No abstract

Tumor cells use preexisting prosurvival signaling pathways to evade the damaging and cytotoxic effects of anticancer agents. Radiation therapy is a primary form of cytotoxic anticancer treatment, but agents that successfully modify the radiation response in vivo are lacking. MicroRNAs (miRNA) are global gene regulators that play critical roles in oncogenesis and have been found to regulate prosurvival pathways. However, there is little understanding of how cellular miRNA expression affects the response of a cancer to cytotoxic therapy and ultimately outcome. The let-7 family of miRNAs regulates expression of oncogenes, such as RAS, and is specifically down-regulated in many cancer subtypes. In fact, low levels of let-7 predict a poor outcome in lung cancer. Here, we report that the let-7 family of miRNAs is overrepresented in a class of miRNAs exhibiting altered expression in response to radiation. More strikingly, we also can create a radiosensitive state when the select let-7 family of miRNAs is overexpressed in vitro in lung cancer cells and in vivo in a Caenorhabditis elegans model of radiation-induced cell death, whereas decreasing their levels causes radioresistance. In C. elegans, we show that this is partly through control of the protooncogene homologue let-60/RAS and genes in the DNA damage response pathway. These findings are the first direct evidence that miRNAs can suppress resistance to anticancer cytotoxic therapy, a common feature of cancer cells, and suggest that miRNAs may be a viable tool to augment current cancer therapies. [Cancer Res 2007;67(23):11111-6]

Doxorubicin (Adriamycin) is an effective anti-cancer drug, but its clinical usage is limited by a dose-dependent cardiotoxicity characterized by widespread sarcomere disarray and loss of myofilaments. Cardiac ankyrin repeat protein (CARP, ANKRD1) is a transcriptional regulatory protein that is extremely susceptible to doxorubicin; however, the mechanism(s) of doxorubicin-induced CARP depletion and its specific role in cardiomyocytes have not been completely defined. We report that doxorubicin treatment in cardiomyocytes resulted in inhibition of CARP transcription, depletion of CARP protein levels, inhibition of myofilament gene transcription, and marked sarcomere disarray. Knockdown of CARP with small interfering RNA (siRNA) similarly inhibited myofilament gene transcription and disrupted cardiomyocyte sarcomere structure. Adenoviral overexpression of CARP, however, was unable to rescue the doxorubicin-induced sarcomere disarray phenotype. Doxorubicin also induced depletion of the cardiac transcription factor GATA4 in cardiomyocytes. CARP expression is regulated in part by GATA4, prompting us to examine the relationship between GATA4 and CARP in cardiomyocytes. We show in co-transfection experiments that GATA4 operates upstream of CARP by activating the proximal CARP promoter. GATA4-siRNA knockdown in cardiomyocytes inhibited CARP expression and myofilament gene transcription, and induced extensive sarcomere disarray. Adenoviral overexpression of GATA4 (AdV-GATA4) in cardiomyocytes prior to doxorubicin exposure maintained GATA4 levels, modestly restored CARP levels, and attenuated sarcomere disarray. Interestingly, siRNA-mediated depletion of CARP completely abolished the Adv-GATA4 rescue of the doxorubicin-induced sarcomere phenotype. These data demonstrate co-dependent roles for GATA4 and CARP in regulating sarcomere gene expression and maintaining sarcomeric organization in cardiomyocytes in culture. The data further suggests that concurrent depletion of GATA4 and CARP in cardiomyocytes by doxorubicin contributes in large part to myofibrillar disarray and the overall pathophysiology of anthracycline cardiomyopathy.

The let-7 family of microRNAs (miRNAs) are important regulators of developmental timing and cell differentiation and are often misexpressed in human cancer. In C. elegans, let-7 controls cell fate transitions from larval stage 4 (L4) to adulthood by post-transcriptionally down-regulating lineage-abnormal 41 (lin-41) and hunchback-like 1 (hbl-1). Primary let-7 (pri-let-7) transcripts are up-regulated in the L3, yet little is known about what controls this transcriptional up-regulation. We sought factors that either turn on let-7 transcription or keep it repressed until the correct time. Here we report that one of let-7's targets, the transcription factor Hunchback-like 1 (HBL-1), is responsible for inhibiting the transcription of let-7 in specific tissues until the L3. hbl-1 is a known developmental timing regulator and inhibits adult development in larval stages. Therefore, one important function of HBL-1 in maintaining larval stage fates is inhibition of let-7. Indeed, our results reveal let-7 as the first known target of the HBL-1 transcription factor in C. elegans and suggest a negative feedback loop mechanism for let-7 and HBL-1 regulation.

No abstract

in myofilament Ca 2þ sensitivity was abolished by the expression of b-Tm. Furthermore, the McTnT 45-74 deletion-induced reduction in cooperativity of force production was more pronounced under a b-Tm background. Thus, our data shows that changes in the isoform expression of Tm modify T1dependent cardiac function, indicating that T1-Tm interactions exert a modulatory role in regulating cardiac thin filament activation.