A systematic CRISPR screen reveals an IL-20/IL20RA-mediated peritoneal immune crosstalk to prevent the ovarian cancer metastasis

AbstractTranscoelomic spread of cancer cells across the peritoneal cavity occurs in most initially diagnosed ovarian cancer (OC) patients and accounts for most cancer-related death. However, how OC cells interact with peritoneal stromal cells to evade the immune surveillance remains largely unexplored. Here, through an in vivo genome-wide CRISPR/Cas9 screen, we identified IL20RA, which decreased dramatically in OC patients during peritoneal metastasis, as a key factor preventing the transcoelomic metastasis of OC. Reconstitution of IL20RA in highly metastatic OC cells greatly suppresses the transcoelomic metastasis. OC cells, when disseminate into the peritoneal cavity, greatly induce peritoneum mesothelial cells to express IL-20 and IL-24, which in turn activate the IL20RA downstream signaling in OC cells to produce mature IL-18, eventually resulting in the polarization of macrophages into the M1-like subtype to clear the cancer cells. Thus, we show an IL-20/IL20RA-mediated crosstalk between OC and mesothelial cells that supports a metastasis-repressing immune microenvironment.

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A systematic CRISPR screen reveals an IL-20/IL20RA-mediated immune crosstalk to prevent the ovarian cancer metastasis

eLife ◽

10.7554/elife.66222 ◽

2021 ◽

Vol 10 ◽

Author(s):

Jia Li ◽

Xuan Qin ◽

Jie Shi ◽

Xiaoshuang Wang ◽

Tong Li ◽

...

Keyword(s):

Ovarian Cancer ◽

Cancer Cells ◽

Peritoneal Cavity ◽

Cancer Metastasis ◽

Immune Surveillance ◽

Mesothelial Cells ◽

Downstream Signaling ◽

Key Factor ◽

Crispr Screen

Transcoelomic spread of cancer cells across the peritoneal cavity occurs in most initially diagnosed ovarian cancer (OC) patients and accounts for most cancer-related death. However, how OC cells interact with peritoneal stromal cells to evade the immune surveillance remains largely unexplored. Here, through an in vivo genome-wide CRISPR/Cas9 screen, we identified IL20RA, which decreased dramatically in OC patients during peritoneal metastasis, as a key factor preventing the transcoelomic metastasis of OC. Reconstitution of IL20RA in highly metastatic OC cells greatly suppresses the transcoelomic metastasis. OC cells, when disseminate into the peritoneal cavity, greatly induce peritoneum mesothelial cells to express IL-20 and IL-24, which in turn activate the IL20RA downstream signaling in OC cells to produce mature IL-18, eventually resulting in the polarization of macrophages into the M1-like subtype to clear the cancer cells. Thus, we show an IL-20/IL20RA-mediated crosstalk between OC and mesothelial cells that supports a metastasis-repressing immune microenvironment.

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LncRNA SPOCD1-AS from ovarian cancer extracellular vesicles remodels mesothelial cells to promote peritoneal metastasis via interacting with G3BP1

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01899-6 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Conghui Wang ◽

Jiaying Wang ◽

Xiameng Shen ◽

Mingyue Li ◽

Yongfang Yue ◽

...

Keyword(s):

Ovarian Cancer ◽

Extracellular Vesicles ◽

Peritoneal Metastasis ◽

Cancer Metastasis ◽

Cancer Therapeutics ◽

Mesothelial Cells ◽

Ovarian Cancer Cells ◽

Adhesion Assay ◽

Mesenchymal Transition

Abstract Background Metastasis is the key cause of death in ovarian cancer patients. To figure out the biological nature of cancer metastasis is essential for developing effective targeted therapy. Here we investigate how long non-coding RNA (lncRNA) SPOCD1-AS from ovarian cancer extracellular vesicles (EVs) remodel mesothelial cells through a mesothelial-to-mesenchymal transition (MMT) manner and facilitate peritoneal metastasis. Methods EVs purified from ovarian cancer cells and ascites of patients were applied to mesothelial cells. The MMT process of mesothelial cells was assessed by morphology observation, western blot analysis, migration assay and adhesion assay. Altered lncRNAs of EV-treated mesothelial cells were screened by RNA sequencing and identified by qRT-PCR. SPOCD1-AS was overexpressed or silenced by overexpression lentivirus or shRNA, respectively. RNA pull-down and RNA immunoprecipitation assays were conducted to reveal the mechanism by which SPOCD1-AS remodeled mesothelial cells. Interfering peptides were synthesized and applied. Ovarian cancer orthotopic implantation mouse model was established in vivo. Results We found that ovarian cancer-secreted EVs could be taken into recipient mesothelial cells, induce the MMT phenotype and enhance cancer cell adhesion to mesothelial cells. Furthermore, SPOCD1-AS embedded in ovarian cancer-secreted EVs was transmitted to mesothelial cells to induce the MMT process and facilitate peritoneal colonization in vitro and in vivo. SPOCD1-AS induced the MMT process of mesothelial cells via interacting with G3BP1 protein. Additionally, G3BP1 interfering peptide based on the F380/F382 residues was able to block SPOCD1-AS/G3BP1 interaction, inhibit the MMT phenotype of mesothelial cells, and diminish peritoneal metastasis in vivo. Conclusions Our findings elucidate the mechanism associated with EVs and their cargos in ovarian cancer peritoneal metastasis and may provide a potential approach for metastatic ovarian cancer therapeutics.

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

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-022-02242-3 ◽

2022 ◽

Vol 41 (1) ◽

Author(s):

Jingjing Zhang ◽

Yun Li ◽

Hua Liu ◽

Jiahui Zhang ◽

Jie Wang ◽

...

Keyword(s):

Ovarian Cancer ◽

Cancer Cells ◽

Cancer Metastasis ◽

Ovarian Cancer Cells ◽

Skov3 Cell ◽

Anoikis Resistance ◽

Primary Tumors ◽

Genome Wide

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.

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Exosomes in ovarian cancer ascites promote epithelial–mesenchymal transition of ovarian cancer cells by delivery of miR-6780b-5p

Cell Death and Disease ◽

10.1038/s41419-021-03490-5 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Jing Cai ◽

Lanqing Gong ◽

Guodong Li ◽

Jing Guo ◽

Xiaoqing Yi ◽

...

Keyword(s):

Ovarian Cancer ◽

Cancer Cells ◽

Tumor Metastasis ◽

Cancer Metastasis ◽

Epithelial Mesenchymal Transition ◽

Ovarian Cancer Cells ◽

Specific Mechanism ◽

Mesenchymal Transition

AbstractThe poor prognosis of ovarian cancer is mainly due to metastasis, and the specific mechanism underlying ovarian cancer metastasis is not clear. Ascites-derived exosomes (ADEs) play an important role in the progression of ovarian cancer, but the mechanism is unknown. Here, we found that ADEs promoted ovarian cancer metastasis not only in vitro but also in vivo. This promotive function was based on epithelial–mesenchymal transition (EMT) of ovarian cancer cells. Bioinformatics analysis of RNA sequencing microarray data indicated that miR-6780b-5p may be the key microRNA (miRNA) in ADEs that facilitates cancer metastasis. Moreover, the expression of exosomal miR-6780b-5p correlated with tumor metastasis in ovarian cancer patients. miR-6780b-5p overexpression promoted and miR-6780b-5p downregulation suppressed EMT of ovarian cancer cells. These results suggest that ADEs transfer miR-6780b-5p to ovarian cancer cells, promoting EMT and finally facilitating ovarian cancer metastasis.

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Host Mesothelin Expression Increases Ovarian Cancer Metastasis in the Peritoneal Microenvironment

International Journal of Molecular Sciences ◽

10.3390/ijms222212443 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12443

Author(s):

Tyvette S. Hilliard ◽

Brooke Kowalski ◽

Kyle Iwamoto ◽

Elizabeth A. Agadi ◽

Yueying Liu ◽

...

Keyword(s):

Ovarian Cancer ◽

Peritoneal Cavity ◽

Cancer Metastasis ◽

Single Cells ◽

Mesothelial Cell ◽

Metastatic Tumor ◽

Tumor Burden ◽

Mesothelial Cells ◽

Stage Disease

Mesothelin (MSLN), a glycoprotein normally expressed by mesothelial cells, is overexpressed in ovarian cancer (OvCa) suggesting a role in tumor progression, although the biological function is not fully understood. OvCa has a high mortality rate due to diagnosis at advanced stage disease with intraperitoneal metastasis. Tumor cells detach from the primary tumor as single cells or multicellular aggregates (MCAs) and attach to the mesothelium of organs within the peritoneal cavity producing widely disseminated secondary lesions. To investigate the role of host MSLN in the peritoneal cavity we used a mouse model with a null mutation in the MSLN gene (MSLNKO). The deletion of host MSLN expression modified the peritoneal ultrastructure resulting in abnormal mesothelial cell surface architecture and altered omental collagen fibril organization. Co-culture of murine OvCa cells with primary mesothelial cells regardless of MSLN expression formed compact MCAs. However, co-culture with MSLNKO mesothelial cells resulted in smaller MCAs. An allograft tumor study, using wild-type mice (MSLNWT) or MSLNKO mice injected intraperitoneally with murine OvCa cells demonstrated a significant decrease in peritoneal metastatic tumor burden in MSLNKO mice compared to MSLNWT mice. Together, these data support a role for host MSLN in the progression of OvCa metastasis.

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Plasma cells shape the mesenchymal identity of ovarian cancers through transfer of exosome-derived microRNAs

Science Advances ◽

10.1126/sciadv.abb0737 ◽

2021 ◽

Vol 7 (9) ◽

pp. eabb0737

Author(s):

Zhengnan Yang ◽

Wei Wang ◽

Linjie Zhao ◽

Xin Wang ◽

Ryan C. Gimple ◽

...

Keyword(s):

Ovarian Cancer ◽

Cancer Cells ◽

Plasma Cell ◽

Plasma Cells ◽

Ovarian Cancer Cells ◽

Mesenchymal Phenotype ◽

Ovarian Cancers ◽

Novel Approach

Ovarian cancer represents a highly lethal disease that poses a substantial burden for females, with four main molecular subtypes carrying distinct clinical outcomes. Here, we demonstrated that plasma cells, a subset of antibody-producing B cells, were enriched in the mesenchymal subtype of high-grade serous ovarian cancers (HGSCs). Plasma cell abundance correlated with the density of mesenchymal cells in clinical specimens of HGSCs. Coculture of nonmesenchymal ovarian cancer cells and plasma cells induced a mesenchymal phenotype of tumor cells in vitro and in vivo. Phenotypic switch was mediated by the transfer of plasma cell–derived exosomes containing miR-330-3p into nonmesenchymal ovarian cancer cells. Exosome-derived miR-330-3p increased expression of junctional adhesion molecule B in a noncanonical fashion. Depletion of plasma cells by bortezomib reversed the mesenchymal characteristics of ovarian cancer and inhibited in vivo tumor growth. Collectively, our work suggests targeting plasma cells may be a novel approach for ovarian cancer therapy.

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Splicing factor USP39 promotes ovarian cancer malignancy through maintaining efficient splicing of oncogenic HMGA2

Cell Death and Disease ◽

10.1038/s41419-021-03581-3 ◽

2021 ◽

Vol 12 (4) ◽

Author(s):

Shourong Wang ◽

Zixiang Wang ◽

Jieyin Li ◽

Junchao Qin ◽

Jianping Song ◽

...

Keyword(s):

Ovarian Cancer ◽

Cancer Cells ◽

Malignant Tumors ◽

Splicing Factor ◽

Ovarian Cancer Cells ◽

Nuclear Speckles ◽

Aberrant Expression ◽

Intergenic Regions

AbstractAberrant expression of splicing factors was found to promote tumorigenesis and the development of human malignant tumors. Nevertheless, the underlying mechanisms and functional relevance remain elusive. We here show that USP39, a component of the spliceosome, is frequently overexpressed in high-grade serous ovarian carcinoma (HGSOC) and that an elevated level of USP39 is associated with a poor prognosis. USP39 promotes proliferation/invasion in vitro and tumor growth in vivo. Importantly, USP39 was transcriptionally activated by the oncogene protein c-MYC in ovarian cancer cells. We further demonstrated that USP39 colocalizes with spliceosome components in nuclear speckles. Transcriptomic analysis revealed that USP39 deletion led to globally impaired splicing that is characterized by skipped exons and overrepresentation of introns and intergenic regions. Furthermore, RNA immunoprecipitation sequencing showed that USP39 preferentially binds to exon-intron regions near 5′ and 3′ splicing sites. In particular, USP39 facilitates efficient splicing of HMGA2 and thereby increases the malignancy of ovarian cancer cells. Taken together, our results indicate that USP39 functions as an oncogenic splicing factor in ovarian cancer and represents a potential target for ovarian cancer therapy.

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The In Vivo Selection Method in Breast Cancer Metastasis

International Journal of Molecular Sciences ◽

10.3390/ijms22041886 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1886

Author(s):

Jun Nakayama ◽

Yuxuan Han ◽

Yuka Kuroiwa ◽

Kazushi Azuma ◽

Yusuke Yamamoto ◽

...

Keyword(s):

Cancer Cells ◽

Cell Lines ◽

Cancer Metastasis ◽

Metastatic Cancer ◽

Expression Patterns ◽

Selection Method ◽

Gene Signatures ◽

In Vivo Selection ◽

Metastatic Cancer Cells

Metastasis is a complex event in cancer progression and causes most deaths from cancer. Repeated transplantation of metastatic cancer cells derived from transplanted murine organs can be used to select the population of highly metastatic cancer cells; this method is called as in vivo selection. The in vivo selection method and highly metastatic cancer cell lines have contributed to reveal the molecular mechanisms of cancer metastasis. Here, we present an overview of the methodology for the in vivo selection method. Recent comparative analysis of the transplantation methods for metastasis have revealed the divergence of metastasis gene signatures. Even cancer cells that metastasize to the same organ show various metastatic cascades and gene expression patterns by changing the transplantation method for the in vivo selection. These findings suggest that the selection of metastasis models for the study of metastasis gene signatures has the potential to influence research results. The study of novel gene signatures that are identified from novel highly metastatic cell lines and patient-derived xenografts (PDXs) will be helpful for understanding the novel mechanisms of metastasis.

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The Epithelial–Mesenchymal Transcription Factor SNAI1 Represses Transcription of the Tumor Suppressor miRNA let-7 in Cancer

Cancers ◽

10.3390/cancers13061469 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1469

Author(s):

Hanmin Wang ◽

Evgeny Chirshev ◽

Nozomi Hojo ◽

Tise Suzuki ◽

Antonella Bertucci ◽

...

Keyword(s):

Ovarian Cancer ◽

Stem Cell ◽

Cancer Cells ◽

Epithelial Mesenchymal Transition ◽

Tumor Burden ◽

Cell Phenotype ◽

Mesenchymal Transition ◽

Carcinoma Cell Lines ◽

Future Work

We aimed to determine the mechanism of epithelial–mesenchymal transition (EMT)-induced stemness in cancer cells. Cancer relapse and metastasis are caused by rare stem-like cells within tumors. Studies of stem cell reprogramming have linked let-7 repression and acquisition of stemness with the EMT factor, SNAI1. The mechanisms for the loss of let-7 in cancer cells are incompletely understood. In four carcinoma cell lines from breast cancer, pancreatic cancer, and ovarian cancer and in ovarian cancer patient-derived cells, we analyzed stem cell phenotype and tumor growth via mRNA, miRNA, and protein expression, spheroid formation, and growth in patient-derived xenografts. We show that treatment with EMT-promoting growth factors or SNAI1 overexpression increased stemness and reduced let-7 expression, while SNAI1 knockdown reduced stemness and restored let-7 expression. Rescue experiments demonstrate that the pro-stemness effects of SNAI1 are mediated via let-7. In vivo, nanoparticle-delivered siRNA successfully knocked down SNAI1 in orthotopic patient-derived xenografts, accompanied by reduced stemness and increased let-7 expression, and reduced tumor burden. Chromatin immunoprecipitation demonstrated that SNAI1 binds the promoters of various let-7 family members, and luciferase assays revealed that SNAI1 represses let-7 transcription. In conclusion, the SNAI1/let-7 axis is an important component of stemness pathways in cancer cells, and this study provides a rationale for future work examining this axis as a potential target for cancer stem cell-specific therapies.

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