Genome-wide CRISPR/Cas9 library screen identifies PCMT1 as a critical driver of ovarian cancer metastasis

Abstract

Background The development of lethal cancer metastasis depends on the dynamic interactions between cancer cells and the tumor microenvironment, both of which are embedded in the extracellular matrix (ECM). The acquisition of resistance to detachment-induced apoptosis, also known as anoikis, is a critical step in the metastatic cascade. Thus, a more in-depth and systematic analysis is needed to identify the key drivers of anoikis resistance. Methods Genome-wide CRISPR/Cas9 knockout screen was used to identify critical drivers of anoikis resistance using SKOV3 cell line and found protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) as a candidate. Quantitative real-time PCR (qRT-PCR) and immune-histochemistry (IHC) were used to measure differentially expressed PCMT1 in primary tissues and metastatic cancer tissues. PCMT1 knockdown/knockout and overexpression were performed to investigate the functional role of PCMT1 in vitro and in vivo. The expression and regulation of PCMT1 and integrin-FAK-Src pathway were evaluated using immunoprecipitation followed by mass spectrometry (IP-MS), western blot analysis and live cell imaging. Results We found that PCMT1 enhanced cell migration, adhesion, and spheroid formation in vitro. Interestingly, PCMT1 was released from ovarian cancer cells, and interacted with the ECM protein LAMB3, which binds to integrin and activates FAK-Src signaling to promote cancer progression. Strikingly, treatment with an antibody against extracellular PCMT1 effectively reduced ovarian cancer cell invasion and adhesion. Our in vivo results indicated that overexpression of PCMT1 led to increased ascites formation and distant metastasis, whereas knockout of PCMT1 had the opposite effect. Importantly, PCMT1 was highly expressed in late-stage metastatic tumors compared to early-stage primary tumors. Conclusions Through systematically identifying the drivers of anoikis resistance, we uncovered the contribution of PCMT1 to focal adhesion (FA) dynamics as well as cancer metastasis. Our study suggested that PCMT1 has the potential to be a therapeutic target in metastatic ovarian cancer.

PCMT1 has Potential Prognostic Value and Promotes Cell Growth and Motility in Breast Cancer

“…Recently, it has been found that PCMT1 is involved in tumor development and progression, and might be a potential biomarker for diagnosis or prognosis of several tumors. For example, PCMT1 expression was signi cantly higher in hepatocellular carcinoma, bladder cancer, advanced-stage lung adenocarcinoma and metastatic ovarian cancer, and higher PCMT1 expression predicted poor prognosis of above malignant tumors [6,7,11,23]. Up to now, although it has showed that PCMT1 mRNA was highly expressed in BC, and the survival of BC patient with higher expression of PCMT1 was signi cantly poorer than those with lower expression [8][9][10].…”

“…The latest research showed that PCMT1 promoted the migration and invasion ability of glioblastoma cells and plays a critical role in the development of glioblastoma [25,26]. Through binding with LAMB3, PCMT1 activated integrin-FAK-Src signaling to enhance ovarian cancer cell adhesion, migration, invasion and metastasis formation [11]. PCMT1 also participated in bladder cancer cell migration and invasion by regulating epithelial-mesenchymal transition-related genes [6].…”

“…Moreover, based on Cox univariate and multivariate regression analysis, the result indicated that the PCMT1 protein high-expression was an independent poorer prognostic factor for BC patients (p = 0.038, HR = 2.068; 95% CI: 1.042-4.104). As previous studies reported, PCMT1 plays an important role in regulating tumor malignant behaviors such as proliferation, migration and invasion [5,6,10,11]. To determine whether PCMT1 in uenced the malignant behaviors of BC cells, we used CRISPR/Cas9 technology to generate the PCMT1 knockout MCF-7 cell line, and western blot assay was performed to verify the knockout effect (Fig.…”

Mechanisms of Cell Adhesion Molecules in Endocrine-Related Cancers: A Concise Outlook

“…Integrin β1/LRRC15 exerts a metastatic invasion role via activation of FAK in patient-derived ovarian cancer xenograft models, inhibited by ABBV-085, an antibody–drug conjugate of LRRC15 ( 195 ). Following binding to integrin, PCMT1 was released from ovarian cancer cells, leading to activated FAK-Src signaling to promote cancer progression and anoikis resistance ( 196 ). Moreover, stiffness-induced autophagy in stromal cells mediated by integrin αV-PTK-AMPKα promotes the growth of adjacent pancreatic stellate cells in vitro and in vivo ( 197 ).…”

Ten Years of CRISPRing Cancers In Vitro

“…Zhang et al, thanks to genome wide CRISPR/Cas9 knockout screen, have identified critical drivers of anoikis, using ovarian cancer cell lines in ultra-low attachment conditions as a model that mimics the situation in which cancer cells shed from the primary attachment. They demonstrated that knockout of Protein-L-Isoaspartate (D-Aspartate) O-methyltransferase ( PCMT1 ), the main driver in anoikis resistance that they identified, caused an increased apoptosis of SKOV3 cells in response to detachment from the ECM, which is correlated with a decreased tumorigenesis and metastasization in the xenograft model [ 110 ].…”