CRC has a high morbidity and mortality rate, which urgently requires a robust molecule to achieve early diagnosis and treatment. The LIMK protein family includes LIMK1 and LIMK2. It was reported that LIMK1 was highly expressed in a variety of tumors and is related to patient prognosis. Although studies have shown that LIMK1 is up-regulated in CRC and causes poor prognosis. And the related mechanism of LIMK1 regulating CRC progression has been studied. But, the relationship between LIMK1 and tumor immune microenvironment in CRC has not been explored. In this article, we not only discuss the relationship between LIMK1 and CRC from several aspects such as expression level in tumor samples, clinicopathological features, but also the correlation with immune cell infiltration and the expression of immune checkpoint genes. First, we confirmed that the expression of LIMK1 mRNA and protein was higher in CRC tissues than in normal tissues. Subsequently, we found that higher LIMK1 expression was correlated with poor prognosis of CRC, regardless of OS or DSS. Moreover, LIMK1 can influence immune cell infiltration and immune checkpoint expression in CRC. In summary, LIMK1 may be a valuable and promising biomarker for the diagnosis of CRC. And our findings laid the foundation of that LIMK1 promotes CRC progression from the mechnisim of regulating tumor immune microenvironment.
LIMK1, a serine protein kinase, is a member of the LIMK kinase family and plays a crucial role in the reorganization of actin and microtubule depolymerization. Currently, research on LIMK1 has also focused on oncology because of its vital role in promoting tumor cell proliferation, invasion, and metastasis. It has been reported that LIMK1 expression was upregulated in several kinds of human cancers especially in highly malignant neoplasm, such as lung adenocarcinoma, breast cancer and prostate cancer[11, 19, 20]. Furthermore, upregulated LIMK1 is often associated with poor patient prognosis. At present, lots of studies provied that LIMK1 was a significent biomarker which portends a poor prognosis in numerous cancer types. A study by Huang et al. indicated that upregulation of LIMK1 was highly associated with lymph node metastasis and shortened biochemical-free survival in prostate cancer. In ovarian carcinoma, it was reported that high levels of LIMK1 indicate poor tumor differentiation and disease severity. In gastric cancer, You et al. confirmed that with the upregulation of LIMK1, the size of the primary tumor was larger and the number of lymph node metastases was greater . Moreover, it has been confirmed that reducing the expression of LIMK1 can delay tumor growth and peritoneal metastasis in vivo. In CRC research, upregulation of LIMK1 enhanced the invasiveness of CRC cells in vitro and in vivo. In the present study, we demonstrated that LIMK1 mRNA is highly expressed in a variety of tumor tissues, including CRC. LIMK1 was highly expressed in tumor tissues with CRC, whether in paired or unpaired samples. Subsequently, we further indicated that the protein expression of LIMK1 was upregulated in CRC tumor tissues compared to that in adjacent tissues. Moreover, we confirmed that the upregulation of LIMK1 was associated with poor survival. Regarding clinicopathological characteristics, a significantly positive correlation was found between LIMK1 and lymphatic invasion, high TNM stage. Thus, LIMK1 might be more advantageous in the detection of metastatic CRC compared to previous screening methods such as CEA, FOBT, CA199, etc. To prove the accuracy and sensitivity of LIMK1 in the diagnosis of CRC, we performed ROC curve analysis. Our results indicated that the AUC value of LIMK1 was obviously high in the detection of CRC, with 98% sensitivity and 81.9% specificity. Although further studies are needed, LIMK1 might serve as a promising marker for identifying CRC with a poor prognosis.
LIMK1 plays an important role in several signaling pathways, especially those related to tumors. In our study, we analyzed the top 10 co-expression genes that were most related to the expression of LIMK1, of which CFL1, RHOB, and RHOC had the highest correlation. In addition, we found that LIMK1 is involved in a variety of biological processes in the following functional annotations. Zeng et al.  found that knockdown of Rho GDP dissociation inhibitor 2 (RhoGDI2) can downregulate the malignant biological behavior of gastric cancer cells via the Rac1/Pak1/LIMK1 pathway. In pancreatic cancer, other researchers have indicated that DEP domain-containing protein 1 B (DEPDC1B) can also stimulate cell migration and invasion through this pathway. In functional annotations of LIMK1, we found that LIMK1 was mainly focused on lamellipodium, ruffle, and cell leading edge in CC. The accumulation of LIMK1 in these cellular components was likely to indicate that it was related to the migration and metastasis of tumor cells. Vainer et al. found VICKZ accumulated at the leading edge of SW480 CRC cells which facilitated the formation of surface morphologies required for cell migration. Rho GTPase-activating protein 5 (ARHGAP5) promoted EMT to accelerate tumor metastasis through regulating RhoA activity in CRC cellwhich corroborated our results of LIMK1 enrichment in MF. And axon guidance, regulation of actin cytoskeleton, and pathogenic E. coli infectionpathways were inovoled in the process of the occurrence or metastasis in CRC. Therefore, combined with our results, we speculate that the upregulated LIMK1 may directly or indirectly affect the biological functions of tumors by regulating these proteins and pathways.
Another novelty of this study was that LIMK1 expression was associated with immune cell infiltration in CRC. Several recent studies have shown that LIMK1 may be involved in the regulation of the immune microenvironment. In T cell immunity, HIV triggers actin polymerization through the LIMK1-cofilin signaling pathway. In NK cells, Duvall et al.  identified LIMK1 as a vital medium to regulate cytoskeletal rearrangement. However, the role of LIMK1 in the immune tumor microenvironment has not been extensively discussed. Only some researchers have found that the expression of LIMK1 is enriched in immune (CMS1) subtypes of CRC. In our study, we found that LIMK1 was associated with multiple tumor-infiltrating immune cells in CRC. Among them, LIMK1 was most closely related to CD4+ T cells, macrophages, and dendritic cells. In further subgroup analysis, we found that LIMK1 had stronger correlations with M2 macrophages and Treg cells, according to the analysis of cell surface markers. Among these markers, FOXP3 and CD163 showed the highest correlation. FOXP3 is a crucial surface protein in Treg cells, which inhibits cytotoxic T cells from attacking tumor cells. Research  showed that macrophages with high expression of CD163 often predicted poor survival prognosis in a variety of tumors. Inspired by immune-related genes, we hypothesized that immune checkpoint genes are related to LIMK1 expression levels. Thus, these results indicate that LIMK1 may be able to regulate the tumor immune microenvironment in some ways. Although more experiments are needed to confirm these speculations, the results suggest that LIMK1 has a significant relationship with immune cell infiltration in CRC.
However, there are several limitations to our study. First, we only used the online shared database to analyze the expression of LIMK1 and clinicopathological features of CRC. We must admit that there are differences in chip consistency in the database. It is important to verify the results using more clinical data. And Zang et al. showed that imbalanced LIMK1 and LIMK2 expression led to CRC progression and metastasis. Thus, it is appropriate to think LIMK1 and LIMK2 as common detection and research targets in future studies of the LIMK family. Second, the relationship between LIMK1 and tumor-infiltrating cells needs to be further confirmed by experiments in vivo or in vitro.
Taken together, we proved that LIMK1 was upregulated in CRC, and its upregulation trend was closely related to tumor lymph node metastasis and pathological staging in this study. Moreover, we verified the potential relationship between LIMK1 and tumor-infiltrating lymphocytes in CRC for the first time. This indicates that LIMK1 is likely to be a powerful indicator for the diagnosis and treatment of CRC.