), lymphovascular invasion (p = 0.032), and increased federation of gynecology and obstetrics (FIGO) stage (p = 0.005). Tumor TRPC1 protein high was linked with shortened accumulating disease‐free survival (DFS) (p = 0.009) and overall survival (OS) (p = 0.026), which were also confirmed by multivariate Cox's regression analysis (both p < 0.050). Further, TRPC1 mRNA validation disclosed that TRPC1 mRNA high was related to shortened accumulating DFS (p = 0.038) and exhibited a correlating trend with declined OS (lacked statistical significance) (p = 0.162). Meanwhile, survival analysis on the data from The Human Protein Atlas (derived from TCGA) also exhibited that TRPC1 mRNA high was correlated with reduced accumulating OS (p < 0.001). Conclusion Our findings support TRPC1 as a prognostic biomarker in resectable EC patients.


| INTRODUC TI ON
Endometrial carcinoma (EC), representing 90% of uterine cancer, is one of the most common gynecologic malignant diseases worldwide, which can be divided into type I (estrogendependent, accounting for 85% of EC cases) and type II (estrogenindependent). 1-3 In China, the incidence of EC is increasing annually with 63,400 new cases recently, which might due to the elevated prevalence of risk factors of EC obesity, hypertension, and diabetes (the main risk factors of EC). 4,5 Currently, the mainstay of treatment in most EC patients is total hysterectomy and bilateral salpingo-oophorectomy; for patients who are not willing/ suitable for surgery, conservative treatment including chemotherapy, radiation therapy, and hormonal therapy, is recommended. 6,7 However, despite the relatively delightful general prognosis of EC patients, a few patients still suffer from a high risk of recurrence and poor prognosis. [8][9][10] Consequently, finding potential biomarkers reflecting the long-term survival can provide some reference for the individualized adjuvant-therapy selection of postoperative EC patients.
Transient receptor potential channel 1 (TRPC1) is a kind of voltage-independent cation channel protein located on the cell membrane, which regulates calcium ions (Ca 2+ ) influx and plays an oncogenic role in some solid cancers through activating calmodulin-mediated phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling axis, such as colorectal cancer, breast cancer, thyroid cancer, and pancreatic cancer. [11][12][13][14] For instance, one study identifies that TRPC1 overexpression promotes tumor growth and metastasis in human colorectal cancer cells. 13 Another study finds that TRPC1 is positively related to the clinical stage and serves as an independent risk factor for metastasis in breast cancer patients. 14 Notably, some studies observe that TRPC1 induces epithelial-mesenchymal transition (EMT) in EC cell lines, which implies that TRPC1 might be involved in EC pathogenesis. 15,16 Nevertheless, the clinical role of TRPC1 in EC management has not been reported yet.
Hence, this study aimed to investigate the association of TRPC1 protein expression with the clinical features and survival of EC patients, then was further validated by TRPC1 mRNA measurement and data from The Human Protein Atlas derived from The Cancer Genome Atlas (TCGA) analysis.

| Patients
This study retrospectively analyzed 176 EC patients who underwent surgical resection between January 2015 and December 2020.
Patients who met the following criteria were eligible for inclusion (a) were diagnosed as EC by histopathological examination; (b) were more than 18 years old; (c) received surgical resection; (d) had avail-

| Detection of TRPC1 protein expression
TRPC1 protein expression was examined by immunohistochemistry (IHC), and the experimentation of the IHC assay was in accord with a previous study. 17 Briefly, the collected FFPE specimens were cut into slides. The slides were depolymerized in xylene, rehydrated, and then heated in a citric acid buffer at pH 6 to expose antigens. Sequentially.
The slides were incubated with goat anti-TRPC1 antibody (dilution 1:150; Abcam) as the primary antibody and rabbit anti-goat IgG (H&L) (dilution 1:2000; Abcam) as the second antibody. After incubation, diaminobenzidine and hematoxylin were used for staining. The results of the IHC assay were semi-quantitatively evaluated using a light microscope by 2 investigators who were blinded to the patient's clinical data according to the density and intensity of stained cells.
The density was scored as 5 grades (score 0-4), and the intensity of stained cells was scored as 4 grades (score 0-3). The final score of the IHC assay was 12, which was a product of the density score and the intensity score. The final score of the IHC assay was a product of the density score and the intensity score, ranging from 0 to 12.

| Clinical characteristics
One hundred and seventy-six EC patients with a mean age of 59.9 ± 9.8 years were recruited in this study, among which, 25

| TRPC1 in patients with different menopausal statuses
The correlation of TRPC1 with the menopausal status of EC patients was relatively weak (r s = −0.395, p = 0.693, Table 2). For further investigating the TRPC1 expression in patients with different menopausal statuses, a subgroup analysis was performed, which showed that TRPC1 protein expression was elevated in tumor tissue compared to normal endometrium of both pre-menopause (IHC score: 5.1 ± 2.9 vs. 2.7 ± 0.9, p = 0.011, Supplementary Figure S1A) and post-menopause (IHC score: 5.4 ± 3.0 vs. 2.7 ± 1.8, p < 0.001, Figure S1B) EC patients.

| Correlation of TRPC1 protein expression with survival
Tumor TRPC1 protein high was linked with shortened accumulating DFS (P = 0.009, Figure 2A) and OS (p = 0.026, Figure 2B)

| Further validation of TRPC1's prognostic value
For validating the prognostic value of TRPC1, this study further de-  Figure 3A).
Tumor TRPC1 mRNA high was related to shortened accumulating DFS (p = 0.038, Figure 3B) and exhibited a correlating trend with declined OS (lacked statistical significance) (p = 0.162, Figure 3C) in EC patients. Increased TRPC1 mRNA expression was associated with lymphovascular invasion (p = 0.039) and elevated FIGO stage (p = 0.049) in EC patients (Supplementary Table S1). Protein Atlas (derived from TCGA), which disclosed that TRPC1 mRNA high was correlated with reduced accumulating OS in EC patients (p < 0.001, Figure 4).

| DISCUSS ION
TRPC1, binding to stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel protein 1 (ORAI1), works as a crucial store-operated calcium ions (Ca 2+ ) channel; meanwhile, its aberrant expression has been identified in several solid cancers. 14,19 For instance, one previous study shows that TRPC1 expression in breast cancer tissues is higher than that in normal breast tissues. 14 Another study discloses that TRPC1 is up-regulated in carcinoma tissue compared with para-carcinoma tissue in non-small cell lung cancer patients. 19  Additional in vivo and in vitro studies were necessary to explore whether TRPC1 could be recognized as a good therapeutic target for EC.
In conclusion, TRPC1 serves as a prognostic biomarker, whose overexpression reflects stromal cervical invasion, lymphovascular invasion, elevated FIGO stage, and poor survival in resectable EC patients.

ACK N OWLED G EM ENTS
None.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

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.