Andrew P. Trotta

Cumulin, an oocyte-secreted heterodimer of the transforming growth factor-β family, is a potent activator of granulosa cells and improves oocyte quality

Background: Cumulin is a newly identified heterodimeric member of the TGF-β family. Results: Mature cumulin potently stimulates granulosa cell signaling and function, whereas pro-cumulin promotes oocyte quality. Conclusion: Formation of cumulin and its potent actions are likely to be central to oocyte paracrine signaling and mammalian fecundity. Significance: The discovery of cumulin provides unique opportunities to improve female fertility in mammals. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-specific growth factors with central roles in mammalian reproduction, regulating species-specific fecundity, ovarian follicular somatic cell differentiation, and oocyte quality. In the human, GDF9 is produced in a latent form, the mechanism of activation being an open question. Here, we produced a range of recombinant GDF9 and BMP15 variants, examined their in silico and physical interactions and their effects on ovarian granulosa cells (GC) and oocytes. We found that the potent synergistic actions of GDF9 and BMP15 on GC can be attributed to the formation of a heterodimer, which we have termed cumulin. Structural modeling of cumulin revealed a dimerization interface identical to homodimeric GDF9 and BMP15, indicating likely formation of a stable complex. This was confirmed by generation of recombinant heterodimeric complexes of pro/mature domains (pro-cumulin) and covalent mature domains (cumulin). Both pro-cumulin and cumulin exhibited highly potent bioactivity on GC, activating both SMAD2/3 and SMAD1/5/8 signaling pathways and promoting proliferation and expression of a set of genes associated with oocyte-regulated GC differentiation. Cumulin was more potent than pro-cumulin, pro-GDF9, pro-BMP15, or the two combined on GC. However, on cumulus-oocyte complexes, pro-cumulin was more effective than all other growth factors at notably improving oocyte quality as assessed by subsequent day 7 embryo development. Our results support a model of activation for human GDF9 dependent on cumulin formation through heterodimerization with BMP15. Oocyte-secreted cumulin is likely to be a central regulator of fertility in mono-ovular mammals.

Mitochondrial dynamics as regulators of cancer biology

Mitochondria are dynamic organelles that supply energy required to drive key cellular processes, such as survival, proliferation, and migration. Critical to all of these processes are changes in mitochondrial architecture, a mechanical mechanism encompassing both fusion and fragmentation (fission) of the mitochondrial network. Changes to mitochondrial shape, size, and localization occur in a regulated manner to maintain energy and metabolic homeostasis, while deregulation of mitochondrial dynamics is associated with the onset of metabolic dysfunction and disease. In cancers, oncogenic signals that drive excessive proliferation, increase intracellular stress, and limit nutrient supply are all able to alter the bioenergetic and biosynthetic requirements of cancer cells. Consequently, mitochondrial function and shape rapidly adapt to these hostile conditions to support cancer cell proliferation and evade activation of cell death programs. In this review, we will discuss the molecular mechanisms governing mitochondrial dynamics and integrate recent insights into how changes in mitochondrial shape affect cellular migration, differentiation, apoptosis, and opportunities for the development of novel targeted cancer therapies.

Hic-5 influences genomic and non-genomic actions of the androgen receptor in prostate myofibroblasts.

There is extensive knowledge of androgen receptor (AR) signaling in cancer cells, but less regarding androgen action in stromal cells of the tumor microenvironment. We report here the genome-wide effects of a stromal cell specific molecular adapter and AR coregulator, hydrogen peroxide-inducible gene 5 (Hic-5/TGFB1I1), on AR function in prostate myofibroblasts. Following androgen stimulation, Hic-5 rapidly translocates to the nucleus, coincident with increased phosphorylation of focal adhesion kinase. As a coregulator, Hic-5 acted to amplify or inhibit regulation of approximately 50% of AR target genes, affected androgen regulation of growth, cell adhesion, motility and invasion. These data suggest Hic-5 as a transferable adaptor between focal adhesions and the nucleus of prostate myofibroblasts, where it acts a key mediator of the specificity and sensitivity of AR signaling. We propose a model in which Hic-5 coordinates AR signaling with adhesion and extracellular matrix contacts to regulate cell behavior in the tumor microenvironment.

A pro-tumourigenic loop at the human prostate tumour interface orchestrated by oestrogen, CXCL12 and mast cell recruitment.

Prostate cancer is hormone‐dependent and regulated by androgens as well as oestrogens. The tumour microenvironment also provides regulatory control, but the balance and interplay between androgens and oestrogens at the human prostate tumour interface is unknown. This study reveals a central and dominant role for oestrogen in the microenvironment, fuelling a pro‐tumourigenic loop of inflammatory cytokines involving recruitment of mast cells by carcinoma‐associated fibroblasts (CAFs). Mast cell numbers were increased in human PCa clinical specimens, specifically within the peritumoural stroma. Human mast cells were also shown to express ERα and ERβ, with oestradiol directly stimulating mast cell proliferation and migration as well as altered cytokine/chemokine expression. There was a significant shift in the oestrogen:androgen balance in CAFs versus normal prostatic fibroblasts (NPFs), with a profound increase to ER:AR expression. Androgen signalling is also reduced in CAFs, while ERα and ERβ transcriptional activity is not, allowing oestrogen to dictate hormone action in the tumour microenvironment. Gene microarray analyses identified CXCL12 as a major oestrogen‐driven target gene in CAFs, and CAFs recruit mast cells via CXCL12 in a CXCR4‐dependent manner. Collectively, these data reveal multicellular oestrogen action in the tumour microenvironment and show dominant oestrogen, rather than androgen, signalling at the prostatic tumour interface. Copyright

Subdomain structure of the co-chaperone SGTA and activity of its androgen receptor client

Ligand-dependent activity of steroid receptors is affected by tetratricopeptide repeat (TPR)-containing co-chaperones, such as small glutamine-rich tetratricopeptide repeat-containing alpha (SGTA). However, the precise mechanisms by which the predominantly cytoplasmic TPR proteins affect downstream transcriptional outcomes of steroid signaling remain unclear. In this study, we assessed how SGTA affects ligand sensitivity and action of the androgen receptor (AR) using a transactivation profiling approach. Deletion mapping coupled with structural prediction, transcriptional assays, and in vivo regulation of AR-responsive promoters were used to assess the role of SGTA domains in AR responses. At subsaturating ligand concentrations of ≤ 0.1 nM 5α-dihydrotestosterone, SGTA overexpression constricted AR activity by an average of 32% (P<0.002) across the majority of androgen-responsive loci tested, as well as on endogenous promoters in vivo. The strength of the SGTA effect was associated with the presence or absence of bioinformatically predicated transcription factor motifs at each site. Homodimerizaion of SGTA, which is thought to be necessary for chaperone complex formation, was found to be dependent on the structural integrity of amino acids 1-80, and a core evolutionary conserved peptide within this region (amino acids 21-40) necessary for an effect of SGTA on the activity of both exogenous and endogenous AR. This study provides new insights into the subdomain structure of SGTA and how SGTA acts as a regulator of AR ligand sensitivity. A change in AR:SGTA ratio will impact the cellular and molecular response of prostate cancer cells to maintain androgenic signals, which may influence tumor progression.

Knockdown of the cochaperone SGTA results in the suppression of androgen and PI3K/Akt signaling and inhibition of prostate cancer cell proliferation

Solid tumors have an increased reliance on Hsp70/Hsp90 molecular chaperones for proliferation, survival and maintenance of intracellular signaling systems. An underinvestigated component of the chaperone system is the tetratricopeptide repeat (TPR)‐containing cochaperone, which coordinates Hsp70/Hsp90 involvement on client proteins as well as having diverse individual actions. A potentially important cochaperone in prostate cancer (PCa) is small glutamine‐rich TPR‐containing protein alpha (SGTA), which interacts with the androgen receptor (AR) and other critical cancer‐related client proteins. In this study, the authors used small interfering RNA coupled with genome‐wide expression profiling to investigate the biological significance of SGTA in PCa and its influence on AR signaling. Knockdown of SGTA for 72 hr in PCa C4‐2B cells significantly altered expression of >1,900 genes (58% decreased) and reduced cell proliferation (p < 0.05). The regulation of 35% of 5α‐dihydrotestosterone (DHT) target genes was affected by SGTA knockdown, with gene‐specific effects on basal or DHT‐induced expression or both. Pathway analysis revealed a role for SGTA in p53, generic PCa and phosphoinositol kinase (PI3K) signaling pathways; the latter evident by a reduction in PI3K subunit p100β levels and decreased phosphorylated Akt. Immunohistochemical analysis of 64 primary advanced PCa samples showed a significant increase in the AR:SGTA ratio in cancerous lesions compared to patient‐matched benign prostatic hyperplasia tissue (p < 0.02). This study not only provides insight into the biological actions of SGTA and its effect on genome‐wide AR transcriptional activity and other therapeutically targeted intracellular signaling pathways but also provides evidence for PCa‐specific alterations in SGTA expression.

The unique transcriptional response produced by concurrent estrogen and progesterone treatment in breast cancer cells results in upregulation of growth factor pathways and switching from a Luminal A to a Basal-like subtype

BackgroundIn breast cancer, progesterone receptor (PR) positivity or abundance is positively associated with survival and treatment response. It was initially believed that PR was a useful diagnostic marker of estrogen receptor activity, but increasingly PR has been recognised to play an important biological role in breast homeostasis, carcinogenesis and metastasis. Although PR expression is almost exclusively observed in estrogen receptor positive tumors, few studies have investigated the cellular mechanisms of PR action in the context of ongoing estrogen signalling.MethodsIn this study, we contrast PR function in estrogen pretreated ZR-75-1 breast cancer cells with vehicle treated ZR-75-1 and T-47D breast cancer cells using expression microarrays and chromatin immunoprecipitation-sequencing.ResultsEstrogen cotreatment caused a dramatic increase in the number of genes regulated by progesterone in ZR-75-1 cells. In T-47D cells that have naturally high levels of PR, estrogen and progesterone cotreatment resulted in a reduction in the number of regulated genes in comparison to treatment with either hormone alone. At a genome level, estrogen pretreatment of ZR-75-1 cells led to a 10-fold increase in the number of PR DNA binding sites detected using ChIP-sequencing. Time course assessment of progesterone regulated genes in the context of estrogen pretreatment highlighted a series of important regulatory pathways, including those driven by epithelial growth factor receptor (EGFR). Importantly, progesterone applied to cells pretreated with estradiol resulted in switching of the PAM50-determined intrinsic breast cancer subtype from Luminal A to Basal-like, and increased the Oncotype DX® Unscaled Recurrence Score.ConclusionEstrogen pretreatment of breast cancer cells increases PR steady state levels, resulting in an unequivocal progesterone response that upregulates key members of growth factor pathways. The transformative changes progesterone exerts on the breast cancer subtype suggest that these subtyping tools should be used with caution in premenopausal women.

Wnt blockade with dickkopf reduces intestinal crypt fission and intestinal growth in infant rats.

Objectives: Intestinal crypt fission peaks during infancy. In human and experimental familial polyposis coli, increased crypt fission is due to activation of Wnt/&bgr;-catenin signalling, but the molecular basis of crypt fission during intestinal growth has not been examined. The aim of this project was to investigate whether crypt fission and intestinal growth are affected by experimental blockade of the Wnt/&bgr;-catenin signalling pathway. Methods: Hooded Wistar rats were given either the Wnt inhibitor, dickkopf (30 and 100 ng), daily or vehicle control intraperitoneally from days 11 to 15 and were killed at day 16. Intestinal morphometry was used to measure villous area, crypt area, percentage of crypt fission, and crypt mitotic count. Intestinal stem cells were assessed by expression of real time-polymerase chain reaction for Lgr5 (a stem cell marker), and the number of &bgr;-catenin–expressing crypts by immunostaining was determined after 100-ng dickkopf treatment. Results: Dickkopf at 30 and 100 ng/day reduced villous area to 71% (P = 0.013) and 29% (P < 0.0001), crypt area to 42% (P = 0.0026) and 30% (P = 0.0067), and crypt fission to 51% (P = 0.006) and 29% (P < 0.0001), respectively, of control values. Mitotic count per crypt did not change. Lgr5 RNA expression and the number of &bgr;-catenin–expressing crypts decreased in dickkopf-treated animals. Conclusions: We conclude that intestinal crypt fission during infancy is mediated by Wnt signalling. It is possible that local treatment with Wnt agonists could be used to increase intestinal growth.

Disruption of mitochondrial electron transport chain function potentiates the pro-apoptotic effects of MAPK inhibition.

The mitochondrial network is a major site of ATP production through the coupled integration of the electron transport chain (ETC) with oxidative phosphorylation. In melanoma arising from the V600E mutation in the kinase v-RAF murine sarcoma viral oncogene homolog B (BRAFV600E), oncogenic signaling enhances glucose-dependent metabolism while reducing mitochondrial ATP production. Likewise, when BRAFV600E is pharmacologically inhibited by targeted therapies (e.g. PLX-4032/vemurafenib), glucose metabolism is reduced, and cells increase mitochondrial ATP production to sustain survival. Therefore, collateral inhibition of oncogenic signaling and mitochondrial respiration may help enhance the therapeutic benefit of targeted therapies. Honokiol (HKL) is a well tolerated small molecule that disrupts mitochondrial function; however, its underlying mechanisms and potential utility with targeted anticancer therapies remain unknown. Using wild-type BRAF and BRAFV600E melanoma model systems, we demonstrate here that HKL administration rapidly reduces mitochondrial respiration by broadly inhibiting ETC complexes I, II, and V, resulting in decreased ATP levels. The subsequent energetic crisis induced two cellular responses involving cyclin-dependent kinases (CDKs). First, loss of CDK1-mediated phosphorylation of the mitochondrial division GTPase dynamin-related protein 1 promoted mitochondrial fusion, thus coupling mitochondrial energetic status and morphology. Second, HKL decreased CDK2 activity, leading to G1 cell cycle arrest. Importantly, although pharmacological inhibition of oncogenic MAPK signaling increased ETC activity, co-treatment with HKL ablated this response and vastly enhanced the rate of apoptosis. Collectively, these findings integrate HKL action with mitochondrial respiration and shape and substantiate a pro-survival role of mitochondrial function in melanoma cells after oncogenic MAPK inhibition.

The prognostic value of stromal FK506-binding protein 1 and androgen receptor in prostate cancer outcome

Improving our ability to predict cancer progression and response to conservative or radical intent therapy is critical if we are to prevent under or over treatment of individual patients. Whereas the majority of solid tumors now have a range of molecular and/or immunological markers to help define prognosis and treatment options, prostate cancer still relies mainly on histological grading and clinical parameters. We have recently reported that androgen receptor (AR) expression in stroma inversely associates with prostate cancer‐specific survival, and that stromal AR reduces metastasis. For this paper, we tested the hypothesis that the AR‐regulated gene FKBP51 could be used as a marker of AR activity to better predict outcome.