Targeting microRNA-mediated gene repression limits adipogenic conversion of skeletal muscle mesenchymal stromal cells.

Intramuscular fatty deposits, which are seen in muscular dystrophies and with aging, negatively affect muscle function. The cells of origin of adipocytes constituting these fatty deposits are mesenchymal stromal cells, fibroadipogenic progenitors (FAPs). We uncover a molecular fate switch, involving miR-206 and the transcription factor Runx1, that controls FAP differentiation to adipocytes. Mice deficient in miR-206 exhibit increased adipogenesis following muscle injury. Adipogenic differentiation of FAPs is abrogated by miR-206 mimics. Using a labeled microRNA (miRNA) pull-down and sequencing (LAMP-seq), we identified Runx1 as a miR-206 target, with miR-206 repressing Runx1 translation. In the absence of miR-206 in FAPs, Runx1 occupancy near transcriptional start sites of adipogenic genes and expression of these genes increase. We demonstrate that miR-206 mimicry in vivo limits intramuscular fatty infiltration. Our results provide insight into the underlying molecular mechanisms of FAP fate determination and formation of harmful fatty deposits in skeletal muscle.

Mesenchymal Stromal Cells Are Required for Regeneration and Homeostatic Maintenance of Skeletal Muscle.

The necessity of mesenchymal stromal cells, called fibroadipogenic progenitors (FAPs), in skeletal muscle regeneration and maintenance remains unestablished. We report the generation of a PDGFRαCreER knockin mouse model that provides a specific means of labeling and targeting FAPs. Depletion of FAPs using Cre-dependent diphtheria toxin expression results in loss of expansion of muscle stem cells (MuSCs) and CD45+ hematopoietic cells after injury and impaired skeletal muscle regeneration. Furthermore, FAP-depleted mice under homeostatic conditions exhibit muscle atrophy and loss of MuSCs, revealing that FAPs are required for the maintenance of both skeletal muscle and the MuSC pool. We also report that local tamoxifen metabolite delivery to target CreER activity in a single muscle, removing potentially confounding systemic effects of ablating PDGFRα+ cells distantly, also causes muscle atrophy. These data establish a critical role of FAPs in skeletal muscle regeneration and maintenance.

DNA mismatch repair-dependent activation of c-Abl/p73alpha/GADD45alpha-mediated apoptosis.

Cells with functional DNA mismatch repair (MMR) stimulate G(2) cell cycle checkpoint arrest and apoptosis in response to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MMR-deficient cells fail to detect MNNG-induced DNA damage, resulting in the survival of "mutator" cells. The retrograde (nucleus-to-cytoplasm) signaling that initiates MMR-dependent G(2) arrest and cell death remains undefined. Since MMR-dependent phosphorylation and stabilization of p53 were noted, we investigated its role(s) in G(2) arrest and apoptosis. Loss of p53 function by E6 expression, dominant-negative p53, or stable p53 knockdown failed to prevent MMR-dependent G(2) arrest, apoptosis, or lethality. MMR-dependent c-Abl-mediated p73alpha and GADD45alpha protein up-regulation after MNNG exposure prompted us to examine c-Abl/p73alpha/GADD45alpha signaling in cell death responses. STI571 (Gleevec, a c-Abl tyrosine kinase inhibitor) and stable c-Abl, p73alpha, and GADD45alpha knockdown prevented MMR-dependent apoptosis. Interestingly, stable p73alpha knockdown blocked MMR-dependent apoptosis, but not G(2) arrest, thereby uncoupling G(2) arrest from lethality. Thus, MMR-dependent intrinsic apoptosis is p53-independent, but stimulated by hMLH1/c-Abl/p73alpha/GADD45alpha retrograde signaling.

Role of c-Abl kinase in DNA mismatch repair-dependent G2 cell cycle checkpoint arrest responses.

Current published data suggest that DNA mismatch repair (MMR) triggers prolonged G(2) cell cycle checkpoint arrest after alkylation damage from N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by activating ATR (ataxia telangiectasia-Rad3-related kinase). However, analyses of isogenic MMR-proficient and MMR-deficient human RKO colon cancer cells revealed that although ATR/Chk1 signaling controlled G(2) arrest in MMR-deficient cells, ATR/Chk1 activation was not involved in MMR-dependent G(2) arrest. Instead, we discovered that disrupting c-Abl activity using STI571 (Gleevec, a c-Abl inhibitor) or stable c-Abl knockdown abolished MMR-dependent p73alpha stabilization, induction of GADD45alpha protein expression, and G(2) arrest. In addition, inhibition of c-Abl also increased the survival of MNNG-exposed MMR-proficient cells to a level comparable with MMR-deficient cells. Furthermore, knocking down GADD45alpha (but not p73alpha) protein levels affected MMR-dependent G(2) arrest responses. Thus, MMR-dependent G(2) arrest responses triggered by MNNG are dependent on a human MLH1/c-Abl/GADD45alpha signaling pathway and activity. Furthermore, our data suggest that caution should be taken with therapies targeting c-Abl kinase because increased survival of mutator phenotypes may be an unwanted consequence.

Mornings with Art, lessons learned: feedback regulation, restriction threshold biology, and redundancy govern molecular stress responses.

Work from the laboratory of Dr. Arthur B. Pardee has highlighted basic principles that govern cellular and molecular biological processes in living cells. Among the most important governing principles in cellular and molecular responses are: (i) threshold "restriction" responses, wherein a level of response is reached and a "point of no return" is achieved; (ii) feedback regulation; and (iii) redundancy. Lessons learned from the molecular biology of cellular stress responses in mammalian cancer versus normal cells after ionizing radiation (IR) or chemotherapeutic agent exposures reveal similar instances of these guiding principles in mammalian cells. Among these are the: (i) induction of cell death responses by beta-lapachone (beta-lap), a naphthoquinone anti-tumor agent that kills cancer cells via an NQO1 (i.e., X-ray-inducible protein-3, xip3)-dependent mechanism; (ii) induction of secretory clusterin (sCLU) in response to TGF-beta1 exposure, and the ability of induced sCLU protein to down-regulate TGF-beta1 signaling; and (iii) induction of DNA mismatch repair-dependent G(2) cell cycle checkpoint responses after exposure to alkylating agents. We have learned these lessons and now adopted strategies to exploit them for improved therapy. These examples will be discussed and compared to the pioneering findings of researchers in the Pardee laboratory over the years.

DNA mismatch repair-dependent response to fluoropyrimidine-generated damage.

Previous studies from our laboratory indicated that expression of the MLH1 DNA mismatch repair (MMR) gene was necessary to restore cytotoxicity and an efficient G(2) arrest in HCT116 human colon cancer cells, as well as Mlh1(-/-) murine embryonic fibroblasts, after treatment with 5-fluoro-2'-deoxyuridine (FdUrd). Here, we show that an identical phenomenon occurred when expression of MSH2, the other major MMR gene, was restored in HEC59 human endometrial carcinoma cells or was present in adenovirus E1A-immortalized Msh2(+/+) (compared with isogenic Msh2(-/-)) murine embryonic stem cells. Because MMR status had little effect on cellular responses (i.e. G(2) arrest and lethality) to the thymidylate synthase inhibitor, Tomudex, and a greater level of [(3)H]FdUrd incorporation into DNA was found in MMR-deficient cells, we concluded that the differential FdUrd cytotoxicity between MMR-competent and MMR-deficient cells was mediated at the level of DNA incorporation. Analyses of ATPase activation suggested that the hMSH2-hMSH6 heterodimer only recognized FdUrd moieties (as the base 5-fluorouracil (FU) in DNA) when mispaired with guanine, but not paired with adenine. Furthermore, analyses of incorporated FdUrd using methyl-CpG-binding domain 4 glycosylase indicated that there was more misincorporated FU:Gua in the DNA of MMR-deficient HCT116 cells. Our data provide the first demonstration that MMR specifically detects FU:Gua (in the first round of DNA replication), signaling a sustained G(2) arrest and lethality.

Role of DNA mismatch repair in apoptotic responses to therapeutic agents.

Deficiencies in DNA mismatch repair (MMR) have been found in both hereditary cancer (i.e., hereditary nonpolyposis colorectal cancer) and sporadic cancers of various tissues. In addition to its primary roles in the correction of DNA replication errors and suppression of recombination, research in the last 10 years has shown that MMR is involved in many other processes, such as interaction with other DNA repair pathways, cell cycle checkpoint regulation, and apoptosis. Indeed, a cell's MMR status can influence its response to a wide variety of chemotherapeutic agents, such as temozolomide (and many other methylating agents), 6-thioguanine, cisplatin, ionizing radiation, etoposide, and 5-fluorouracil. For this reason, identification of a tumor's MMR deficiency (as indicated by the presence of microsatellite instability) is being utilized more and more as a prognostic indicator in the clinic. Here, we describe the basic mechanisms of MMR and apoptosis and investigate the literature examining the influence of MMR status on the apoptotic response following treatment with various therapeutic agents. Furthermore, using isogenic MMR-deficient (HCT116) and MMR-proficient (HCT116 3-6) cells, we demonstrate that there is no enhanced apoptosis in MMR-proficient cells following treatment with 5-fluoro-2'-deoxyuridine. In fact, apoptosis accounts for only a small portion of the induced cell death response.

A role for DNA mismatch repair in sensing and responding to fluoropyrimidine damage.

The phenomenon of damage tolerance, whereby cells incur DNA lesions that are nonlethal, largely ignored, but highly mutagenic, appears to play a key role in carcinogenesis. Typically, these lesions are generated by alkylation of DNA or incorporation of base analogues. This tolerance is usually a result of the loss of specific DNA repair processes, most often DNA mismatch repair (MMR). The availability of genetically matched MMR-deficient and -corrected cell systems allows dissection of the consequences of this unrepaired damage in carcinogenesis as well as the elucidation of cell cycle checkpoint responses and cell death consequences. Recent data indicate that MMR plays an important role in detecting damage caused by fluorinated pyrimidines (FPs) and represents a repair system that is probably not the primary system for detecting damage caused by these agents, but may be an important system for correcting key mutagenic lesions that could initiate carcinogenesis. In fact, clinical studies have shown that there is no benefit of FP-based adjuvant chemotherapy in colon cancer patients exhibiting microsatellite instability, a hallmark of MMR deficiency. MMR-mediated damage tolerance and futile cycle repair processes are discussed, as well as possible strategies using FPs to exploit these systems for improved anticancer therapy.