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RNA sequencing of Cd44-positive and Cd44-negative livers isolated from Nf2 - mutant mice

ABSTRACT: Primary liver cancer is the third leading cause of cancer related death worldwide. An increasing body of evidence suggests that the Hippo tumor suppressor pathway plays a critical role in restricting cell proliferation and determining cell fate during physiological and pathological processes in the liver. Merlin (Moesin-Ezrin-Radixin-likeprotein), encoded by the NF2 (neurofibromatosis type 2) gene is an upstream regulator of the Hippo signaling pathway. Targeting of Merlin to the plasma membrane seems to be critical for fulfilling its major tumor-suppressive functions; this is facilitated by interactions with membrane-associated proteins, including CD44 (clusterofdifferentiation44). Indeed, mutations within the CD44-binding domain of Merlin were reported in many human cancers. In this study, we evaluated the relative contribution of CD44- and Merlin-dependent processes to the development and progression of liver tumors. To this end, mice with liver-specific deletion of the Nf2 gene were crossed with Cd44-deleted mice, and subjected to extensive histological, biochemical and molecular analyses. In addition, cells were isolated from mutant livers and subjected to in vitro assays. Deletion of Nf2 in the liver led to substantial liver enlargement and generation of hepatocellular carcinomas (HCCs), intrahepatic cholangiocarcinomas (iCCAs) as well as mixed hepatocellular cholangiocarcinomas. Deletion of Cd44 had no influence on liver size or primary liver tumor development but it significantly inhibited metastasis formation in Nf2-mutant mice. CD44 upregulates expression of integrin β2 and promotes transendothelial migration of liver cancer cells which may facilitate metastatic spreading. Altogether, our results suggest that CD44 may be a promising target for interfering with metastatic spreading of liver cancer.

ORGANISM(S): Mus musculus

PROVIDER: GSE228630 | GEO | 2023/05/17

REPOSITORIES: GEO

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Project description:Osteosarcoma is the most common type of pediatric bone tumor. Despite great advances in chem-otherapy during the past decades the survival rates of osteosarcoma patients remains usatisfacto-ry. Drug resistance is one of the main reasons leading to treatment failure and poor progno-sis. Previous reports correlated expression of cluster of differentiation 44 (CD44) with drug re-sistance and poor survival of osteosarcoma patients, however the underlying mechanisms are poorly defined. Here, we investigated the role of CD44 in the regulation of drug chemoresistance using osteosarcoma cells isolated from mice carrying a mutation of the tumor suppressor neurofi-bromatosis type 2 (Nf2) gene. CD44 expression was knocked-down in the cells using CRISPR/Cas9 approach. Subsequently, CD44 isoforms and mutants were re-introduced to investigate CD44-dependent processes. Sensitivity to doxorubicin was analyzed in the osteosarcoma cells with modified CD44 expression by immunoblot, colony formation- and WST-1 assay. To dissect the molecular alterations induced by deletion of Cd44, RNA sequencing was performed on Cd44-positive and Cd44-negative primary osteosarcoma tissues isolated from Nf2-mutant mice. Subsequently, expression of candidate genes was evaluated by quantitative reverse transcription PCR (qRT-PCR). Our results indicate that CD44 increases the resistance of osteosarcoma cells to doxorubicin by up-regulating the levels of multidrug resistance (MDR) 1 protein expression, and suggest the role of proteolytically released CD44 intracellular domain, and hyaluronan interac-tions in this process. Moreover, high throughput sequencing analysis identified differential regula-tion of several apoptosis-related genes in Cd44-positive and -negative primary osteosarcomas, in-cluding p53 apoptosis effector related to PMP-22 (Perp). Deletion of CD44 in osteosarcoma cells led to doxorubicin-dependent p53 activation and a profound increase in Perp mRNA expression. Overall, our results suggest that CD44 might be an important regulator of drug resistance and suggest that targeting CD44 can sensitize osteosarcoma to standard chemotherapy.

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RNA sequencing of Cd44-positive and Cd44-negative livers isolated from Nf2 - mutant mice

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