HDAC 1 and 6 modulate cell invasion and migration in clear cell renal cell carcinoma

Indexación: Web of ScienceBackground: Class I histone deacetylases (HDACs) have been reported to be overexpressed in clear cell renal cell carcinoma (ccRCC), whereas the expression of class II HDACs is unknown. Methods: Four isogenic cell lines C2/C2VHL and 786-O/786-OVHL with differential VHL expression are used in our studies. Cobalt chloride is used to mimic hypoxia in vitro. HIF-2 alpha knockdowns in C2 and 786-O cells is used to evaluate the effect on HDAC 1 expression and activity. Invasion and migration assays are used to investigate the role of HDAC 1 and HDAC 6 expression in ccRCC cells. Comparisons are made between experimental groups using the paired T-test, the two-sample Student's T-test or one-way ANOVA, as appropriate. ccRCC and the TCGA dataset are used to observe the clinical correlation between HDAC 1 and HDAC 6 overexpression and overall and progression free survival. Results: Our analysis of tumor and matched non-tumor tissues from radical nephrectomies showed overexpression of class I and II HDACs (HDAC6 only in a subset of patients). In vitro, both HDAC1 and HDAC6 over-expression increased cell invasion and motility, respectively, in ccRCC cells. HDAC1 regulated invasiveness by increasing matrix metalloproteinase (MMP) expression. Furthermore, hypoxia stimulation in VHL-reconstituted cell lines increased HIF isoforms and HDAC1 expression. Presence of hypoxia response elements in the HDAC1 promoter along with chromatin immunoprecipitation data suggests that HIF-2 alpha is a transcriptional regulator of HDAC1 gene. Conversely, HDAC6 and estrogen receptor alpha (ER alpha) were co-localized in cytoplasm of ccRCC cells and HDAC6 enhanced cell motility by decreasing acetylated alpha-tubulin expression, and this biological effect was attenuated by either biochemical or pharmacological inhibition. Finally, analysis of human ccRCC specimens revealed positive correlation between HIF isoforms and HDAC. HDAC1 mRNA upregulation was associated with worse overall survival in the TCGA dataset. Conclusions: Taking together, these results suggest that HDAC1 and HDAC6 may play a role in ccRCC biology and could represent rational therapeutic targets.http://bmccancer.biomedcentral.com/articles/10.1186/s12885-016-2604-

Generation of a syngeneic orthotopic transplant model of prostate cancer metastasis. Oncoscience

ABSTRACT Progression to metastatic disease is the primary cause of mortality in men with prostate cancer (PCa). Mouse models which progress with spontaneous metastasis are limited. Such models would allow for extensive studies of molecular mechanisms of metastasis, and more definite pre-clinical therapy trials. Orthotopic murine models have been described; however a limiting biology of these models is their lack of an intact immune system. Within, we describe the development of an androgen sensitive and castrate resistant tractable orthotopic murine syngeneic (immune competent) model of prostate cancer. Both models develop primary tumors which spontaneously progress to metastatic disease in lymph tissue. These models will allow for more complete mechanistic and therapeutic studies in a short time period

Inhibition of Hsp90 augments docetaxel therapy in castrate resistant prostate cancer.

First line treatment of patients with castrate resistant prostate cancer (CRPC) primarily involves administration of docetaxel chemotherapy. Unfortunately, resistance to docetaxel therapy is an ultimate occurrence. Alterations in androgen receptor (AR) expression and signaling are associated mechanisms underlying resistance to docetaxel treatment in CRPC. Heat shock protein 90 (Hsp90) is a molecular chaperone, which regulates the activation, maturation and stability of critical signaling proteins involved in prostate cancer, including the AR. This knowledge and recent advances in compound design and development have highlighted Hsp90 as an attractive therapeutic target for the treatment of CRPC. We recently reported the development of a MYC-CaP castrate resistant (MYC-CaP/CR) transplant tumor model, which expresses amplified wild type AR. Within, we report that a second generation Hsp90 inhibitor, NVP-AUY922, inhibits cell growth and significantly induces cell death in MYC-CaP/CR and Pten-CaP/cE2 cell lines. NVP-AUY922 induced proteasome degradation of AR, though interestingly does not require loss of AR protein to inhibit AR transcriptional activity. Further, NVP-AUY922 increased docetaxel toxicity in MYC-CaP/CR and Pten-CaP/cE2 cell lines in vitro. Finally, NVP-AUY922/docetaxel combination therapy in mice bearing MYC-CaP/CR tumors resulted in greater anti-tumor activity compared to single treatment. This study demonstrates that NVP-AUY922 elicits potent activity towards AR signaling and augments chemotherapy response in a mouse model of CRPC, providing rationale for the continued clinical development of Hsp90 inhibitors in clinical trials for treatment of CRPC patients

AUY922 effects on AR expression and activity in castrate resistant prostate cancer cells.

<p>(<b>A</b>) MYC-CaP castrate resistant cell were treated for 48 hr with increasing concentrations of AUY922 or increasing concentrations of AUY922 concurrently with the proteasome inhibitor MG132 [0.5 µM]. Expression of AR protein was assessed by western blot. (<b>B</b>) MYC-CaP/CR cell lines with stable transfection of an AR reporter plasmid (ARE-Luc) were incubated in androgen depleted cell culture conditions for 6 hours. Cells were treated concurrently with 1 nM R1881 and indicated concentrations of AUY922 overnight or pre-treated with 1 nM R1881 for 4 hr before adding the indicated concentrations of AUY992 Luminescence intensity was measured and quantitated. Columns represent 3 independent experiments; mean ±SE. (<b>C</b>) Pten-CaP/cE2 cells were treated with increasing concentrations of AUY922 for 48 hr. Expression of AR protein was assessed by western blot. Transcriptional activity of AR was performed by measuring FKBP5 mRNA expression level. Cells were incubated in androgen depleted cell culture conditions for 6 hours, and then treated concurrently with 1 nM R1881 and indicated concentrations of AUY922 overnight. Experiments represent 3 independent experiments, mean ±SE. (<b>D</b>) MYC-CaP/CR and Pten-CaP/cE2 cells were treated concurrently with 5 µg/ml cycloheximide and increasing concentrations of AUY922. Expression of AR protein was assessed by western blot. GAPDH served as protein loading control. The densitometry was performed by image j analysis. The numbers were shown below the bands relative to the control cells.</p

Docetaxel treatment of castrate resistant prostate cancer tumor models <i>in vivo</i>.

<p>(<b>A</b>) MYC-CaP/CR cells (5×10⁶), (<b>B</b>) LuCaP23.1 AI tumor chunk (∼5 mm²) and (<b>C</b>) PC3 cells (5×10⁶) were injected or placed subcutaneously into the flank of castrated male FVB (MYC-CaP/CR) or SCID (LuCaP23.1 AI and PC3) mice. Treatment was initiated when tumors measured approximately 50 mm². Docetaxel was received from the pharmacy at RPCI as an aqueous solution (20 mg/ml) and diluted to 1 mg/ml in 1× PBS daily. Mice were treated with docetaxel doses of 10, 25 and 50 mg/kg weekly by intra-peritoneal (i.p.) injection for duration of 2 cycles (MYC-CaP/CR) or 3 cycles (LuCaP23.1 AI and PC3). Tumor growth was monitored by serial caliper measurements bi-weekly. Tumor size was calculated by L × W. All treatment groups consisted of 3–4 mice. Each treatment group was normalized to the pretreatment measurements and converted to percent tumor growth. Each point represents mean tumor size ±SE. (D–F) All mice were weighed 2 times/week to monitor docetaxel toxicity. Toxicity was considered to be occurring when mouse body weight was reduced ≥20%. Each column is represented by 3–4 individual mice/treatment and represented as percent bodyweight change compared to pre-treatment measurements, mean ±SE.</p

Castrate resistant prostate cancer cell line sensitivity to AUY922 treatment.

<p>(<b>A</b>) Chemical structure of NVP-AUY922. (<b>B</b>) Castrate resistant (MYC-CaP/CR and Pten-CaP/cE2) cell lines were treated with indicated concentrations of AUY922 for 48h. Adherent cells were fixed with 70% EtOH. Cell growth was assessed by staining fixed cells with crystal violet and measuring absorbance at 570 nm. Each point was normalized to the untreated control (0) and is represented by 3 independent experiments, mean ±SE. (<b>C</b>) MYC-CaP/CR and Pten-CaP/cE2 cells were treated with indicated concentrations of AUY922 for 48h. Adherent and non-adherent cells were collected and washed in 1x PBS. Cells were incubated with propidium iodide and cell death was assessed by flow cytometry. Each point represents mean ±SE of 3 independent experiments. * indicates p<0.05 compared to untreated control (0), by two-tailed t-test.</p

AUY922 and docetaxel combination therapy significantly attenuate AR transcriptional activity and increase tumor cell death <i>in vivo</i>.

<p>(<b>A</b>) Representative c-MYC immunostaining. Positive nuclear staining for c-MYC was quantified using aperio imagescope analysis of 6 random fields at x40 magnification. Scale bar = 500 µM. Two-tailed t-test: **** p<0.0001 AUY922 versus vehicle; ** p = 0.006 AUY922 versus docetaxel; * p = 0.03 combination versus docetaxel. (<b>B</b>) Representative cleaved caspase-3 immunostaining. Positive nuclear staining for cleaved caspase-3 was quantified using aperio imagescope analysis of 6 random fields at x20 magnification. Scale bar = 500 µM. Two-tailed t-test: **** p<0.0001 combination versus single treatments.</p

Response of castrate resistant prostate cancer cell lines to docetaxel treatment.

<p>Castrate resistant cell lines were treated with indicated concentrations of docetaxel and/or AUY922 for 48 h. MYC-CaP/CR (A) and Pten-CaP/cE2 (B) cell lines were treated with indicated concentrations of docetaxel for 48 h. Adherent cells were fixed with 70% EtOH. Cell growth was assessed by staining fixed cells with crystal violet and measuring absorbance at 570 nm. MYC-CaP/CR (C) and Pten-CaP/cE2 (D) cells were treated with indicated concentrations of AUY922 and/or docetaxel for 48 h. Cells were trypinized and washed in 1x PBS and incubated with propidium iodide (PI). Percentages of dead cells (PI positive cells) were determined by flow cytometry. Columns represent the mean ±SE, * indicates significantly greater in the combination compared to treatment with each drug alone (p = 0.03 as determined by one-way ANOVA).</p

CFS-1686 causes cell cycle arrest at intra-S phase by interference of interaction of topoisomerase 1 with DNA.

CFS-1686 (chemical name (E)-N-(2-(diethylamino)ethyl)-4-(2-(2-(5-nitrofuran-2-yl)vinyl)quinolin-4-ylamino)benzamide) inhibits cell proliferation and triggers late apoptosis in prostate cancer cell lines. Comparing the effect of CFS-1686 on cell cycle progression with the topoisomerase 1 inhibitor camptothecin revealed that CFS-1686 and camptothecin reduced DNA synthesis in S-phase, resulting in cell cycle arrest at the intra-S phase and G1-S boundary, respectively. The DNA damage in CFS-1686 and camptothecin treated cells was evaluated by the level of ATM phosphorylation, γH2AX, and γH2AX foci, showing that camptothecin was more effective than CFS-1686. However, despite its lower DNA damage capacity, CFS-1686 demonstrated 4-fold higher inhibition of topoisomerase 1 than camptothecin in a DNA relaxation assay. Unlike camptothecin, CFS-1686 demonstrated no activity on topoisomerase 1 in a DNA cleavage assay, but nevertheless it reduced the camptothecin-induced DNA cleavage of topoisomerase 1 in a dose-dependent manner. Our results indicate that CFS-1686 might bind to topoisomerase 1 to inhibit this enzyme from interacting with DNA relaxation activity, unlike campothecin's induction of a topoisomerase 1-DNA cleavage complex. Finally, we used a computer docking strategy to localize the potential binding site of CFS-1686 to topoisomerase 1, further indicating that CFS-1686 might inhibit the binding of Top1 to DNA