Introduction
Uterine corpus endometrial carcinoma (UCEC) is the most common gynecologic malignancy in plenty of countries, 9 out of 10 women with early-stage disease present with the symptom of postmenopausal bleeding [
1]. Over 50,000 women die from UCEC every year in the world [
2]. UCSC disrupts a lot of signal pathways including PI3K/PTEN/AKT/mTOR pathway [
3]. PI3K pathway serves a pivotal function that may have potential for defining targeted therapy for the treatment of grade 3 UCEC [
4]. The main component of PI3K pathway, PIK3CA, is a strong prognostic biomarker in UCEC and associates with disease-specific mortality [
5]. PIK3CA missense mutation is associated with unfavorable outcome in endometrioid carcinoma [
6,
7]. PIK3CA and PIK3R1 mutations were frequent and showed a strong tendency for mutual exclusivity in UCSC [
8]. The previous work on molecular mechanism of UCEC has indicated that PIK3CA played a crucial role in development of tumor.
Tumor-associated neutrophils (TANs) are phenotypically distinct from circulating neutrophils in terms of their surface protein composition and cytokines/chemokine activity, modulate the tumor immune microenvironment [
9]. TANs can perform pro-tumoral functions, strengthening tumor cell invasion and metastasis, angiogenesis, and extracellular matrix remodeling, while inhibiting the antitumoral immune surveillance [
10]. TANs recruit macrophages and Treg cells to hepatocellular carcinoma to promote their growth, progression, and resistance to sorafenib [
11], which implied the regulated role of TANs in tumor immune microenvironment. High levels of TANs have been associated with a poor prognosis in different malignances [
12]. TANs are an important component of the immune cell infiltrate in colorectal cancer and assessment of TAN infiltration may help identify patients likely to benefit from 5-FU-based chemotherapy [
13]. Furthermore, TANs can also inhibit metastatic seeding in the lung cancer through hydrogen peroxide generation [
14]. In general, the significant function of TANs has been verified in various researches.
However, until now no field research on what effects and internal mechanism PIK3CA has on TANs in UCEC have been reported. On the basis of existing literature data, we carried out studies in an effort to clarify the crucial role of PIK3CA in UCEC immune microenvironment.
Discussion
UCEC is one of the most common cancer in female worldwide. Determining what factors cause UCEC progressing predominantly has been a subject of intense interest. Meanwhile, PIK3CA was proven to be a significant molecule during the progression of UCEC. Our result showed PIK3CA expressed differently, ranging from medium to high, in UCEC patients, which suggested tumor progression could be largely influenced PIK3CA expression quantity. PIK3CA, as expected, could change the outcome of UCEC patients, leading to shorter survival time in high PIK3CA group.
A lot of genes related to PIK3CA found in our research has been reported to correlate with other cancer types, which proved the importance of PIK3CA. PIK3R4 mutation was discovered in metastatic melanoma [
22], suggested an important function in melanoma. Meanwhile, copy number gains of PIK3CA and PIK3R4 were associated with decreased survival in ovarian cancer [
23]. PIK3R4 might alter PIK3CA expression in ovarian cancer. SENP2 have not been reported in UCEC related research. However, it may play a crucial role in hypoxia-induced lung cancer progression [
24] and diagnosis or therapeutic targets for breast cancer treatment [
25]. TBCCD1, a key regulator of centrosome positioning and consequently of internal cell organization, is a new centrosomal protein [
26], and it has not been mentioned in any cancer.
Multiple kinds of cancer including thyroid cancer, prostate cancer, non-small cell lung cancer, glioma, colorectal cancer, chronic and acute myeloid leukemia, endometrial cancer and renal cell carcinoma seemed to affect by PIK3CA expression, some of which was demonstrated in a number of studies. PIK3CA may play a significant role in tumor cell proliferation, invasion, metastasis, apoptosis or cell cycle among different cancer types. It is worthwhile mentioning that PIK3CA probably regulates some common molecules or pathways in different cancer. Targeting PIK3CA to investigate and develop drugs may be a new way for better treatment of cancer, including UCEC.
Immunotherapy of cancer has become the center of attention these years. Most researches were related to T cell or B cell, whereas few reports were related to TANs. However, TANs played an important part in immune microenvironment of many cancer types. It should be pointed out that no one has investigated TANs in UCEC. Our findings suggest that fraction of TANs are significantly altered by PIK3CA expression in UCEC, which is crucial for further studies of immune microenvironment and immunotherapy in UCEC.
The novelty of the study is that it was the first time and research to find out intrinsic mechanism in which PIK3CA influence TANs in UCEC. Our result implied the potential role of PIK3CA or other components of PI3K pathway in tumor immune microenvironment and may open a new approach for clinical anti-tumor research and anti-tumor drug development.
The shortcoming of our research was that we have not tested results in vivo owing to limitation of experimental conditions. However, we proposed a new aspect in UCEC immune infiltration status, which was influenced by PIK3CA expression.
Further effort is required to confirm the specific mechanism PIK3CA engaged to alter the fraction of TANs in UCEC. There is thereby an urgent need but it is still a significant challenge to uncover the role in immune system in UCEC.
Method
Acquisition and processing of TCGA UCEC patients’ mRNA-seq data.
mRNA-seq FPKM data of TCGA UCEC patients was downloaded from The Cancer Genome Atlas. All UCEC patients were divided into two subgroups according to the expression of PIK3CA. PIK3CA low group contained 255 patients, whereas PIK3CA high group contained 256 patients. DEGs were calculated by setting log
2(Fold change) as ±1 and FDR as 0.01. Utilizing ImageGP (
http://www.ehbio.com/ImageGP/) drew Volcano plot. Heatmap was generated by R (3.3.2) pheatmap package.
GO and KEGG enrichment analysis and GSEA.
Top 500 DEGs selected to process GO and KEGG enrichment analysis. GO and KEGG enrichment analysis were performed and visualized on website (
http://enrich.shbio.com/index/ga.asp). Gene Set Enrichment Analysis (GSEA) is a computational method that determines whether an a priori defined set of genes shows statistically significant, concordant differences between two biological states. UCEC mRNA-seq data were divided into PIK3CA low group and PIK3CA high group As mentioned and performed GSEA. Several pathways were selected to analyze including Wnt Pathway, JNK MAPK Pathway, MTOR Pathway, CD40 Pathway using GSEA. NES and FDR were selected to value the pathway enrichment in each group. GSEA was performed using the GSEA 3.0 software.
Immune infiltration analysis
Immune infiltration status was analyzed by TIMER (
https://cistrome.shinyapps.io/timer/), visualized by GraphPad Prism 8. We download TCGA tumor immune estimation data from website and filtered the UCEC PIK3CA low group and PIK3CA high group data for immune infiltration analysis. Percents of B cells, CD4 T cells, CD8 T cells, TANs, macrophages and dendritic cells in PIK3CA low group and PIK3CA high group were compared using t test.
Survival analysis
Survival analysis of 542 TCGA UCEC patients was performed on Kaplan-Meier Survival Analysis [
15] (
http://kmplot.com/analysis/). 542 patients were divided into two subgroups, PIK3CA low group (
n = 213) and PIK3CA high group (
n = 329). Survival of UCEC patients with high TANs and low TANs was illustrated with the help of TCGA UCEC clinical follow-up information.
Immunohistochemical images acquisition
Images of PIK3CA antibody stained UCEC tissue were obtained on Human Protein Atlas (
https://www.proteinatlas.org/). The total number of UCEC patients screened for the analysis was 11. The histological subtypes of analyzed pathological section were endometrium adenocarcinoma.
cBioPortal
One hundred seventy mutation sites in TCGA UCEC patients PIK3CA gene were analyzed by cBioPortal (
http://www.cbioportal.org/), so as to survival status.
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