Lysophosphatidic Acid Receptor 6: A Prognostic Biomarker for Lung Adenocarcinoma via Correlating Immune Infiltration

Abstract

Background

LPAR6 is the most recently determined GPCR of LPA, and very few of study have demonstrated the performance of LPAR6 in cancers. Moreover, the relationship of LPAR6 to prognosis potential and tumor infiltration immune cells in different cancers still unclarified.

Methods

The mRNA expression of LPAR6 and its clinical characteristics were evaluated on various databases. The association between LPAR6 and immune infiltrates of various types of cancer were investigated via TIMER. IHC for LPAR6 in LUAD and LUSC tissue microarray with patients’ information was detected.

Results

We constructed a systematic prognostic landscape in various types of cancer base on the mRNA expression level. We enclosed that higher LPAR6 expression level was associated with better OS in some types of malignancy. Moreover, LPAR6 significantly affects the prognostic potential of various cancers in TCGA, especially in lung cancer. Tissue microarray’s results demonstrated that higher protein level of LPAR6 was correlated with better overall survival of LUAD rather than LUSC cohorts. Further research found that the underlying mechanism of this phenome might be the expression level of LPAR6 was positively associated with infiltrating statuses of devious immunocytes in LUAD rather than in LUSC, that is, LPAR6 expression potentially contributes to the activation and recruiting of CD8 + T, naive T, effector T cell and natural killer cell and inactivates Tregs, decrease T cell exhaustion and regulate T-helper cells in LUAD.

Conclusions

Our discovery implies that LPAR6 is associated with prognostic potential and immune-infiltrating levels in LUAD. These discoveries imply that LPAR6 could be a promising biomarker for indicating prognosis potential and immune infiltration level in LUAD cohorts.

1. Introduction

Lung cancer is one of the most common malignancies around the world, and metastasis is a crucial biological procedure leading to a poor prognosis [1]. It is the top one diagnosed malignancy in China and the second most common malignancy in the U.S., also the leading cause of cancer-related deaths both in China and the U.S. [2]. Scientists have made great efforts to treat various types of lung cancer, but there is still a large amount of time and effort to do. According to histopathological classification, lung cancer could be catalogued into two broad subtypes, non-small-cell lung cancer (NSCLC) and small cell lung cancer (SCLC), and the NSCLC is more prevalent [3]. Lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) are the first and the second most common subtype NSCLC respectively [4]. Surgery is the primary treatment option during the early stages of the NSCLC, while in late stages, surgery is combined with chemotherapies, and/or radiotherapy [4]. However, despite these treatment procedures, the prognosis of the patients remains not good, also the post-treatment recurrence is the main cause of the disease, the total 5-year survival rate for all stages is only 16.6% [4].

NSCLC has been regarded as a kind of non-immunogenic disease in the past twenty years. However, more and more knowledge of tumor immune interactions has opposed this model in lung cancer and other types of malignancy. Immune-related interaction mechanisms act as a crucial role in oncogenesis and development, and immune therapy is considered a promising approach for cancer treatment [4, 5], based on this, scientists are attempting to employ the body’s own immune system to fight and prevent malignancies [6]. Recently, immunotherapies, including adoptive cell transfers, monoclonal antibodies and vaccines, have become more and more applied to the clinic treatment of many types of cancers, for example, melanoma, and more recently for lung cancer [7]. During the last decade, the finding of antibodies that target the immune checkpoints has revolutionized the treatment of NSCLC, such as PD-1 and PD-L1 [8], and these two therapy approaches (PD-1 and PD-L1), has demonstrated promising anti-tumor performance in NSCLC and melanoma [9–11]. In addition, more and more research has demonstrated that TILs (tumor-infiltrating lymphocytes) play a key role in modulating the response to chemotherapy and heighten the clinical prognosis potential of various types of cancer [12, 13], such as tumor-associated macrophages (TAMs) [14–16] and tumor-infiltrating neutrophils (TINs), they also associate with the prognosis [17–20]. So, it is an essential and urgent requirement for the explanations of the immunophenotypes of tumor immune interactions and the identification of new immune therapy targets for lung cancers.

LPA is a kind of lipid that involved in the proliferation of tumor cells via its G-protein coupled (GPC) receptors [21, 22] and one of their receptors-LPAR6 is a newly identified receptor of LPA [23, 24], and it has been demonstrated to be related to many types of tumor, including prostate [25], liver [26, 27], colorectal [28, 29] and pancreatic cancer [30]. But the function of LPAR6 remains highly controversial since in colorectal cancer, the scientists found that LPAR6 might act as a tumor suppressor whereas act as a facilitator in the other types of tumors [25–27, 30, 31]. All these indicate that LPAR6 plays a key role in cancer, but the relationship between LPAR6 and tumor biology and the underlying mechanism involved is still not well understood.

Bioinformatics is an emerging procedure that supports us to make full usage of numerous high throughput data to analyze the level of specific genes in various types of cancer [31]. So in this work, we investigated the mRNA expression level of LPAR6 and the correlation with prognosis patterns of cancer patients in databases. In addition, we analyzed the correlation of LPAR6 with tumor-infiltrating immune cells (TIICs) in various tumor microenvironments via TIMER. Moreover, IHC staining for LPAR6 in two separate lung cancer cohorts with patients’ information was detected to analyze the correlation of the expression of LPAR6 and the clinicopathological parameters of lung cancer.

All these discoveries shed light on the crucial role of LPAR6 in lung cancers as well as provide a potential correlation and the mechanism involved between LPAR6 and tumor-immune interactions.

2. Materials And Methods

3. Results

4. Discussion

LPA receptors are GPCR that bind to the LPA and trigger multiple downstreaming cellular responses, including cell proliferation, cytoskeletal rearrangements, apoptosis and motility [48–50]. Previously, five LPA receptors (LPAR1-5) are well characterized and extensively studied [51]. LPAR6 is a recently determined GPCR, alias as ARWH1, HYPT8, LAH3, P2RY5, at first was considered as purinergic receptor P2Y5 that involved in inherited hair loss [23, 52]. Although LPAR6 has not been extensively studied, it was reported that the LPAR6 suppresses tumor cell migration in colorectal cancer [28], and the expression of LPAR6 was decreased in P53-mutated cases [29]. It was also reported that the LPA axis plays an important role in HCC by recruiting and trans-differentiating of peritumoral fibroblasts into TAMs [53, 54]. This offers scientists a promising hint that LPAR6 is involved in the TME. Immunotherapy is a new genre of treatment for patients and has a tightly association with TME [29].

In this study, we announced that different expression levels of LPAR6 are associated with the prognostic potential in various cancer types. Higher level of LPAR6 is associated with a better prognosis in three types of cancers, including liver cancer, lung cancer and breast cancer. Moreover, our data demonstrated that the immune infiltration levels and diverse immune marker sets of the different subtypes of lung cancers (LUAD and LUSC) are associated with the expression level of LPAR6. To this end, our study provides insights into elucidating the potential role of LPAR6 in tumor immunology and its usage as a biomarker and novel therapy target for LUAD.

In this work, we determined the LPAR6 expression levels and constructed a systematic prognostic landscape in various types of cancers by using independent datasets in Oncomine and 33 type cancers of TCGA data in GEPIA2. The variation expression level of LPAR6 between cancer and normal tissues was observed in many cancer types. Based on the Oncomine database, we found that LPAR6, compared to normal tissues, was highly expressed in brain and CNS, kidney, gastric cancer, leukemia, lymphoma, liver and pancreatic cancer while some data sets showed that LPAR6 has a lower mRNA expression level in bladder, breast, cervical, colorectal, esophageal, lung and prostate cancer (Fig. 1A). However, the redetermination of the TCGA data demonstrated that LPAR6 expression was higher expressed in ESCA, KIRC, KIRP and THCA, but significantly lower expressed in BLCA, COAD, BRCA, HNSC, KICH, PRAD, LUAD, UCEC, READ and slightly lower in LIHC compared with adjacent normal tissues (Fig. 1B). The vary in the expression levels of LPAR6 in different types of cancer among various databases might be a reflection in data collection approaches and underlying mechanisms involved in different biological properties. Nevertheless, in these databases, we found similar prognostic associations between LPAR6 expression in bladder, breast, cervical, colorectal, esophageal, lung and prostate cancers. Investigation of the TCGA database enclosed that the higher LPAR6 expression level is correlated with better prognostic potential in ACC, LGG, SKCM (Supplementary-Figure2). Furthermore, the determination of patient cohorts from PrognoScan and Kaplan-Meier Plotter demonstrated a high level of LPAR6 expression is correlated with better prognosis in breast, lung, bladder, colorectal, eye and ovarian cancer (Fig. 2). In two datasets of PrognoScan, high LPAR6 expression levels could be considered as an independent risk factor for better prognosis in LUAD. Moreover, a high level of LPAR6 expression was shown to be correlated with a better prognosis of LUAD in the early stage with the lowest HR [0.27 (0.17–0.42)] for a better OS when LPAR6 was highly expressed in LUAD, rather than in LUSC. These together strongly suggest that LPAR6 could be a prognostic biomarker in LUAD.

Another crucial aspect of this work is that the mRNA expression level of LPAR6 is correlated with diverse immune infiltration levels in cancer, especially in LUAD. Here, we demonstrate that there’s a strong positive correlation between the infiltration level of T cells (CD8 + and CD4+), neutrophils, macrophages and DCs and LPAR6 expression in LUAD (Figs. 3A, 3C). Moreover, the correlation patterns of the infiltration level are different in two kinds of lung cancers (LUAD and LUSC). The correlation between LPAR6 expression and the marker genes of immune cells implicates the role of LPAR6 in regulating tumor immunology in these types of cancers. A possible explanation for this striking effect might be that LPAR6 orchestrates the function of multiple immune marker gene sets. This supports the argument that the LPAR6 expression levels are important contributors to human malignancies and indicating the prognosis of specific types of cancer.

Firstly, gene markers of M1 macrophages such as PTGS2 and IRF5 show significant correlations with LPAR6 expression in LUAD respectively (Tables 2). Since macrophages are functionally plastic cells. Type 1 macrophages (M1) producing type 1 cytokines prevent tumors from developing, whereas type 2 macrophages (M2) inducing type 2 cytokines facilitate tumor growth. Especially in the tumor tissue of LUAD, both NOS2 and IRF5 show significant correlations with LPAR6 expression and PTGS2 shows a significant correlation with LPAR6 expression in the tumor tissue (Supplementary-Table 4). These results reveal the potential regulating role of LPAR6 in de-polarization macrophages against tumor that activated macrophages can be re-polarized into opposite functional phenotypes by microenvironmental modifications and then inhibit tumor growth.

Secondly, our results indicated that LPAR6 has the potential to activate CD8 + T cell, naive T-cell, effector T-cell and natural killer cell and inactivate Tregs, and decrease T cell exhaustion. CD8A, a crucial surface protein on T cells, is highly correlated with LPAR6 expression in LUAD which are types of cancers with better prognosis. And CD8A did not demonstrate a significant correlation pattern in LUSC (Table 3). This pattern also occurs with the general T cell markers such as CD3D, CD3E, CD2 and most markers of naive T-cell, effector T-cell, effector memory T-cell and natural killer cells. Such as LEF1 which has been proved to be a predictor of better treatment response in AML, because high LEF1 expression level was associated with favorable relapse-free survival in patients and predicted a significantly better overall survival for AML patients [55].

Thirdly, different correlation patterns can be found between LPAR6 expression and the regulation of several markers of T helper cells (Th1, Th2, Tfh, and Th17) in these different cancers. IFN-g is a Th1 cytokine with both pro-and anti-cancer properties [56] which are highly correlated with LPAR6 expression in LUAD, whereas did not demonstrate significant correlations in LUSC (Table 3). IL-13 is an important immunoregulatory cytokine mainly produced by activated type II helper T cells and is widely involved in tumorigenesis and development, fibrosis and inflammation [57, 58]. We found that IL-13 is highly correlated with LPAR6 expression in LUAD, but did not demonstrate significant correlations in LUSC (adjusted by purity), and a similar situation is in the IL-21. So these would be explanations that why LPAR6 indication poor prognosis in LUSC and a better prognosis in LUAD.

All these correlations above could be indications of a potential mechanism where LPAR6 regulates T cell functions in LUAD. Together these findings suggest that the LPAR6 plays an important role in the recruitment and regulation of effective T cells infiltrating in LUAD leading to a better prognosis.

5. Conclusions

In this study, we offered a potential explanation for the mechanism that why LPAR6 expression correlates with immune infiltration level and better prognostic potential in some specific types of cancer, especially in LUSC. Hence, the interactions between LPAR6 and the immunocytes in the tumor microenvironment could be a potential mechanism for the correlation of LPAR6 expression with immune infiltration level and better prognosis in LUAD patients.

Abbreviations

Declarations

Ethics approval and consent to participate

This project was permitted by Independent Ethics Committee of Shanghai Jiao Tong University School of Medicine.

Consent for publication

Not applicable

Availability of data and materials 

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Conflict of interest

The authors declare there is none conflicts of interest. This research conforms to all the laws and ethical guidelines that apply in the country where I carried it out.

Authors' contributions

J. H. contributed to idea, conception, and study design. J. H. collected and analyzed the datasets, performed TMA analysis. J. H., M.M. and R.G. wrote the manuscript and generating the figures. H. W. revised and proofread the article.

Funding

This project is funded by the grants of National Natural Science Foundation of China (NSFC 81702730) and Start-up Plan for New Young Teacher of SHSMU (KJ30214190026) of JH and National Natural Science Foundation of China (NSFC 81630086), National Key Research & Development Program of China (2018YFC2000700) of HW.

Acknowledgments

We would like to thank to the funder.

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