A catalog of curated breast cancer genes.

PURPOSE: Decades of research have identified multiple genetic variants associated with breast cancer etiology. However, there is no database that archives breast cancer genes and variants responsible for predisposition. We set out to build a dynamic repository of curated breast cancer genes. METHODS: A comprehensive literature search was performed in PubMed and Google Scholar, followed by data extraction and harmonization for downstream analysis. RESULTS: Using a subset of 345 studies, we cataloged 652 breast cancer-associated loci across the genome. A majority of these were present in the non-coding region (i.e., intergenic (101) and intronic (345)), whereas only 158 were located within an exon. Using the odds ratio, we identified 429 loci to increase the disease risk and 198 to confer protection against breast cancer, whereas 25 were identified to both increase disease risk and confer protection against breast cancer. Chromosomal ideogram analysis indicated that chromosomes 17 and 19 have the highest density of breast cancer loci. We manually annotated and collated breast cancer genes in which a previous association between rare-monogenic variant and breast cancer has been documented. Finally, network and functional enrichment analysis revealed that steroid metabolism and DNA repair pathways were predominant among breast cancer genes and variants. CONCLUSIONS: We have built an online interactive catalog of curated breast cancer genes ( https://cbcg.dk ). This will expedite clinical diagnostics and support the ongoing efforts in managing breast cancer etiology. Moreover, the database will serve as an essential repository when designing new breast cancer multigene panels.

Identification of E6AP-independent degradation targets of HPV E6.

The high-risk Human Papillomavirus (HPV) E6 oncoprotein is known to contribute to human malignancy by targeting several of its cellular substrates through the ubiquitin-mediated degradation pathway. Previous studies have revealed that E6 interacts with the E6AP ubiquitin-protein ligase and directs its ubiquitylation activity toward several specific cellular proteins, one of the most important of which is p53. However, the role of E6AP in the degradation of many other E6 substrates is still ambiguous because loss of E6AP also induces a loss of E6 expression. To examine this further, we used CRISPR-edited E6AP knockout cells to perform E6 degradation assays in the presence of a catalytically inactive mutant form of E6AP, thus ensuring the stabilization of E6 but with the ligase itself being functionally inactive. Using this system, we found that E6 can mediate the degradation of several PDZ domain-containing proteins independently of E6AP ubiquitin ligase activity. This study thus opens up ways to investigate other possible components of the cellular ubiquitin proteasome pathway that E6 might utilize to target these substrates.

The Human Papillomavirus E6 PDZ Binding Motif Links DNA Damage Response Signaling to E6 Inhibition of p53 Transcriptional Activity.

The presence of a PDZ binding motif (PBM) in the human papillomavirus (HPV) E6 oncoprotein appears to be a characteristic marker of high oncogenic potential and confers interaction with a number of different cellular PDZ domain-containing substrates. The E6 PBM is also subject to phosphorylation, resulting in inhibition of E6 PDZ binding activity and instead allowing E6 to associate with 14-3-3 proteins. In this study, we analyzed the conditions under which the E6 PBM is phosphorylated. We demonstrate that in normal cycling cells, the levels of E6 phosphorylation are very low. However, following exposure of cells to oxidative stress or the induction of DNA damage, there is a striking increase in the levels of E6 phosphorylation. Depending on the specific stimulus, this phosphorylation of E6 can involve the ATM/ATR pathway and is performed primarily through Chk1, although the Chk2 pathway is also involved indirectly through activation of protein kinase A (PKA). To understand the biological relevance of these phospho-modifications of E6, we analyzed their effects upon the ability of E6 to inhibit p53 transcriptional activity. We show that an intact E6 phospho-acceptor site plays an essential role in the ability of E6 to inhibit p53 transcriptional activity on a subset of p53-responsive promoters in a manner that is independent of E6's ability to direct p53 degradation. These results are, to our knowledge, the first example of a DNA damage response controlling PBM-PDZ recognition. This study also provides links between the DNA damage response, the regulation of E6 PBM function, and the inhibition of p53 activity and begins to explain how HPV-infected cells remain within the cell cycle, despite activation of DNA damage response pathways during productive virus infections.IMPORTANCE The cancer-causing HPV E6 oncoproteins all possess a PDZ binding motif at their extreme carboxy termini. Depending upon whether this motif is phosphorylated, E6 can recognize PDZ domain-containing proteins or members of the 14-3-3 family of proteins. We show here that DNA damage response pathways directly signal to the E6 PBM, resulting in Chk1- and Chk2-driven phosphorylation. This phosphorylation is particularly pronounced following treatment of cells with a variety of different chemotherapeutic drugs. A direct functional consequence of this signaling is to confer an enhanced ability upon E6 to inhibit p53 transcriptional activity in a proteasome-independent but phosphorylation-dependent manner. These results are the first example of DNA damage signaling pathways regulating PBM-PDZ interactions and provide the mechanistic link between E6 PBM function and perturbation of p53 activity.

Infectivity of adeno-associated virus serotypes in mouse testis.

BACKGROUND: Recombinant adeno-associated viruses (AAVs) are emerging as favoured transgene delivery vectors for both research applications and gene therapy. In this context, a thorough investigation of the potential of various AAV serotypes to transduce specific cell types is valuable. Here, we rigorously tested the infectivity of a number of AAV serotypes in murine testis by direct testicular injection. RESULTS: We report the tropism of serotypes AAV2, 5, 8, 9 and AAVrh10 in mouse testis. We reveal unique infectivity of AAV2 and AAV9, which preferentially target intertubular testosterone-producing Leydig cells. Remarkably, AAV2 TM, a mutant for capsid designed to increase transduction, displayed a dramatic alteration in tropism; it infiltrated seminiferous tubules unlike wildtype AAV2 and transduced Sertoli cells. However, none of the AAVs tested infected spermatogonial cells. CONCLUSIONS: In spite of direct testicular injection, none of the tested AAVs appeared to infect sperm progenitors as assayed by reporter expression. This lends support to the current view that AAVs are safe gene-therapy vehicles. However, testing the presence of rAAV genomic DNA in germ cells is necessary to assess the risk of individual serotypes.

Human Papillomavirus 16 (HPV-16), HPV-18, and HPV-31 E6 Override the Normal Phosphoregulation of E6AP Enzymatic Activity.

The human papillomavirus (HPV) E6 oncoproteins recruit the cellular ubiquitin ligase E6AP/UBE3A to target cellular substrates for proteasome-mediated degradation, and one consequence of this activity is the E6 stimulation of E6AP autoubiquitination and degradation. Recent studies identified an autism-linked mutation within E6AP at T485, which was identified as a protein kinase A phosphoacceptor site and which could directly regulate E6AP ubiquitin ligase activity. In this study, we have analyzed how T485-mediated regulation of E6AP might affect E6 targeting of some of its known substrates. We show that modulation of T485 has no effect on the ability of E6 to direct either p53 or Dlg for degradation. Furthermore, T485 regulation has no effect on HPV-16 or HPV-31 E6-induced autodegradation of E6AP but does affect HPV-18 E6-induced autodegradation of E6AP. In cells derived from cervical cancers, we find low levels of both phosphorylated and nonphosphorylated E6AP in the nucleus. However, ablation of E6 results in a dramatic accumulation of phospho-E6AP in the cytoplasm, whereas nonphosphorylated E6AP accumulates primarily in the nucleus. Interestingly, E6AP phosphorylation at T485 confers association with 14-3-3 proteins, and this interaction seems to be important, in part, for the ability of E6 to recruit phospho-E6AP into the nucleus. These results demonstrate that HPV E6 overrides the normal phosphoregulation of E6AP, both in terms of its enzymatic activity and its subcellular distribution.IMPORTANCE Recent reports demonstrate the importance of phosphoregulation of E6AP for its normal enzymatic activity. Here, we show that HPV E6 is capable of overriding this regulation and can promote degradation of p53 and Dlg regardless of the phosphorylation status of E6AP. Furthermore, E6 interaction with E6AP also significantly alters how E6AP is subject to autodegradation and suggests that this is not a simple stimulation of an already-existing activity but rather a redirection of E6AP activity toward itself. Furthermore, E6-mediated regulation of the subcellular distribution of phospho-E6AP appears to be dependent, in part, upon the 14-3-3 family of proteins.

Molecular dissection of the E6 PBM identifies essential residues regulating Chk1 phosphorylation and subsequent 14-3-3 recognition.

Previous studies have shown that the high-risk HPV E6 oncoprotein PDZ binding motifs (PBMs) can interact with PDZ proteins or members of the 14-3-3 family, depending upon the E6 phosphorylation status. However, different HPV E6 oncoproteins are subjected to phosphorylation by different cellular kinases. We have therefore been interested in determining whether we can dissect E6's PDZ and 14-3-3 interactions at the molecular level. Using HPV-18 E6, we have found that its Chk1 phosphorylation requires residues both upstream and downstream of the phospho-acceptor site, in addition to the Chk1 consensus recognition motif. Furthermore, we demonstrate that different high-risk HPV E6 types are differentially phosphorylated by Chk1 kinases, potentially due to the differences in their carboxy-terminal residues, as they are critical for kinase recognition. Moreover, differences in the E6 phosphorylation levels of different HR HPV types directly link to their ability to interact with different 14-3-3 isoforms, based on their phospho-status. Interestingly, 14-3-3 recognition appears to be less dependent upon the precise sequence constraints of the E6 carboxy terminal region, whilst minor amino acid variations have a major impact upon PDZ recognition. These results demonstrate that changes in E6 phospho-status during the life cycle or during malignant progression will modulate E6 interactions and, potentially, inversely regulate the levels of PDZ and 14-3-3 proteins.

Inhibition of kinase IKKß suppresses cellular abnormalities induced by the human papillomavirus oncoprotein HPV 18E6.

Human papillomavirus (HPV) is the leading cause of cervical cancer and has been implicated in several other cancer types including vaginal, vulvar, penile, and oropharyngeal cancers. Despite the recent availability of a vaccine, there are still over 310,000 deaths each year worldwide. Current treatments for HPV-mediated cancers show limited efficacy, and would benefit from improved understanding of disease mechanisms. Recently, we developed a Drosophila 'HPV 18 E6' model that displayed loss of cellular morphology and polarity, junctional disorganization, and degradation of the major E6 target Magi; we further provided evidence that mechanisms underlying HPV E6-induced cellular abnormalities are conserved between humans and flies. Here, we report a functional genetic screen of the Drosophila kinome that identified IKK[Formula: see text]-a regulator of NF-κB-as an enhancer of E6-induced cellular defects. We demonstrate that inhibition of IKK[Formula: see text] reduces Magi degradation and that this effect correlates with hyperphosphorylation of E6. Further, the reduction in IKK[Formula: see text] suppressed the cellular transformation caused by the cooperative action of HPVE6 and the oncogenic Ras. Finally, we demonstrate that the interaction between IKK[Formula: see text] and E6 is conserved in human cells: inhibition of IKK[Formula: see text] blocked the growth of cervical cancer cells, suggesting that IKK[Formula: see text] may serve as a novel therapeutic target for HPV-mediated cancers.

RhoC regulates radioresistance via crosstalk of ROCK2 with the DNA repair machinery in cervical cancer.

BACKGROUND: Radioresistance remains a challenge to the successful treatment of various tumors. Intrinsic factors like alterations in signaling pathways regulate response to radiation. RhoC, which has been shown to modulate several tumor phenotypes has been investigated in this report for its role in radioresistance. In vitro and clinical sample-based studies have been performed to understand its contribution to radiation response in cervical cancer and this is the first report to establish the role of RhoC and its effector ROCK2 in cervical cancer radiation response. METHODS: Biochemical, transcriptomic and immunological approaches including flow cytometry and immunofluorescence were used to understand the role of RhoC and ROCK2. RhoC variants, siRNA and chemical inhibitors were used to alter the function of RhoC and ROCK2. Transcriptomic profiling was performed to understand the gene expression pattern of the cells. Live sorting using an intracellular antigen has been developed to isolate the cells for transcriptomic studies. RESULTS: Enhanced expression of RhoC conferred radioprotection on the tumor cells while inhibition of RhoC resulted in sensitization of cells to radiation. The RhoC overexpressing cells had a better DNA repair machinery as observed using transcriptomic analysis. Similarly, overexpression of ROCK2, protected tumor cells against radiation while its inhibition increased radiosensitivity in vitro. Further investigations revealed that ROCK2 inhibition abolished the radioresistance phenotype, conferred by RhoC on SiHa cells, confirming that it is a downstream effector of RhoC in this context. Additionally, transcriptional analysis of the live sorted ROCK2 high and ROCK2 low expressing SiHa cells revealed an upregulation of the DNA repair pathway proteins. Consequently, inhibition of ROCK2 resulted in reduced expression of pH2Ax and MRN complex proteins, critical to repair of double strand breaks. Clinical sample-based studies also demonstrated that ROCK2 inhibition sensitizes tumor cells to irradiation. CONCLUSIONS: Our data primarily indicates that RhoC and ROCK2 signaling is important for the radioresistance phenotype in cervical cancer tumor cells and is regulated via association of ROCK2 with the proteins of DNA repair pathway involving pH2Ax, MRE11 and RAD50 proteins, partly offering insights into the mechanism of radioresistance in tumor cells. These findings highlight RhoC-ROCK2 signaling involvement in DNA repair and urge the need for development of these molecules as targets to alleviate the non-responsiveness of cervical cancer tumor cells to irradiation treatment.

The Human Papillomavirus E6 PDZ Binding Motif: From Life Cycle to Malignancy.

Cancer-causing HPV E6 oncoproteins are characterized by the presence of a PDZ binding motif (PBM) at their extreme carboxy terminus. It was long thought that this region of E6 had a sole function to confer interaction with a defined set of cellular substrates. However, more recent studies have shown that the E6 PBM has a complex pattern of regulation, whereby phosphorylation within the PBM can regulate interaction with two classes of cellular proteins: those containing PDZ domains and the members of the 14-3-3 family of proteins. In this review, we explore the roles that the PBM and its ligands play in the virus life cycle, and subsequently how these can inadvertently contribute towards the development of malignancy. We also explore how subtle alterations in cellular signal transduction pathways might result in aberrant E6 phosphorylation, which in turn might contribute towards disease progression.