PAX7 is a required target for microRNA-206-induced differentiation of fusion-negative rhabdomyosarcoma

Abstract

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. RMS can be parsed based on clinical outcome into two subtypes, fusion-positive RMS (FP-RMS) or fusion-negative RMS (FN-RMS) based on the presence or absence of either PAX3-FOXO1 or PAX7-FOXO1 gene fusions. In both RMS subtypes, tumor cells show histology and a gene expression pattern resembling that of developmentally arrested skeletal muscle. Differentiation therapy is an attractive approach to embryonal tumors of childhood including RMS; however, agents to drive RMS differentiation have not entered the clinic and their mechanisms remain unclear. MicroRNA-206 (miR-206) expression increases through normal muscle development and has decreased levels in RMS compared with normal skeletal muscle. Increasing miR-206 expression drives differentiation of RMS, but the target genes responsible for the relief of the development arrest are largely unknown. Using a combinatorial approach with gene and proteomic profiling coupled with genetic rescue, we identified key miR-206 targets responsible for the FN-RMS differentiation blockade, PAX7, PAX3, NOTCH3, and CCND2. Specifically, we determined that PAX7 downregulation is necessary for miR-206-induced cell cycle exit and myogenic differentiation in FN-RMS but not in FP-RMS. Gene knockdown of targets necessary for miR-206-induced differentiation alone or in combination was not sufficient to phenocopy the differentiation phenotype from miR-206, thus illustrating that miR-206 replacement offers the ability to modulate a complex network of genes responsible for the developmental arrest in FN-RMS. Genetic deletion of miR-206 in a mouse model of FN-RMS accelerated and exacerbated tumor development, indicating that both in vitro and in vivo miR-206 acts as a tumor suppressor in FN-RMS at least partially through downregulation of PAX7. Collectively, our results illustrate that miR-206 relieves the differentiation arrest in FN-RMS and suggests that miR-206 replacement could be a potential therapeutic differentiation strategy.

PAX3-FOXO1 drives miR-486-5p and represses miR-221 contributing to pathogenesis of alveolar rhabdomyosarcoma

“…In FP-RMS, we identified CCND2, CDK6, and ERBB3 as novel targets of miR-221. Interestingly, CCND2 is the predominant D-Type Cyclin expressed in RMS and its downregulation by miR-1/206 decreases RMS proliferation [ 34 , 55 , 56 ]. Furthermore, Cyclin D and CDK4/6 activity is tightly regulated in myoblast proliferation and differentiation [ 57 , 58 ] and overexpressed/hyperactive in cancer and RMS [ 33 ].…”

“…Cells were from the following sources: RD (CCL-136) and Rh30 (CRL-2061) from ATCC (Manassas, VA, USA), Rh3, RH4, Rh28, and Rh41 (Gerard Grosveld, St. Jude), 293T (Martine F. Roussel, St. Jude), and LHCN-M2 (Woodring Wright, University of Texas Southwestern Medical Center) [ 73 ]. Cells were authenticated with short tandem repeat profiling (Supplementary Table S2 ) [ 56 ].…”

Biallelic Dicer1 Loss Mediated by aP2-Cre Drives Angiosarcoma

“…Immunoblots were performed on total cell lysates prepared in RIPA buffer as described previously (25). Blots were probed with antibodies detailed in Supplementary Table S4.…”

miR‐206 inhibits the growth of hepatocellular carcinoma cells via targeting CDK9

“…miR‐206, a member of miR‐1 family, is specifically expressed in skeletal muscle and play an important role in skeletal muscle development, function, and pathology . Furthermore, miR‐206 is involved in the pathogenesis of various diseases including pulmonary disease , heart failure , Alzheimer's disease , and various types of cancers . It is frequently downregulated in some cancer tissues, and functions as a cell cycle regulator and tumor suppressor in clear‐cell renal cell carcinoma , gastric cancer , and rhabdomyosarcoma , indicating that miR‐206 plays an important role in the development and progression of cancer .…”

“…Furthermore, miR‐206 is involved in the pathogenesis of various diseases including pulmonary disease , heart failure , Alzheimer's disease , and various types of cancers . It is frequently downregulated in some cancer tissues, and functions as a cell cycle regulator and tumor suppressor in clear‐cell renal cell carcinoma , gastric cancer , and rhabdomyosarcoma , indicating that miR‐206 plays an important role in the development and progression of cancer . Previous reports have shown that miR‐206 is significantly decreased in HCC tissues, and overexpression of miRNA‐206 can promote apoptosis, induce cell cycle arrest, and inhibit proliferation, invasion, and migration of HCC cells .…”