As one of the major components of TME, the ECM plays multiple crucial roles during tumorigenesis. The dysregulation of ECM is a remarkable feature of cancer. Growing studies have shown that ECM-related proteins may modulate the migration and invasion of cancer cells through related signaling pathways.5,6 More importantly, the deposition, reconstruction, and cross-linking of ECM can reprogram the local microenvironment and regulate the pro- and antitumor immune responses upon the stimulation of different ECM-related proteins, leading to aberrant mechanotransduction and further malignant transformation.5,7,28 As a special type of ECM, BM also presents the major barrier cancer cells have to overcome multiple times to form metastases.8,9 Therefore, a comprehensive understanding of the dysregulation of the BM in the TME would contribute to the discovery of promising therapeutic targets for cancer treatment.
FRAS1, comprising 4010 amino acids, is encoded by the FRAS1 gene located at the chromosome 4q21.21. FREM1 comprises 2197 residues and resides on chromosome 9p22.3. In between, FREM2 consists of 3160 amino acids and is encoded by the FREM2 gene located at the chromosome 13q13.3. FRAS1 and FREM2 are produced by epithelial cells as membrane proteins, while FREM1 is produced by mesenchymal cells as a secreted protein.11 These three proteins meet together at BM and form an independent ternary complex, supposedly having a similar function to collagen VII. FRAS1/FREM share common polypeptide repetitive motifs with possible interactive and organizing functions, and contribute to embryonic epithelial–mesenchymal integrity.10,11
FRAS1/FREM family are involved in the progression of several cancers. Zhan et al.15 reported that FRAS1 knockdown reduced non-small cell lung cancer A549 cells migration and invasion ability and Umeda16 reported that knockout of FRAS1 inhibited liver metastasis of gastric cancer. The analysis of a xenograft model of human endometrial cancer found that FRAS1 might also serve as a potential diagnostic marker.29 FREM1 was reported to be associated with the favorable prognosis of breast carcinoma patients and FREM2 was reported to be associated with favorable prognosis of patients with isocitrate dehydrogenase (IDH)- wild-type glioblastoma.17,18 Additionally, the polymorphism of FRAS1 was involved in various malignancies. For example, miR-1 targeting FRAS1 was downregulated in sunitinib resistance renal cancer cell.30 The rs1910301, the promoter region of FRAS1, was found to be a candidate SNP associated with lethal prostate cancer.31 The rs150303591, the nearby genomic loci of FRAS1 was also involved in drug resistance of ovarian cancer to carboplatin.32 Yet, none of them in KIRC has been systematically studied.
Our study revealed that the mRNA and protein expression levels of FRAS1/FREM1 were significantly downregulated in KIRC tissues than in normal tissues. Besides, FRAS1, FREM1 and FREM2 mRNA and protein expressions were correlated with the clinicopathological characteristics (pathological stage, grade and tumor metastasis status) of the patients with KIRC. The expression level of FRAS1 and FREM2 were low in the matestasis of KIRC tissues and might be associated with the number of pulmonary matastases. Interestingly, FRAS1 and FREM2 expression decreased in the stage I and stage II patients who had a high propensity to metastasise, which means FRAS1 and FREM2 could be tumor metastasis markers of KIRC. In addition, we report that FRAS1/FREM expression correlated with the prognosis of KIRC. Elevated FRAS1, FREM1 and FREM2 expression levels were found to be significantly related to a better OS, DSS and PFS in KIRC. Multivariate Cox proportional-hazards regression adjusted for clinical parameters (age, gender, race, TNM stage and grade) still suggested that FRAS1 and FREM2 could be independent risk factors for better prognosis. Thus, these results above indicated that FRAS1 and FREM2 might be used as potential biomarkers of diagnosis and prognosis in KIRC.
TMB is a potential pan-cancer predictive biomarker of immune checkpoint inhibitor response in most cancers.25 MSI and MMR deficiency could also serve as an potential biomarker and predict the efficacy of immune-checkpoint inhibitors (ICI).24 Our results show that FRAS1/FREM expression were positively correlated with essential MMR signatures in KIRC. In addation, FRAS1 and FREM2 expression were positively correlated with high MSI in KIRC, while FREM1 expression was negatively correlated with low TMB in KIRC. However, the correlation coefficients between FRAS1/FREM and TMB, as well as MSI, were below 0.5, suggesting that they were not sufficient to independently predict the patient’s response to immune checkpoint blockade efficacy.
Tumor immune infiltrating cells migrate from blood to tumor tissues and can antagonize or promote tumor occurrence and development. Cancer immunotherapy activates the immune system to specifically target malignant cells33. Previous research has often focused on CD8 + cytotoxic T cells, however, CD4 + T cells have gained attention in the field, as they are not only essential to promote help to CD8 + T cells, but are also able to kill tumor cells via MHC-class II dependent recognition or via the activation of other immune cells such as macrophages. Therefore, immunotherapy approaches have shifted from only stimulating CD8 + T cells to targeting and assessing both, CD4 + and CD8 + T cell subsets and increasing numbers of clinical studies have demonstrated that targeting CD4 + T cells is safe and effective26,34,35. Our study reveal that FRAS1/FREM might play an important part in recruitment and regulation of immune infiltrating cells, especially CD4 + T cells, its corresponding subsets (Th1, Th2, Tfh and Tregs) and macrophages. It will be interesting to investigate whether FRAS1/FREM might serve as new targets for the development of various cancer immunotherapies.
Another main finding of our study is that multiple factors such as genetic changes, epigenetic regulation, transcriptional regulation and translation regulation can synergistically be the potential mechanisms through which FRAS1/FREM exerts antitumor effects. As an a central epigenetic modification of the human genome, the changes of DNA methylation in cancer have been heralded as promising targets for the development of powerful diagnostic, prognostic, and predictive biomarkers.27,36,37 Our study explored the relationship between FRAS1, FREM1 and FREM2 promoter methylation and cancer for the first time. We found that FRAS1 and FREM2 expression were significantly correlated with DNA promoter methylation and three DNA methyltransferases (DNMT1, DNMT2 and DNMT3A). This may partially explain the differential mRNA expression of FRAS1/FREM between tumor tissues and normal tissues, while the somatic mutation rates of them in KIRC were low. More importantly, the correlations of FRAS1 and FREM2 single CpG methylation levels with clinical prognosis revealed that they could serve as biomarkers of prognosis in patients with KIRC. Consistent with the conclusion stated above, the results of BP and MF by GSEA also revealed that FRAS1, FREM1 and FREM2 were mainly enriched in the process of transcriptional regulation, post-transcriptional regulation, and regulation of specific epigenetic modification enzymatic activities.
The oncogenic developmental signalling pathways such as the Notch, WNT, Hedgehog and Hippo are crucial for the development Cancer stem cells (CSC), which have important roles in tumour development, relapse and metastasis. As such, therapeutics targeting the above pathways are prime targets for anti-CSC therapy— with some success in certain tumors38,39. The crosstalk between the above pathways and other tumorigenic pathways (e.g. NF-κB, KRAS–RAF–MAPK and PI3K–AKT–mTOR) have also hinted at their profoundly complex roles in cancer38,40. Moreover, the tumor microenvironment and the ECM can regulate cell metabolism, such as glucose metabolism and lipid synthesis6,7. Indeed, aberrant activation of PI3K/AKT and Ras signaling pathways can facilitate constant glucose uptake, mTOR can also induce anabolic processes such as protein, nucleotide, and lipid biosynthesis7. Our results of KEGG and Hallmark by GSEA revealed that FRAS1, FREM1 and FREM2 can potentially impact cancer etiology or pathogenesis by functioning in oncogenic signalling pathways (TGF-β signaling, PI3K AKT mTOR signaling, Wnt-β catenin signaling, Kras signaling, Hedgehog signaling and Notch signaling) and metabolic pathways (Heme metabolism, Fatty acid metabolism, Bile acid metabolism, Xenobiotic metabolism). A better understanding of the interplay between FRAS1/FREM and the tumor microenvironment might be the key to unlock a new era of oncological treatments and proposes new therapeutic targets for KIRC.
However, there are limitations to our study. First, while bioinformatics has the advantages of large sample size, simplicity and low cost, the biases caused by the confounders might exist. Second, our analyses are mainly based on TCGA database and some results are further validated by GEO database and CPTAC database, even so, further experiments in vivo and in vitro should be still needed to confirm it clearly. Third, although a serious of function annotations and enrichment analysis were investigated in our study, the detailed molecular biological mechanisms of FRAS1/FREM in KIRC need to be further validated.
In summary, our first pan-cancer analyses indicated that the FRAS1/FREM genes and proteins are differentially expressed between tumor and normal tissues. Moreover, elevated FRAS1/FREM expression levels are significantly correlated with cancer progression (pathological stage, pathological grade and tumor metastasis status), poor survival (OS, DSS and PFS), immune infiltrations (MMR, TMB, MSI and CD4 + T cells subsets) and DNA methylation in KIRC patients, sharing the potential as efficient markers of the prognostic value of KIRC and potential targets in the development of anti-KIRC therapeutics.