Identification of PTPLAD1 as a Novel Tumor Suppressor and its Implication in Metastatic Colon Cancer Therapy

Background: Colon cancer is one of the most common malignant cancers, and cancer metastasis always leads to a failure of clinical treatment. Although there have been many studies on the process of colon cancer progression, the detailed mechanism of colon cancer metastasis still remains unclear, and more effective drugs targeting colon cancer metastasis are urgently needed. This study aims to explore novel effectors involved in colon cancer metastasis and screen out potential targeted drug for colon cancer therapy.Methods: Mass spectrometry and bioinformatics analyses are performed to present the proteomics variation between two colon cancer cell lines with different invasion abilities. Boyden chamber invasion assay (in vitro) and experimental metastasis assay in mice (in vivo) are performed to explore the role of protein tyrosine phosphatase-like A domain containing 1 (PTPLAD1) in colon cancer metastasis. Western blotting and qRT-PCR assays are performed to analyze the expression of proteins and mRNA of related signaling cascades. Co-immunoprecipitation (Co-IP) and confocal assays are conducted to examine the proteins interacted with PTPLAD1. Chromatin-immunoprecipitation (ChIP) assay is fulfilled to evaluate the relationship of PTPLAD1 expression and histone H3K9 acetylation. Enzyme-linked immuno sorbent assay (ELISA) screening system are used to screen out the small molecular inhibitor that mimics the effect of PTPLAD1 on suppressing colon cancer metastasis.Results: Our results identify that PTPLAD1 is significantly downregulated in the highly invasive cell lines, and PTPLAD1 suppresses colon cancer metastasis by interacting with prohibitin (PHB) and prohibiting the activation of PHB/C-Raf1 (Raf)/ extracellular signal-regulated kinase (ERK)/Snail signaling pathway. Moreover, the expression of PTPLAD1 is modulated through the acetylation of histone H3K9. Besides, we identify a small molecule named avobenzone, once used to protect skin from ultraviolet damage, that can disrupt the interaction of PHB and Raf, significantly abrogate the activation of downstream signaling cascades and prohibit colon cancer metastasis.Conclusions: Collectively, our study not only identifies PTPLAD1 as a novel tumor suppressor and clarifies its role in suppressing colon cancer metastasis, but also provides a potential targeted drug for metastatic colon cancer therapy.

NSUN2-Mediated Splice Site m5C Methylation Modification Promotes the Progression of Colon Cancer Through Modulating the Discrepant Cleavage of tRNA-Arg

Background: Hypoxia is a key driving factor for the tumour microenvironment restructuring, which leading to the variation of gene expression profiling in cancer cells. Increasing evidence reveals the initial action of hypoxia in the epitranscriptomics including RNA methylation. The role of tRNA-derived fragments (tRFs) in regulating tumour metastasis potential has attracted attention. Methods: The expression of tRFs in colon cancer cells under hypoxia were evaluated based on full-transcript sequencing and bioinformatics analysis and their effects on colon cancer metastasis were detected by transwell assays. The role of C34 was verified by introducing mutation and artificial m5C modification. The effects of NSUN2 on the biological characteristics of colon cancer cells were investigated on the basis of gain-of-function and loss-of function analyses. Lung metastasis model further uncovered the roles of NSUN2 and key tRF in tumour progression. Assays of RNA immunoprecipitation-qPCR (RIP-qPCR) were performed to identify that NSUN2 is a key methyltransferase for cysteine modification at C34 of tRNA-Arg.Results: The present study verified the up-expression of tRF (tRF-20-MEJB5Y13) and down-expression of tRFs (tRF-20-M0NK5Y93 and tRF-21-3OPP6N7KE) in colon cancer cells under hypoxia, and all of them were derived from different tRNA-Arg. Contradictory effects of these three tRNA-Arg-derived tRFs on metastatic potential of colon cancer were demonstrated in this study. The sequence differences and the key nucleotide bases of tRNA with the methylation modification potential among the source tRNAs were analysed. Notably, our data identified C34 of tRNA-Arg as a key site that may play an important role in hypoxia-mediated tRNA-Arg discrepant cleavage. We further investigated that NSUN2 mediated specific site methylation of tRNA-Arg at C34, thereby protecting tRNA from cleavage by endonuclease and subsequently promoting the colon cancer metastasis both in vitro and in vivo. Conclusion: The present study elaborates on the precise regulatory mechanism of m5C methylation and the role in the selective cleavage of tRNA from the perspective of noncoding RNA methylation epitranscriptomics, providing a novel insight into the molecular basis of selective expression of tRFs in colon cancer under hypoxia.

Exosomal lncRNA PVT1/VEGFA Axis Promotes Colon Cancer Metastasis and Stemness by Downregulation of Tumor Suppressor miR-152-3p

Background. Treating advanced colon cancer remains challenging in clinical settings because of the development of drug resistance and distant metastasis. Mechanisms underlying the metastasis of colon cancer are complex and unclear. Methods. Computational analysis was performed to determine genes associated with the exosomal long noncoding (lncRNA) plasmacytoma variant translocation 1 (PVT1)/vascular endothelial growth factor A (VEGFA) axis in patients with colon cancer. The biological importance of the exosomal lncRNA PVT1/VEGFA axis was examined in vitro by using HCT116 and LoVo cell lines and in vivo by using a patient-derived xenograft (PDX) mouse model through knockdown (by silencing of PVT1) and overexpression (by adding serum exosomes isolated from patients with distant metastasis (M-exo)). Results. The in silico analysis demonstrated that PVT1 overexpression was associated with poor prognosis and increased expression of metastatic markers such as VEGFA and epidermal growth factor receptor (EGFR). This finding was further validated in a small cohort of patients with colon cancer in whom increased PVT1 expression was correlated with colon cancer incidence, disease recurrence, and distant metastasis. M-exo were enriched with PVT1 and VEGFA, and both migratory and invasive abilities of colon cancer cell lines increased when they were cocultured with M-exo. The metastasis-promoting effect was accompanied by increased expression of Twist1, vimentin, and MMP2. M-exo promoted metastasis in PDX mice. In vitro silencing of PVT1 reduced colon tumorigenic properties including migratory, invasive, colony forming, and tumorsphere generation abilities. Further analysis revealed that PVT1, VEGFA, and EGFR interact with and are regulated by miR-152-3p. Increased miR-152-3p expression reduced tumorigenesis, where increased tumorigenesis was observed when miR-152-3p expression was downregulated. Conclusion. Exosomal PVT1 promotes colon cancer metastasis through its association with EGFR and VEGFA expression. miR-152-3p targets both PVT1 and VEGFA, and this regulatory pathway can be explored for drug development and as a prognostic biomarker.

LAPAROSCOPIC LIVER RESECTION VIA LAPAROSCOPY: EXPERIENCE IN OUR CENTRE

INTRODUCTION Laparoscopic liver surgery has undergone a great evolution in recent years, allowing increasingly complex resections without increasing complications and with evident postoperative benefits. Our purpose is to analyze our initial experience in this type of resection. MATERIAL AND METHODS We performed a retrospective observational study analyzing 41 patients who underwent liver resections by laparoscopy in our centre from March 2019 to January 2020. RESULTS The mean age of the patients was 64.5 years (SD 11) with a mean BMI of 27.56 (from 4.59). The most common surgical indication was colon cancer metastasis (41.4%), followed by hepatocarcinoma (36.6%) and usually single lesions (75.6%). The procedure passed without complications except in the case of 5 patients who required conversion to open surgery (12.5%) and 3 patients (7.5%) who required intraoperative transfusion. During the postoperative period 4 patients (9.8%) presented complications and all of them were classified as Clavien-Dindo II. We only reported 1 death (2.4%) in > 90 days, which was not related to the reason for surgery. No reinterventions were necessary during admission and there were no readmissions in the first 30 days after discharge, CONCLUSIONS Laparoscopic liver surgery is technically demanding and requires previous experience in open surgery, as well as specific training, which makes a regulated implementation of the technique necessary.

Inactivation of E-cadherin by Trop-2 drives colon cancer metastasis.

105 Background: Tumor metastasis is the main cause of death of colon cancer patients and the biggest hurdle for cancer cure. We set to identify decisive drivers and of pivotal therapy targets for colon cancer metastasis. Methods: IHC analysis quantified the expression of target molecules in primary tumors and metastases. Cell-cell adhesion capacity was assessed in vitro and in HCT116 colon cancer cell spheroids. Pre-clinical models of orthotopic growth of KM12SM colon cancer cells and metastatic diffusion to the liver were utilized to assess metastatic spreading force of wtTrop-2 and of the constitutively-active, tail-less form of Trop-2 (Δcyto). Xenotransplant and metastasis transcriptomes were analyzed for differential induction of EMT determinants. Kaplan–Meier plots were used to illustrate survival and metastatic relapse in independent case series of colon cancer patients. Results: wtTrop-2 was shown to induce wound-healing. ΔcytoTrop-2 further increased cell migration ability. Both wtTrop-2 and ΔcytoTrop-2 induced resistance to apoptosis in vitro and in vivo. wtTrop-2 strikingly increased the metastatic capacity of KM12SM cells, raising metastasis rates from 45% for control cells to 90% for wtTrop-2 transfectants. The constitutively-active ΔcytoTrop-2 further boosted metastatic spreading, with metastatic livers reaching up to four times their normal size. Cancer metastases revealed high levels of E-cadherin, in the absence of transcriptional down-regulation. EMT transcription factors were largely missing from Trop-2-activated cells. Rather, binding to Trop-2 was shown to cause the release of E-cadherin from the cytoskeleton, loss of cell-cell adhesion and activation of β-catenin. This global, Trop-2/E-cadherin/β-catenin-driven pro-metastatic program was recapitulated in colon cancer patients and was shown to impact on colon cancer metastatic relapse and overall patient survival. Conclusions: We identify Trop-2-driven functional inactivation of E-cadherin as a widespread driver of metastatic diffusion in colon cancer, opening novel avenues for personalized diagnostic procedures and anti-cancer therapies. [Table: see text]

Inhibitory Effect of lncRNA GHRLOS on Colon Cancer Metastasis and its Molecular Mechanism

Background: LncRNA GHRLOS is a long non-coding RNAs (lncRNAs). Microarray detection for expression profile revealed that lncRNA GHRLOS was significantly under-expressed in colon cancer tissues, while its role in a tumor remained unclear.