MDM4 Isoform Expression in Melanoma Supports an Oncogenic Role for MDM4-A.

The p53 tumor suppressor integrates upstream signals such as DNA damage and active oncogenes to initiate cell cycle arrest or apoptosis. This response is critical to halting inappropriate growth signals. As such, p53 activity is lost in cancer. In melanoma, however, the p53 gene is intact in a reported 94% of human cases. Rather than direct mutation, p53 is held inactive through interaction with inhibitory proteins. Here, we examine the expression of the two primary inhibitors of p53, MDM2 and MDM4, in genomic databases and biopsy specimens. We find that MDM4 is frequently overexpressed. Moreover, changes in splicing of MDM4 occur frequently and early in melanomagenesis. These changes in splicing must be considered in the design of therapeutic inhibitors of the MDM2/4 proteins for melanoma.

The atypical MAPK ERK3 potently suppresses melanoma cell growth and invasiveness.

Mitogen-activated protein kinase 6 (MAPK6) represents an atypical MAPK also known as extracellular signal-regulated kinase 3 (ERK3), which has been shown to play roles in cell motility and metastasis. ERK3 promotes migration and invasion of lung cancer cells and head and neck cancer cells by regulating the expression and/or activity of proteins involved in cancer progression. For instance, ERK3 upregulates matrix metallopeptidases and thereby promotes cancer cell invasiveness, and it phosphorylates tyrosyl-DNA phosphodiesterase 2, thereby enhancing chemoresistance in lung cancer. Here we discovered that ERK3 plays a converse role in melanoma. We observed that BRAF, an oncogenic Ser/Thr kinase, upregulates ERK3 expression levels by increasing both ERK3 messenger RNA levels and protein stability. Interestingly, although BRAF's kinase activity was required for upregulating ERK3 gene transcription, BRAF stabilized ERK3 protein in a kinase-independent fashion. We further demonstrate that ERK3 inhibits the migration, proliferation and colony formation of melanoma cells. In line with this, high level of ERK3 predicted increased survival among patients with melanomas. Taken together, these results indicate that ERK3 acts as a potent suppressor of melanoma cell growth and invasiveness.

Girardia dorotocephala transcriptome sequence, assembly, and validation through characterization of piwi homologs and stem cell progeny markers.

Planarian flatworms are popular models for the study of regeneration and stem cell biology in vivo. Technical advances and increased availability of genetic information have fueled the discovery of molecules responsible for stem cell pluripotency and regeneration in flatworms. Unfortunately, most of the planarian research performed worldwide utilizes species that are not natural habitants of North America, which limits their availability to newcomer laboratories and impedes their distribution for educational activities. In order to circumvent these limitations and increase the genetic information available for comparative studies, we sequenced the transcriptome of Girardia dorotocephala, a planarian species pandemic and commercially available in North America. A total of 254,802,670 paired sequence reads were obtained from RNA extracted from intact individuals, regenerating fragments, as well as freshly excised auricles of a clonal line of G. dorotocephala (MA-C2), and used for de novo assembly of its transcriptome. The resulting transcriptome draft was validated through functional analysis of genetic markers of stem cells and their progeny in G. dorotocephala. Akin to orthologs in other planarian species, G. dorotocephala Piwi1 (GdPiwi1) was found to be a robust marker of the planarian stem cell population and GdPiwi2 an essential component for stem cell-driven regeneration. Identification of G. dorotocephala homologs of the early stem cell descendent marker PROG-1 revealed a family of lysine-rich proteins expressed during epithelial cell differentiation. Sequences from the MA-C2 transcriptome were found to be 98-99% identical to nucleotide sequences from G. dorotocephala populations with different chromosomal number, demonstrating strong conservation regardless of karyotype evolution. Altogether, this work establishes G. dorotocephala as a viable and accessible option for analysis of gene function in North America.

Vulvodynia: What We Know and Where We Should Be Going.

OBJECTIVE: The aim of the study was to review the current nomenclature and literature examining microbiome cytokine, genomic, proteomic, and glycomic molecular biomarkers in identifying markers related to the understanding of the pathophysiology and diagnosis of vulvodynia (VVD). MATERIALS AND METHODS: Computerized searches of MEDLINE and PubMed were conducted focused on terminology, classification, and "omics" variations of VVD. Specific MESH terms used were VVD, vestibulodynia, metagenomics, vaginal fungi, cytokines, gene, protein, inflammation, glycomic, proteomic, secretomic, and genomic from 2001 to 2016. Using combined VVD and vestibulodynia MESH terms, 7 references were identified related to vaginal fungi, 15 to cytokines, 18 to gene, 43 to protein, 38 to inflammation, and 2 to genomic. References from identified publications were manually searched and cross-referenced to identify additional relevant articles. A narrative synthesis of the articles was conducted; however, meta-analysis was not conducted because of substantial heterogeneity in the studies and limited numbers of control-matched studies. RESULTS: Varying definitions of VVD complicate a meta-analysis, and standard definitions will better allow for comparisons of studies and enhance the applicability of evidence to patient populations. Although data are still limited, genomic and molecular diagnostic testings continue to be investigated as potential tools for the diagnosis of VVD. CONCLUSIONS: Standardized nomenclature will allow for comparability of studies and progress in research related to the pathophysiology of VVD and to facilitate clinical decision making and treatment choices. Although the current understanding of the pathogenesis of VVD is limited, there are new opportunities to explore potential diagnostic markers differences in women with VVD, which may lead to targeted therapy.

Sarcoptes scabiei mites modulate gene expression in human skin equivalents.

The ectoparasitic mite, Sarcoptes scabiei that burrows in the epidermis of mammalian skin has a long co-evolution with its hosts. Phenotypic studies show that the mites have the ability to modulate cytokine secretion and expression of cell adhesion molecules in cells of the skin and other cells of the innate and adaptive immune systems that may assist the mites to survive in the skin. The purpose of this study was to identify genes in keratinocytes and fibroblasts in human skin equivalents (HSEs) that changed expression in response to the burrowing of live scabies mites. Overall, of the more than 25,800 genes measured, 189 genes were up-regulated >2-fold in response to scabies mite burrowing while 152 genes were down-regulated to the same degree. HSEs differentially expressed large numbers of genes that were related to host protective responses including those involved in immune response, defense response, cytokine activity, taxis, response to other organisms, and cell adhesion. Genes for the expression of interleukin-1α (IL-1α) precursor, IL-1ß, granulocyte/macrophage-colony stimulating factor (GM-CSF) precursor, and G-CSF precursor were up-regulated 2.8- to 7.4-fold, paralleling cytokine secretion profiles. A large number of genes involved in epithelium development and keratinization were also differentially expressed in response to live scabies mites. Thus, these skin cells are directly responding as expected in an inflammatory response to products of the mites and the disruption of the skin's protective barrier caused by burrowing. This suggests that in vivo the interplay among these skin cells and other cell types, including Langerhans cells, dendritic cells, lymphocytes and endothelial cells, is responsible for depressing the host's protective response allowing these mites to survive in the skin.

MicroRNA-34a modulates MDM4 expression via a target site in the open reading frame.

BACKGROUND: MDM4, also called MDMX or HDMX in humans, is an important negative regulator of the p53 tumor suppressor. MDM4 is overexpressed in about 17% of all cancers and more frequently in some types, such as colon cancer or retinoblastoma. MDM4 is known to be post-translationally regulated by MDM2-mediated ubiquitination to decrease its protein levels in response to genotoxic stress, resulting in accumulation and activation of p53. At the transcriptional level, MDM4 gene regulation has been less clearly understood. We have reported that DNA damage triggers loss of MDM4 mRNA and a concurrent increase in p53 activity. These experiments attempt to determine a mechanism for down-regulation of MDM4 mRNA. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that MDM4 mRNA is a target of hsa-mir-34a (miR-34a). MDM4 mRNA contains a lengthy 3' untranslated region; however, we find that it is a miR-34a site within the open reading frame (ORF) of exon 11 that is responsible for the repression. Overexpression of miR-34a, but not a mutant miR-34a, is sufficient to decrease MDM4 mRNA levels to an extent identical to those of known miR-34a target genes. Likewise, MDM4 protein levels are decreased by miR-34a overexpression. Inhibition of endogenous miR-34a increased expression of miR-34a target genes and MDM4. A portion of MDM4 exon 11 containing this 8mer-A1 miR-34a site fused to a luciferase reporter gene is sufficient to confer responsiveness, being inhibited by additional expression of exogenous mir-34a and activated by inhibition of miR-34a. CONCLUSIONS/SIGNIFICANCE: These data establish a mechanism for the observed DNA damage-induced negative regulation of MDM4 and potentially provide a novel means to manipulate MDM4 expression without introducing DNA damage.

Regulation of MDM4.

Mouse double minute 4 (MDM4), also known as MDMX or HDMX (human MDMX), is a critical negative regulator of the tumor suppressor p53. Under normal growth conditions, MDM4 contributes to the repression of p53 activity. Upon DNA damage, it becomes important to down-regulate MDM4 to allow a full p53 response. Here, the mechanisms by which MDM4 activity is controlled are reviewed and discussed, starting with alterations in copy number, then control of transcription, mRNA stability, translation, and finally post-translational interactions, modifications, localization, and targeting by recently developed drugs.

Int7G24A variant of transforming growth factor-beta receptor type I is associated with invasive breast cancer.

PURPOSE: The transforming growth factor-beta (TGF-beta) signaling pathway has been frequently implicated in breast cancer. An intronic variant (Int7G24A) of TGF-beta receptor type I (TGFBR1) is associated with kidney and bladder cancers in our recent study. We hypothesize that this germline variant may be involved in development and progression of breast cancer. EXPERIMENTAL DESIGN: Case-control studies were designed from archived paraffin-embedded tissue specimens from the same geographic area with a homogenous ethnic population. We analyzed 223 patients (25 with preinvasive tumors and 198 with invasive and metastatic breast cancers) and 153 noncancer controls. The Int7G24A was identified by PCR-RFLP. Another germline deletion (TGFBR1*6A) and somatic mutations in the TGFBR1 were also analyzed by PCR and single-strand conformational polymorphism. RESULTS: The Int7G24A allele was evident in 32% of patients with preinvasive neoplasms and 48% of patients with invasive breast cancers compared with 26% controls (P = 0.00008). In addition, 11 (5.6%) homozygous Int7G24A carriers were found in patients with invasive breast cancers, whereas only 3 (2%) homozygous carriers were found in the control group. The TGFBR1*6A allele was not significantly associated with breast cancer patients and only one somatic mutation was found in 71 breast cancers. CONCLUSION: These data suggest that the germline Int7G24A variant may represent a risk factor for invasive breast cancer and a marker for breast cancer progression. A separate study with a larger sample size is warranted to validate the association of the Int7G24A with human breast cancer.

RB reversibly inhibits DNA replication via two temporally distinct mechanisms.

The retinoblastoma (RB) tumor suppressor is a critical negative regulator of cellular proliferation. Repression of E2F-dependent transcription has been implicated as the mechanism through which RB inhibits cell cycle progression. However, recent data have suggested that the direct interaction of RB with replication factors or sites of DNA synthesis may contribute to its ability to inhibit S phase. Here we show that RB does not exert a cis-acting effect on DNA replication. Furthermore, the localization of RB was distinct from replication foci in proliferating cells. While RB activation strongly attenuated the RNA levels of multiple replication factors, their protein expression was not diminished coincident with cell cycle arrest. During the first 24 h of RB activation, components of the prereplication complex, initiation factors, and the clamp loader complex (replication factor C) remained tethered to chromatin. In contrast, the association of PCNA and downstream components of the processive replication machinery was specifically disrupted. This signaling from RB occurred in a manner dependent on E2F-mediated transcriptional repression. Following long-term activation of RB, we observed the attenuation of multiple replication factors, the complete cessation of DNA synthesis, and impaired replicative capacity in vitro. Therefore, functional distinctions exist between the "chronic" RB-mediated arrest state and the "acute" arrest state. Strikingly, attenuation of RB activity reversed both acute and chronic replication blocks. Thus, continued RB action is required for the maintenance of two kinetically and functionally distinct modes of replication inhibition.

Unbiased analysis of RB-mediated transcriptional repression identifies novel targets and distinctions from E2F action.

The retinoblastoma tumor suppressor, RB, is thought to inhibit cell cycle progression through transcriptional repression. E2F-regulated genes have been viewed as presumptive targets of RB-mediated repression. However, we found that specific E2F targets were not regulated in a consistent manner by the action of a RB allele that is refractory to cyclin-dependent kinase/cyclin-mediated phosphorylation (PSM-RB) when compared with E2F2 overproduction. Therefore, we used Affymetrix GeneChips as an unbiased approach to identify RB targets. We found that expression of PSM-RB significantly attenuates >200 targets, the majority of which are involved in cell cycle control (DNA replication or G2-M), DNA repair, or transcription/chromatin structure. The observed repression was due to the action of RB and not merely a manifestation of altered cell cycle distribution. Additionally, the majority of RB repression targets were confirmed through the blockade of endogenous RB phosphorylation via p16ink4a overexpression. Thus, these results have utility in assigning RB pathway activation in more complex systems of cell cycle inhibition (e.g., mitogen withdrawal, senescence, or DNA damage checkpoint). As expected, a significant fraction of RB-repressed genes have promoters that are bound/regulated by E2F family members. However, targets were identified that are distinct from genes known to be stimulated by overexpression of specific E2F proteins. Moreover, the relative action of RB versus E2F2 overexpression on specific genes demonstrates that a simple opposition model does not explain the relative contribution of RB to gene regulation. Thus, this study provides the first unbiased description of RB-repressed genes, thereby delineating new aspects of RB-mediated transcriptional control and novel targets involved in diverse cellular processes.

Retinoblastoma tumor suppressor targets dNTP metabolism to regulate DNA replication.

The retinoblastoma tumor suppressor, RB, is a negative regulator of the cell cycle that is inactivated in the majority of human tumors. Cell cycle inhibition elicited by RB has been attributed to the attenuation of CDK2 activity. Although ectopic cyclins partially overcome RB-mediated S-phase arrest at the replication fork, DNA replication remains inhibited and cells fail to progress to G(2) phase. These data suggest that RB regulates an additional execution point in S phase. We observed that constitutively active RB attenuates the expression of specific dNTP synthetic enzymes: dihydrofolate reductase, ribonucleotide reductase (RNR) subunits R1/R2, and thymidylate synthase (TS). Activation of endogenous RB and related proteins by p16ink4a yielded similar effects on enzyme expression. Conversely, targeted disruption of RB resulted in increased metabolic protein levels (dihydrofolate reductase, TS, RNR-R2) and conferred resistance to the effect of TS or RNR inhibitors that diminish available dNTPs. Analysis of dNTP pools during RB-mediated cell cycle arrest revealed significant depletion, concurrent with the loss of TS and RNR protein. Importantly, the effect of active RB on cell cycle position and available dNTPs was comparable to that observed with specific antimetabolites. Together, these results show that RB-mediated transcriptional repression attenuates available dNTP pools to control S-phase progression. Thus, RB employs both canonical cyclin-dependent kinase/cyclin regulation and metabolic regulation as a means to limit proliferation, underscoring its potency in tumor suppression.

Active RB elicits late G1/S inhibition.

The retinoblastoma tumor suppressor protein (RB) is activated/dephosphorylated to mediate cell cycle inhibition in response to antimitogenic signals. To elucidate the mode of RB action at this critical transition, we utilized cell lines that can be induced to express a constitutively active allele of RB (PSM-RB). As expected, induction of PSM-RB, but not wild-type protein (WT), inhibited progression into S phase. It has been well documented that active RB inhibits E2F reporter activity, and this observation was confirmed upon induction of PSM-RB. Additionally, active RB inhibited E2F-2-mediated stimulation of cyclin E. By contrast, PSM-RB did not affect the mRNA or protein levels of endogenous cyclin E when mediating cell cycle inhibition. Similarly, there was no observable effect on cyclin E protein levels when p16ink4a was utilized to activate endogenous RB. CDK2/cyclin E complex formation was not disrupted and cyclin E-associated kinase activity was retained in the presence of PSM-RB. Additionally, centrosome duplication, a CDK2/cyclin E-dependent event, was not altered in the presence of active RB. Together, these data indicate that active RB does not block the G1/S transition through inhibition of cyclin E expression or activity. In contrast, PSM-RB leads to a dramatic reduction in cyclin A protein levels by coordinate transcriptional repression and degradation. This attenuation of cyclin A protein correlates with cell cycle inhibition. These studies indicate that RB inhibits cell cycle progression by targeting CDK2/cyclin A-dependent events at the G1/S transition to inhibit cell cycle progression.