Shiwani Limbu, Kara E. McCloskey
Abstract
Lung cancer makes up one-fourth of all cancer-related mortality with the highest mortality rate among all cancers. Despite recent scientific advancements in cancer therapeutics, the 5-year survival rate of lung adenocarcinoma (LUAD) cancer patients remains below 15 percent. It has been suggested that the high mortality rate of LUAD is linked to the acquisition of progenitor-like cells with stem-like characteristics that assist the whole tumor in regulating immune cell infiltration. To examine this hypothesis further, this study mined several databases to explore the presence of stemness-related genes and miRNAs in LUAD cancers. We examine their association with immune and accessory cell infiltration rates and patient survival. We found 3 stem cell-related genes, ORC1L, KIF20A, and DLGAP5, present in LUAD that also correlate with changes in immune infiltration rates and reduced patient survival rates. Additionally, the modulation in myeloid-derived suppressor cell (MDSC) infiltration and miRNA hsa-mir-1247-3p mediated targeting of tumor suppressor SLC24A4 and oncogenes RAB3B and HJURP appears to primarily regulate LUAD patient survival. Given these findings, hsa-mir-1247-3p and/or its associated gene targets may offer a promising avenue to enhance patient survivability.
Introduction
Lung cancer corresponds to 25% of all cancer deaths [1], the greatest mortality rate among all cancers [2]. The two most common types of lung cancer are lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) [3], with LUAD recently replacing squamous cell cancer as the most prevalent non-small cell cancer in the United States. Despite recent scientific advancements in the development of anti-cancer therapeutics, the 5-year survival rate of LUAD cancer patients remains less than 12% to 15% [4]. One hypothesis for the high mortality rate of LUAD is its ability to evade the body’s immune system [5]. Cancer progression is marked by the de-differentiation of tissue-specific cells and acquisition of progenitor-like cells with stem-like characteristics. These cancer stem cells (CSC) [6] are then able to assist the whole tumor [7] with proliferation, progression and propagation [8]. Moreover, stem cells and CSCs in cancers correlate with the rate and type of immune responses [9], tumor metastasis [10], multi-drug resistance [11], and low cancer patient survival [12, 13].
Methods
Data collection
We used 109 stem cell-related genes indicating tumor stemness from cancer transcriptomes using single-sample gene set enrichment analysis (ssGSEA) from cancer transcriptomes [9]. Gene and miRNA expression data of 537 LUAD tumor and 59 normal samples were collected from The Cancer Genome Atlas (TCGA) database [45], and two more microarray gene expression datasets were obtained from GEO database [46] (GSE ID: GSE40419 and GSE31210) and used as a validation cohort for gene differential expression test. Here GSE40419 contains 87 tumor samples and 77 normal samples, whereas GSE31210 contains 226 tumor samples and 20 normal samples. Tumor patient cancer stage (I, II, III, and IV), patient age, and LUAD specific patients survival duration (in months) were collected from cBioPortal [47].
Results
Stemness genes associated with cancer patient survival
Out of total 109 stemness related genes obtained from Miranda et al., 2019 [9], four (ORC1L, KIF20A, DLGAP5, and RAB3B) genes were found to be differentially expressed in LUAD cancer using datasets TCGA, GSE40419, and GSE31210, hazard ratio > 1. Benjamini and Hochberg (BH) adjusted p-value ≤ 0.05 (Table 1). Due to their likely role in promoting cancer progression, ORC1L, KIF20A, DLGAP5, and RAB3B were selected as marker genes for downstream analysis.
Stemness genes and immune cell infiltration
Three stemness-related gene (ORC1L, KIF20A, DLGAP5) positively correlated (> 0.6) with the infiltration rate of Th2 and MDSC. ORC1L negatively correlated with hematopoietic stem cell (HSC) infiltration rate (< -0.6) (Fig 1). Cox-proportional hazard Model-based survival analysis coefficients obtained from Timer2.0 indicated a higher infiltration rate of both Th2 and MDSCs in three genes (ORC1L, KIF20A, and DLCAP5) corresponding to a lower patient survival rate. RAB3B did not exhibit a correlation with any immune and accessory cell infiltration rate within the correlation cutoff range. Moreover, higher infiltration rate of HSCs corresponded to higher patient survival rate (Table 2).
Discussion
Using predefined stemness-related genes and cross-referencing these against the LUAD patients, we found 15 differentially expressed stemness-related genes in LUAD. Of these, four genes (ORC1L, KIF20A, DLGAP5, and RAB3B) negatively correlated with the probability of patient survival (Table 1 and Fig 1). We then examined the immune cell infiltration in LUAD cancer patients and found a decreased infiltration rate of HSCs and increased infiltration of MDSC and Th2 cells correlating with decreased survival probability correlating with three genes (ORC1L, KIF20A, and DLGAP5—Fig 1). Lastly, the examination of stemness-related genes and immune cell infiltration rates in LAUD patients linked the increase in expression of ORC1L with the negatively correlated infiltration rate of HSC (Fig 2C) but did not suggest a strong correlation between Th2 infiltration or other differentially expressed stemness-related genes. ORC1L is a highly conserved six subunit protein, which plays an important role in regulating initiation of DNA replication process during cell division [64] and organ development [65]. Recently, aberrations in ORC1L have been implicated in breast cancer [66], cervical cancer [67], as well as LUAD cancer progression [68].
Conclusions
As expected, our results also agreed with previous studies [22, 25–27, 30] showing that an increase in infiltration of MDSC and Th2 decreased the survival probability of cancer patients in LUAD (Fig 2). Uniquely, we found an increase in expression of ORC1L, KIF20A, and DLGAP5 stemness-related genes also positively correlated with MDSC and Th2 infiltration rates in LUAD cancer (Fig 1). Myeloid-derived suppressor cells (MDSCs) comprise a varied collection of undeveloped myeloid cells and play a crucial role in negatively modulating the immune response [71]. This regulation, combined with immune infiltration rates of T-helper Th2 cells, contributes to the advancement of tumors [71], the formation of pre-metastatic niches [72], and a decrease in the effectiveness of immunotherapy [71].
Citation: Limbu S, McCloskey KE (2023) Stemness genes and miR-1247-3p expression associate with clinicopathological parameters and prognosis in lung adenocarcinoma. PLoS ONE 18(11): e0294171. https://doi.org/10.1371/journal.pone.0294171
Editor: Hyun-Sung Lee, Baylor College of Medicine, UNITED STATES
Received: February 27, 2023; Accepted: October 26, 2023; Published: November 10, 2023
Copyright: © 2023 Limbu, McCloskey. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The data underlying the results presented in the study are available from https://github.com/Sashoss/LUAD_PLOS_data.
Funding: This work was supported by the Cancer Research Coordinating Committee (CRCC) award # C23CR5706.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: CSC, cancer stem cell; HSC, hematopoietic stem cell; LFC, Log2 Fold Change; LUAD, Lung adenocarcinoma; mRNA, messenger RNA; miRNA, microRNA; MDSC, myeloid-derived suppressor cells; TME, tumor microenvironment; Th2, Th2 T cell CD4+; TCGA, The Cancer Genome Atlas
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0294171#abstract0
