E2F1 confers anticancer drug resistance by targeting ABC transporter family members and Bcl-2 via the p73/DNp73-miR-205 circuitry

Resistance to anti-neoplastic agents is the major cause of therapy failure, leading to disease recurrence and metastasis. E2F1 is a strong inducer of apoptosis in response to DNA damage through its capacity to activate p53/p73 death pathways. Recent evidence, however, showed that E2F1, which is aberrantly expressed in advanced malignant melanomas together with antagonistic p73 family members, drives cancer progression. Investigating mechanisms responsible for dysregulated E2F1 losing its apoptotic function, we searched for genomic signatures in primary and late clinical tumor stages to allow the prediction of downstream effectors associated with apoptosis resistance and survival of aggressive melanoma cells. We identified miR-205 as specific target of p73 and found that upon genotoxic stress, its expression is sufficiently abrogated by endogenous DNp73. Significantly, metastatic cells can be rescued from drug resistance by selective knockdown of DNp73 or overexpression of miR-205 in p73-depleted cells, leading to increased apoptosis and the reduction of tumor growth in vivo. Our data delineate an autoregulatory circuit, involving high levels of E2F1 and DNp73 to downregulate miR-205, which, in turn, controls E2F1 accumulation. Finally, drug resistance associated to this genetic signature is mediated by removing the inhibitory effect of miR-205 on the expression of Bcl-2 and the ATP-binding cassette transporters A2 (ABCA2) and A5 (ABCA5) related to multi-drug resistance and malignant progression. These results define the E2F1-p73/DNp73-miR-205 axis as a crucial mechanism for chemoresistance and, thus, as a target for metastasis prevention.     

miRNA-205 suppresses melanoma cell proliferation and induces senescence via regulation of E2F1 protein

MicroRNAs (miRNAs) regulate gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Here, we report that expression of miR-205 is significantly suppressed in melanoma specimens when compared with nevi and is correlated inversely with melanoma progression. miRNA target databases predicted E2F1 and E2F5 as putative targets. The expression levels of E2F1 and E2F5 were correlated inversely with that of miR-205 in melanoma cell lines. miR-205 significantly suppressed the luciferase activity of reporter plasmids containing the 3'-UTR sequences complementary to either E2F1 or E2F5. Overexpression of miR-205 in melanoma cells reduced E2F1 and E2F5 protein levels. The proliferative capacity of melanoma cells was suppressed by miR-205 and mediated by E2F-regulated AKT phosphorylation. miR-205 overexpression resulted in induction of apoptosis, as evidenced by increased cleaved caspase-3, poly-(ADP-ribose) polymerase, and cytochrome c release. Stable overexpression of miR-205 suppressed melanoma cell proliferation, colony formation, and tumor cell growth in vivo and induced a senescence phenotype accompanied by elevated expression of p16INK4A and other markers for senescence. E2F1 overexpression in miR-205-expressing cells partially reversed the effects on melanoma cell growth and senescence. These results demonstrate a novel role for miR-205 as a tumor suppressor in melanoma.     

Regulation of HSF1-responsive gene expression by N-terminal truncated form of p73alpha

DNp73 is a transactivation domain (TAD)-truncated form of p73. The ability of DNp73alpha to regulate gene expression was examined using reporter assays with luciferase gene constructs. Among various promoter-regulated reporter genes tested, heat shock factor (HSF)-responsive gene expression was selectively activated by DNp73alpha, but not by other p73-isoforms with TAD and DNp73beta. Deletion of TAD endowed p73alpha with the ability to activate HSF-responsive gene expression, but deletion of N-terminal proline-rich domain (PRD) rendered both DNp73alpha and the TAD-deleted p73alpha inactive. Considering the inability of DNp73beta, which is the C-terminus-truncated form of DNp73alpha, to function, these results indicate that both the PRD and C-terminus are necessary for DNp73alpha to be able to activate the HSF-dependent gene expression. In addition to the reporter gene expression, both DNp73alpha and TAD-deleted p73alpha activated the expression of an endogenous gene, hsp70, corresponding with an increase in the active form of HSF1. Taken together, these results demonstrate that TAD-truncated p73alpha can activate HSF-dependent gene expression via induction of active HSF1.     

Hypoxia-induced DNp73 stabilization regulates Vegf-A expression and tumor angiogenesis similar to TAp73

P73, the homolog of p53, exists in 2 major forms: either as a pro-apoptotic TAp73 or an amino-terminally truncated DNp73, the latter lacking the first transactivation domain. While TAp73s tumor suppressive functions have been established, DNp73 is an anti-apoptotic protein conferring chemoresistance and is associated with poor survival. However, both forms are variably overexpressed in many human cancers. In this context, we have recently demonstrated that TAp73 is stabilized by hypoxia, a tumor-relevant condition that is associated with cell survival, via HIF-1α-mediated suppression of Siah1 E3 ligase that degrades TAp73. Consequently, hypoxic signals lead to TAp73-mediated activation of several angiogenic genes and blood vessel formation, thereby supporting tumorigenesis. We show here that, similar to TAp73, DNp73 is stabilized by hypoxia in a HIF-1α-dependent manner, which otherwise is degraded by Siah1. Moreover, DNp73 is capable of inducing the expression of Vegf-A, the prototypic angiogenic gene, and loss of DNp73 expression results in reduction in tumor vasculature and size. These data therefore indicate a common mode of regulation for both p73 forms by hypoxia, resulting in the promotion of angiogenesis and tumor growth, highlighting common functionality of these antagonistic proteins under specific physiological contexts.     

DNp73 a matter of cancer: Mechanisms and clinical implications

The p53 family proteins carry on a wide spectrum of biological functions from differentiation, cell cycle arrest, apoptosis, and chemosensitivity of tumors. NH2-terminally truncated p73 (referred to as DNp73) acts as a potent inhibitor of all these tumor suppressor properties, implying that it has oncogenic functions in human tumorigenesis. This was favored by the observation that high DNp73 expression levels in a variety of cancers are associated with adverse clinico-pathological characteristics and the response failure to chemotherapy. The actual challenge is the deciphering of the molecular mechanisms by which DNp73 promotes malignancy and to unravel the regulatory pathways for controlling TP73 isoform expression. This review is focused on recent findings leaving no doubt that N-terminally truncated p73 proteins are operative during oncogenesis, thus underscoring its significance as a marker for disease severity in patients and as target for cancer therapy.     

Mechanisms,function and clinical applications of DNp73

p73, has two distinct promoters, which allow the formation of two protein isoforms: full-length transactivating (TA) p73 and an N-terminally truncated p73 species (referred to as DNp73) that lacks the N-terminal transactivating domain. Although the exact cellular function of DNp73 is unclear, the high expression levels of the genes have been observed in a variety of human cancers and cancer cell lines and have been connected to pro-tumor activities. Hence the aim of this review is to summarize DNp73 expression status in cancer in the current literature. Furthermore, we also focused on recent findings of DNp73 related to the biological functions from apoptosis, chemosensitivity, radiosensitibity, differentiation, development, etc. Thus this review highlights the significance of DNp73 as a marker for disease severity in patients and as target for cancer therapy.     

Downregulation of miR-205 and miR-31 confers resistance to chemotherapy-induced apoptosis in prostate cancer cells

Advanced prostate cancers are known to acquire not only invasive capabilities but also significant resistance to chemotherapy-induced apoptosis. To understand how microRNAs (miRNAs) may contribute to prostate cancer resistance to apoptosis, we compared microRNA expression profiles of a benign prostate cancer cell line WPE1-NA22 and a highly malignant WPE1-NB26 cell line (derived from a common lineage). We found that miR-205 and miR-31 are significantly downregulated in WPE1-NB26 cells, as well as in other cell lines representing advanced-stage prostate cancers. Antiapoptotic genes BCL2L2 (encoding Bcl-w) and E2F6 are identified as the targets of miR-205 and miR-31, respectively. By downregulating Bcl-w and E2F6, miR-205 and miR-31 promote chemotherapeutic agents-induced apoptosis in prostate cancer cells. The promoter region of the miR-205 gene was cloned and was found to be hypermethylated in cell lines derived from advanced prostate cancers, contributing to the downregulation of the gene. Treatment with DNA methylation inhibitor 5-aza-2′-deoxycytidine induced miR-205 expression, downregulated Bcl-w, and sensitized prostate cancer cells to chemotherapy-induced apoptosis. Thus, downregulation of miR-205 and miR-31 has an important role in apoptosis resistance in advanced prostate cancer.     

DNp73 a matter of cancer: mechanisms and clinical implications.

The p53 family proteins carry on a wide spectrum of biological functions from differentiation, cell cycle arrest, apoptosis, and chemosensitivity of tumors. NH2-terminally truncated p73 (referred to as DNp73) acts as a potent inhibitor of all these tumor suppressor properties, implying that it has oncogenic functions in human tumorigenesis. This was favored by the observation that high DNp73 expression levels in a variety of cancers are associated with adverse clinico-pathological characteristics and the response failure to chemotherapy. The actual challenge is the deciphering of the molecular mechanisms by which DNp73 promotes malignancy and to unravel the regulatory pathways for controlling TP73 isoform expression. This review is focused on recent findings leaving no doubt that N-terminally truncated p73 proteins are operative during oncogenesis, thus underscoring its significance as a marker for disease severity in patients and as target for cancer therapy.     

MCPH1/BRIT1 cooperates with E2F1 in the activation of checkpoint, DNA repair and apoptosis

Microcephalin (MCPH1) has a crucial role in the DNA damage response by promoting the expression of Checkpoint kinase 1 (CHK1) and Breast cancer susceptibility gene 1 (BRCA1); however, the mechanism of this regulation remains unclear. Here, we show that MCPH1 regulates CHK1 and BRCA1 through the interaction with E2F1 on the promoters of both genes. MCPH1 also regulates other E2F target genes involved in DNA repair and apoptosis such as RAD51, DDB2, TOPBP1, p73 and caspases. MCPH1 interacts with E2F1 on the p73 promoter, and regulates p73 induction and E2F1-induced apoptosis as a result of DNA damage. MCPH1 forms oligomers through the second and third BRCT domains. An MCPH1 mutant containing only its oligomerization domain has a dominant-negative role by blocking MCPH1 binding to E2F1. It also inhibits p73 induction in DNA damage and E2F1-dependent apoptosis. Taken together, MCPH1 cooperates with E2F1 to regulate genes involved in DNA repair, checkpoint and apoptosis, and might participate in the maintenance of genomic integrity.     

MiR-221 promotes trastuzumab-resistance and metastasis in HER2-positive breast cancers by targeting PTEN

HER2-overexpressing breast cancers are characterized by frequent distant metastasis and often develop resistance after short-term effective treatment with the monoclonal antibody drug, trastuzumab. Here, we found that the oncogenic miRNA, miR-221, inhibited apoptosis, induced trastuzumab resistance and promoted metastasis of HER2-positive breast cancers. The tumor suppressor PTEN was identified as a miR-221 target; overexpression of PTEN abrogated the aforementioned miR-221-induced malignant phenotypes of the cells. These findings indicate that miR-221 may promote trastuzumab resistance and metastasis of HER2-positive breast cancers by targeting PTEN, suggesting its role as a potential biomarker for progression and poor prognosis, and as a novel target for trastuzumab-combined treatment of breast cancers. [BMB Reports 2014; 47(5): 268-273].