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

Jingjing Zhang,Weiwei Feng,Jinyan Ma,Hua Liu,Yun Li,Rutao Cui,Qian Li,Jie Wang,Liangzhe Wang,Jiahui Zhang,Yingjie Xu,Xiang Yu,Wen Yang,Yu Zhang,Jiahui Wang,Masha Huang,Jia Xia

doi:10.1186/s13046-022-02242-3

Abstract

BackgroundThe 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.MethodsGenome-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.ResultsWe 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.ConclusionsThrough 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.

Highlights

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)
Through systematically identifying the drivers of anoikis resistance, we uncovered the contribution of Protein-L-isoaspartate O-methyltransferase (PCMT1) to focal adhesion (FA) dynamics as well as cancer metastasis
These findings collectively demonstrate that focal adhesion kinase (FAK) and LAMB3 are required in PCMT1-induced cell migration and spheroid formation. (See figure on page.) Fig. 5 Cell invasion and adhesion are enhanced by supernatant from PCMT1-OE cells and inhibited by a PCMT1 blocking antibody

Summary

Introduction

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). Cancer metastasis is a multistep and highly selective process that involves dissociation of tumor cells from the primary site, anchorage-independent growth, apoptosis evasion, cell migration, invasion of surrounding tissues, intravasation into the circulation, extravasation and colonization to distant sites [1]. During this process, tumor cells are in a dynamic state and susceptible to anoikis in response to extracellular matrix (ECM) detachment. The dynamic interplay between tumor cells and the microenvironment critically impacts the process of metastatic progression It is unclear how disseminate solid tumor cells acquire the essential ability to survive under “anchorage-independent” (“spheroid”) growth conditions. Ovarian cancer cells shedding from the primary site are able to survive in vivo single cells or free-floating clumps in the ascites fluid, which may be a consequence of anoikis resistance [2, 3]

Methods

Results

Discussion

Conclusion