Deletion Hotspots in AMACR Promoter CpG Island Are cis-Regulatory Elements Controlling the Gene Expression in the Colon

Alpha-methylacyl-coenzyme A racemase (AMACR) regulates peroxisomal β-oxidation of phytol-derived, branched-chain fatty acids from red meat and dairy products — suspected risk factors for colon carcinoma (CCa). AMACR was first found overexpressed in prostate cancer but not in benign glands and is now an established diagnostic marker for prostate cancer. Aberrant expression of AMACR was recently reported in Cca; however, little is known about how this gene is abnormally activated in cancer. By using a panel of immunostained-laser-capture-microdissected clinical samples comprising the entire colon adenoma–carcinoma sequence, we show that deregulation of AMACR during colon carcinogenesis involves two nonrandom events, resulting in the mutually exclusive existence of double-deletion at CG3 and CG10 and deletion of CG12-16 in a newly identified CpG island within the core promoter of AMACR. The double-deletion at CG3 and CG10 was found to be a somatic lesion. It existed in histologically normal colonic glands and tubular adenomas with low AMACR expression and was absent in villous adenomas and all CCas expressing variable levels of AMACR. In contrast, deletion of CG12-16 was shown to be a constitutional allele with a frequency of 43% in a general population. Its prevalence reached 89% in moderately differentiated CCas strongly expressing AMACR but only existed at 14% in poorly differentiated CCas expressing little or no AMACR. The DNA sequences housing these deletions were found to be putative cis-regulatory elements for Sp1 at CG3 and CG10, and ZNF202 at CG12-16. Chromatin immunoprecipitation, siRNA knockdown, gel shift assay, ectopic expression, and promoter analyses supported the regulation by Sp1 and ZNF202 of AMACR gene expression in an opposite manner. Our findings identified key in vivo events and novel transcription factors responsible for AMACR regulation in CCas and suggested these AMACR deletions may have diagnostic/prognostic value for colon carcinogenesis

Estrogen receptor beta expression in prostate adenocarcinoma

<p>Abstract</p> <p>Background</p> <p>Prostate cancer is the most commonly diagnosed cancer in men and the second leading cause of cancer death in men. Estrogen induction of cell proliferation is a crucial step in carcinogenesis of gynecologic target tissues, and there are many studies recently done, showing that prostate cancer growth is also influenced by estrogen. The characterization of estrogen receptor beta (ER-b) brought new insight into the mechanisms underlying estrogen signalling. In the present study, we investigated the expression of estrogen receptor-b (ER-b) in human prostate cancer tissues.</p> <p>Methods</p> <p>We selected 52 paraffin-embedded blocks of prostate needle biopsies in a cross-sectional study to determine frequency and rate of ER-b expression in different grades of prostate adenocarcinoma according to Gleason grading system. Immunohistochemical staining of tissue sections by monoclonal anti ER-b antibody was performed using an Envision method visualising system.</p> <p>Results</p> <p>ER-b expression was seen in tumoral cells of prostatic carcinoma in all 29 cases with low and intermediate tumors (100%) and 19 of 23 cases with high grade tumor (83%). Mean rate of ER-b expression in low & intermediate grade cancers was 68.41% (SD = 25.63) whereas high grade cancers showed 49.48% rate of expression (SD = 28.79).</p> <p>Conclusions</p> <p>ER-b expression is reduced in high grade prostate cancers compared to low & intermediate grade ones (<it>P </it>value 0.027).</p

Cryptosporidium Priming Is More Effective than Vaccine for Protection against Cryptosporidiosis in a Murine Protein Malnutrition Model

Cryptosporidium is a major cause of severe diarrhea, especially in malnourished children. Using a murine model of C. parvum oocyst challenge that recapitulates clinical features of severe cryptosporidiosis during malnutrition, we interrogated the effect of protein malnutrition (PM) on primary and secondary responses to C. parvum challenge, and tested the differential ability of mucosal priming strategies to overcome the PM-induced susceptibility. We determined that while PM fundamentally alters systemic and mucosal primary immune responses to Cryptosporidium, priming with C. parvum (106 oocysts) provides robust protective immunity against re-challenge despite ongoing PM. C. parvum priming restores mucosal Th1-type effectors (CD3+CD8+CD103+ T-cells) and cytokines (IFNγ, and IL12p40) that otherwise decrease with ongoing PM. Vaccination strategies with Cryptosporidium antigens expressed in the S. Typhi vector 908htr, however, do not enhance Th1-type responses to C. parvum challenge during PM, even though vaccination strongly boosts immunity in challenged fully nourished hosts. Remote non-specific exposures to the attenuated S. Typhi vector alone or the TLR9 agonist CpG ODN-1668 can partially attenuate C. parvum severity during PM, but neither as effectively as viable C. parvum priming. We conclude that although PM interferes with basal and vaccine-boosted immune responses to C. parvum, sustained reductions in disease severity are possible through mucosal activators of host defenses, and specifically C. parvum priming can elicit impressively robust Th1-type protective immunity despite ongoing protein malnutrition. These findings add insight into potential correlates of Cryptosporidium immunity and future vaccine strategies in malnourished children

Phase I Evaluation of STA-1474, a Prodrug of the Novel HSP90 Inhibitor Ganetespib, in Dogs with Spontaneous Cancer

The novel water soluble compound STA-1474 is metabolized to ganetespib (formerly STA-9090), a potent HSP90 inhibitor previously shown to kill canine tumor cell lines in vitro and inhibit tumor growth in the setting of murine xenografts. The purpose of the following study was to extend these observations and investigate the safety and efficacy of STA-1474 in dogs with spontaneous tumors.This was a Phase 1 trial in which dogs with spontaneous tumors received STA-1474 under one of three different dosing schemes. Pharmacokinetics, toxicities, biomarker changes, and tumor responses were assessed. Twenty-five dogs with a variety of cancers were enrolled. Toxicities were primarily gastrointestinal in nature consisting of diarrhea, vomiting, inappetence and lethargy. Upregulation of HSP70 protein expression was noted in both tumor specimens and PBMCs within 7 hours following drug administration. Measurable objective responses were observed in dogs with malignant mast cell disease (n = 3), osteosarcoma (n = 1), melanoma (n = 1) and thyroid carcinoma (n = 1), for a response rate of 24% (6/25). Stable disease (>10 weeks) was seen in 3 dogs, for a resultant overall biological activity of 36% (9/25).This study provides evidence that STA-1474 exhibits biologic activity in a relevant large animal model of cancer. Given the similarities of canine and human cancers with respect to tumor biology and HSP90 activation, it is likely that STA-1474 and ganetespib will demonstrate comparable anti-cancer activity in human patients

Combination treatment with doxorubicin and gamitrinib synergistically augments anticancer activity through enhanced activation of Bim

Background: A common approach to cancer therapy in clinical practice is the combination of several drugs to boost the anticancer activity of available drugs while suppressing their unwanted side effects. In this regard, we examined the efficacy of combination treatment with the widely-used genotoxic drug doxorubicin and the mitochondriotoxic Hsp90 inhibitor gamitrinib to exploit disparate stress signaling pathways for cancer therapy.Methods: The cytotoxicity of the drugs as single agents or in combination against several cancer cell types was analyzed by MTT assay and the synergism of the drug combination was evaluated by calculating the combination index. To understand the molecular mechanism of the drug synergism, stress signaling pathways were analyzed after drug combination. Two xenograft models with breast and prostate cancer cells were used to evaluate anticancer activity of the drug combination in vivo. Cardiotoxicity was assessed by tissue histology and serum creatine phosphokinase concentration.Results: Gamitrinib sensitized various human cancer cells to doxorubicin treatment, and combination treatment with the two drugs synergistically increased apoptosis. The cytotoxicity of the drug combination involved activation and mitochondrial accumulation of the proapoptotic Bcl-2 family member Bim. Activation of Bim was associated with increased expression of the proapoptotic transcription factor C/EBP-homologous protein and enhanced activation of the stress kinase c-Jun N-terminal kinase. Combined drug treatment with doxorubicin and gamitrinib dramatically reduced in vivo tumor growth in prostate and breast xenograft models without increasing cardiotoxicity.Conclusions: The drug combination showed synergistic anticancer activities toward various cancer cells without aggravating the cardiotoxic side effects of doxorubicin, suggesting that the full therapeutic potential of doxorubicin can be unleashed through combination with gamitrinib.open

Targeted inhibition of mitochondrial Hsp90 suppresses localised and metastatic prostate cancer growth in a genetic mouse model of disease

BACKGROUND: The molecular chaperone heat shock protein-90 (Hsp90) is a promising cancer drug target, but current Hsp90-based therapy has so far shown limited activity in the clinic. METHODS: We tested the efficacy of a novel mitochondrial-targeted, small-molecule Hsp90 inhibitor, Gamitrinib (GA mitochondrial matrix inhibitor), in the Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model. The TRAMP mice receiving 3-week or 5-week systemic treatment with Gamitrinib were evaluated for localised or metastatic prostate cancer, prostatic intraepithelial neoplasia (PIN) or localised inflammation using magnetic resonance imaging, histology and immunohistochemistry. Treatment safety was assessed histologically in organs collected at the end of treatment. The effect of Gamitrinib on mitochondrial dysfunction was studied in RM1 cells isolated from TRAMP tumours. RESULTS: Systemic administration of Gamitrinib to TRAMP mice inhibited the formation of localised prostate tumours of neuroendocrine or adenocarcinoma origin, as well as metastatic prostate cancer to abdominal lymph nodes and liver. The Gamitrinib treatment had no effect on PIN or prostatic inflammation, and caused no significant animal weight loss or organ toxicity. Mechanistically, Gamitrinib triggered acute mitochondrial dysfunction in RM1 cells, with loss of organelle inner membrane potential and release of cytochrome-c in the cytosol. CONCLUSIONS: The Gamitrinib has pre-clinical activity and favourable tolerability in a genetic model of localised and metastatic prostate cancer in immunocompetent mice. Selective targeting of mitochondrial Hsp90 could provide novel molecular therapy for patients with advanced prostate cancer

Relationship of coregulator and oestrogen receptor isoform expression to de novo tamoxifen resistance in human breast cancer

This study addresses the hypothesis that altered expression of oestrogen receptor-beta and/or altered relative expression of coactivators and corepressors of oestrogen receptors are associated with and may be mechanisms of de novo tamoxifen resistance in oestrogen receptor positive breast cancer. All cases were oestrogen receptor +, node negative, primary breast tumours from patients who later had no disease progression (tamoxifen sensitive) or whose disease progressed while on tamoxifen (tamoxifen resistant). Using an antibody to oestrogen receptor-beta that detects multiple forms of this protein (total) but not an antibody that detects only full-length oestrogen receptor-beta 1, it was found that high total oestrogen receptor beta protein expressors were more frequently observed in tamoxifen sensitive tumours than resistant tumours (Fisher's exact test, P=0.046). However, no significant differences in the relative expression of oestrogen receptor β2, oestrogen receptor β5 and full-length oestrogen receptor β1 RNA in the tamoxifen sensitive and resistant groups were found. Also, when the relative expression of two known coactivators, steroid receptor RNA activator and amplified in breast cancer 1 RNA to the known corepressor, repressor of oestrogen receptor activity RNA, was examined, no significant differences between the tamoxifen sensitive and resistant groups were found. Altogether, there is little evidence for altered coregulators expression in breast tumours that are de novo tamoxifen resistant. However, our data provide preliminary evidence that the expression of oestrogen receptor β protein isoforms may differ in primary tumours of breast cancer patients who prove to have differential sensitivity to tamoxifen therapy

Hsa-miRNA-765 as a key mediator for inhibiting growth, migration and invasion in fulvestrant-treated prostate cancer

Fulvestrant (ICI-182,780) has recently been shown to effectively suppress prostate cancer cell growth in vitro and in vivo. But it is unclear whether microRNAs play a role in regulating oncogene expression in fulvestrant-treated prostate cancer. Here, this study reports hsa-miR-765 as the first fulvestrant-driven, ERβ-regulated miRNA exhibiting significant tumor suppressor activities like fulvestrant, against prostate cancer cell growth via blockage of cell-cycle progression at the G2/M transition, and cell migration and invasion possibly via reduction of filopodia/intense stress-fiber formation. Fulvestrant was shown to upregulate hsa-miR-765 expression through recruitment of ERβ to the 5′-regulatory-region of hsa-miR-765. HMGA1, an oncogenic protein in prostate cancer, was identified as a downstream target of hsa-miR-765 and fulvestrant in cell-based experiments and a clinical study. Both the antiestrogen and the hsa-miR-765 mimic suppressed HMGA1 protein expression. In a neo-adjuvant study, levels of hsa-miR-765 were increased and HMGA1 expression was almost completely lost in prostate cancer specimens from patients treated with a single dose (250 mg) of fulvestrant 28 days before prostatectomy. These findings reveal a novel fulvestrant signaling cascade involving ERβ-mediated transcriptional upregulation of hsa-miR-765 that suppresses HMGA1 protein expression as part of the mechanism underlying the tumor suppressor action of fulvestrant in prostate cancer. © 2014 Leung et al

High Diversity of Cryptosporidium Subgenotypes Identified in Malaysian HIV/AIDS Individuals Targeting gp60 Gene

BACKGROUND: Currently, there is a lack of vital information in the genetic makeup of Cryptosporidium especially in developing countries. The present study aimed at determining the genotypes and subgenotypes of Cryptosporidium in hospitalized Malaysian human immunodeficiency virus (HIV) positive patients. METHODOLOGY/PRINCIPAL FINDINGS: In this study, 346 faecal samples collected from Malaysian HIV positive patients were genetically analysed via PCR targeting the 60 kDa glycoprotein (gp60) gene. Eighteen (5.2% of 346) isolates were determined as Cryptosporidium positive with 72.2% (of 18) identified as Cryptosporidium parvum whilst 27.7% as Cryptosporidium hominis. Further gp60 analysis revealed C. parvum belonging to subgenotypes IIaA13G1R1 (2 isolates), IIaA13G2R1 (2 isolates), IIaA14G2R1 (3 isolates), IIaA15G2R1 (5 isolates) and IIdA15G1R1 (1 isolate). C. hominis was represented by subgenotypes IaA14R1 (2 isolates), IaA18R1 (1 isolate) and IbA10G2R2 (2 isolates). CONCLUSIONS/SIGNIFICANCE: These findings highlighted the presence of high diversity of Cryptosporidium subgenotypes among Malaysian HIV infected individuals. The predominance of the C. parvum subgenotypes signified the possibility of zoonotic as well as anthroponotic transmissions of cryptosporidiosis in HIV infected individuals