Role of INSL4 Signaling in Sustaining the Growth and Viability of LKB1-Inactivated Lung Cancer

Abstract

Background The LKB1 tumor suppressor gene is commonly inactivated in non-small cell lung carcinomas (NSCLC), a major form of lung cancer. Targeted therapies for LKB1-inactivated lung cancer are currently unavailable. Identification of critical signaling components downstream of LKB1 inactivation has the potential to uncover rational therapeutic targets. Here we investigated the role of INSL4, a member of the insulin/IGF/relaxin superfamily, in LKB1-inactivated NSCLCs. Methods INSL4 expression was analyzed using global transcriptome profiling, quantitative reverse transcription PCR, western blotting, enzyme-linked immunosorbent assay, and RNA in situ hybridization in human NSCLC cell lines and tumor specimens. INSL4 gene expression and clinical data from The Cancer Genome Atlas lung adenocarcinomas (n = 515) were analyzed using log-rank and Fisher exact tests. INSL4 functions were studied using short hairpin RNA (shRNA) knockdown, overexpression, transcriptome profiling, cell growth, and survival assays in vitro and in vivo. All statistical tests were two-sided. Results INSL4 was identified as a novel downstream target of LKB1 deficiency and its expression was induced through aberrant CRTC-CREB activation. INSL4 was highly induced in LKB1-deficient NSCLC cells (up to 543-fold) and 9 of 41 primary tumors, although undetectable in all normal tissues except the placenta. Lung adenocarcinomas from The Cancer Genome Atlas with high and low INSL4 expression (with the top 10th percentile as cutoff) showed statistically significant differences for advanced tumor stage (P < .001), lymph node metastasis (P = .001), and tumor size (P = .01). The INSL4-high group showed worse survival than the INSL4-low group (P < .001). Sustained INSL4 expression was required for the growth and viability of LKB1-inactivated NSCLC cells in vitro and in a mouse xenograft model (n = 5 mice per group). Expression profiling revealed INSL4 as a critical regulator of cell cycle, growth, and survival. Conclusions LKB1 deficiency induces an autocrine INSL4 signaling that critically supports the growth and survival of lung cancer cells. Therefore, aberrant INSL4 signaling is a promising therapeutic target for LKB1-deficient lung cancers.

Identification of an immune prognostic 11-gene signature for lung adenocarcinoma

“…HLA-DOB, which is anchored in the membrane, are expressed in antigen presenting cells. A research reported that aberrant INSL4 signaling may act as a promising therapeutic target for LKB1-deficient NSCLC ( Yang et al, 2018 ). Accolla et al, (2019) constructed the vaccination with CIITA-tumour cells to facilitate the triggering and persistence of anti-tumour cells to enhance the immune system.…”

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“…INSL4 upregulation was previously identified in a breast cancer cell subclone with increased invasiveness through in vitro selection ( Brandt et al, 2002 , 2005 ). INSL4 was also discovered as a novel target downstream of LKB1 deficiency ( Yang et al, 2018 ). INSL4 signaling is a potential vital for LKB1-inactivated non-small-cell lung carcinoma, and its introduction helps to develop novel and effective anti-tumor strategies ( Yang et al, 2018 ).…”

“…INSL4 was also discovered as a novel target downstream of LKB1 deficiency ( Yang et al, 2018 ). INSL4 signaling is a potential vital for LKB1-inactivated non-small-cell lung carcinoma, and its introduction helps to develop novel and effective anti-tumor strategies ( Yang et al, 2018 ). SCN7A encodes a voltage-dependent sodium channel of the excitable membrane, and it is reported to be downregulated in colorectal carcinoma at the mRNA level ( Ostasiewicz et al, 2016 ).…”

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“…The Ki-67 IHC staining was performed by the Molecular Pathology Core at University of Florida as previously described. 57 Briefly, xenograft tumors were fixed in 4% paraformaldehyde followed by graded ethanol dehydration and paraffin embedding. The sections (4 µm) were de-paraffinized with xylene and re-hydrated using ethanol of decreasing concentration until only water was used.…”