Colorectal cancer inhibition by BET inhibitor JQ1 is MYC-independent and not improved by nanoencapsulation.

Bromodomain and extraterminal domain protein inhibitors (BETi) for cancer treatment did not convince during their first clinical trials. Their epigenetic mechanism of action is still not well understood, even if MYC is generally considered as its main downstream target. In this context, we intended to assess two new nanoformulations of the BETi JQ1 for the treatment of colorectal cancer (CRC). JQ1 was encapsulated at 10 mg/mL in lipid nanocapsules (LNC) or polymeric micelles (PM), both compatible for an intravenous administration. Their effect was compared with free JQ1 on several CRC cell lines in vitro and with daily intraperitoneal cyclodextrin (CD)-loaded JQ1 on the CT26 CRC tumor model in vivo. We showed that LNC preferentially accumulated in tumor, liver, and lymph nodes. LNC-JQ1 and CD-JQ1 similarly delayed tumor growth and increased median survival from 15 to 23 or 20.5 days. JQ1 altered MYC in only two among four CRC cell lines. This MYC-independence found in CT26 was confirmed in vivo by PCR and immunohistochemistry. The main explanation of the JQ1 anticancer effect was an increase in apoptosis. The investigation of its impact on the tumor microenvironment did not show significant effects. Finally, JQ1 association with irinotecan did not synergize in vivo with JQ1 nanoformulations. In conclusion, we demonstrated that the JQ1 anticancer effect was not improved by nanoencapsulation even if their tumor delivery was probably higher. MYC inhibition was not associated to JQ1 efficacy in the case of the CT26 CRC murine model.

Identification of ZBTB18 as a novel colorectal tumor suppressor gene through genome-wide promoter hypermethylation analysis.

BACKGROUND: Cancer initiation and progression are driven by genetic and epigenetic changes. Although genome/exome sequencing has significantly contributed to the characterization of the genetic driver alterations, further investigation is required to systematically identify cancer driver genes regulated by promoter hypermethylation. RESULTS: Using genome-wide analysis of promoter methylation in 45 colorectal cancer cell lines, we found that higher overall methylation levels were associated with microsatellite instability (MSI), faster proliferation and absence of APC mutations. Because epigenetically silenced genes could represent important oncogenic drivers, we used mRNA expression profiling of colorectal cancer cell lines and primary tumors to identify a subset of 382 (3.9%) genes for which promoter methylation was negatively associated with gene expression. Remarkably, a significant enrichment in zinc finger proteins was observed, including the transcriptional repressor ZBTB18. Re-introduction of ZBTB18 in colon cancer cells significantly reduced proliferation in vitro and in a subcutaneous xenograft mouse model. Moreover, immunohistochemical analysis revealed that ZBTB18 is frequently lost or reduced in colorectal tumors, and reduced ZBTB18 expression was found to be associated with lymph node metastasis and shorter survival of patients with locally advanced colorectal cancer. CONCLUSIONS: We identified a set of 382 genes putatively silenced by promoter methylation in colorectal cancer that could significantly contribute to the oncogenic process. Moreover, as a proof of concept, we demonstrate that the epigenetically silenced gene ZBTB18 has tumor suppressor activity and is a novel prognostic marker for patients with locally advanced colorectal cancer.

STAT3 inhibition with galiellalactone effectively targets the prostate cancer stem-like cell population.

Cancer stem cells (CSCs) are a small subpopulation of quiescent cells with the potential to differentiate into tumor cells. CSCs are involved in tumor initiation and progression and contribute to treatment failure through their intrinsic resistance to chemo- or radiotherapy, thus representing a substantial concern for cancer treatment. Prostate CSCs' activity has been shown to be regulated by the transcription factor Signal Transducer and Activator of Transcription 3 (STAT3). Here we investigated the effect of galiellalactone (GL), a direct STAT3 inhibitor, on CSCs derived from prostate cancer patients, on docetaxel-resistant spheres with stem cell characteristics, on CSCs obtained from the DU145 cell line in vitro and on DU145 tumors in vivo. We found that GL significantly reduced the viability of docetaxel-resistant and patient-derived spheres. Moreover, CSCs isolated from DU145 cells were sensitive to low concentrations of GL, and the treatment with GL suppressed their viability and their ability to form colonies and spheres. STAT3 inhibition down regulated transcriptional targets of STAT3 in these cells, indicating STAT3 activity in CSCs. Our results indicate that GL can target the prostate stem cell niche in patient-derived cells, in docetaxel-resistant spheres and in an in vitro model. We conclude that GL represents a promising therapeutic approach for prostate cancer patients, as it reduces the viability of prostate cancer-therapy-resistant cells in both CSCs and non-CSC populations.

Autophagy inhibition as a promising therapeutic target for laryngeal cancer.

To identify the putative relevance of autophagy in laryngeal cancer, we performed an immunohistochemistry study to analyze the expression of the proteins involved in this process, namely, LC3, ATG5 and p62/SQSTM1. Additionally, Prostate tumor-overexpressed gene 1 protein (PTOV1) was included due to its potential relevance in laryngeal cancer. Moreover, as cancer resistance might involve autophagy in some circumstances, we studied the intrinsic drug resistance capacity of primary tumor cultures derived from 13 laryngeal cancer biopsies and their expression levels of LC3, ATG5, p62 and PTOV1. Overall, our results suggest that (i) cytoplasmic p62 and PTOV1 can be considered prognostic markers in laryngeal cancer, (ii) the acquisition of resistance seems to be related to PTOV1 and autophagy-related protein overexpression, (iii) by increasing autophagy, PTOV1 might contribute to resistance in this model and (iv) the expression of autophagy-related proteins could classify a subgroup of laryngeal cancer patients who will benefit from a therapy based upon autophagy inhibition. Our study suggests that autophagy inhibition with hydroxychloroquine could be a promising strategy for laryngeal cancer patients, particularly those patients with high resistance to the CDDP treatment that in addition have autophagy upregulation.

A novel DNA-binding motif in prostate tumor overexpressed-1 (PTOV1) required for the expression of ALDH1A1 and CCNG2 in cancer cells.

PTOV1 is a transcription and translation regulator and a promoter of cancer progression. Its overexpression in prostate cancer induces transcription of drug resistance and self-renewal genes, and docetaxel resistance. Here we studied PTOV1 ability to directly activate the transcription of ALDH1A1 and CCNG2 by binding to specific promoter sequences. Chromatin immunoprecipitation and electrophoretic mobility shift assays identified a DNA-binding motif inside the PTOV-A domain with similarities to known AT-hooks that specifically interacts with ALDH1A1 and CCNG2 promoters. Mutation of this AT-hook-like sequence significantly decreased the expression of ALDH1A1 and CCNG2 promoted by PTOV1. Immunohistochemistry revealed the association of PTOV1 with mitotic chromosomes in high grade prostate, colon, bladder, and breast carcinomas. Overexpression of PTOV1, ALDH1A1, and CCNG2 significantly correlated with poor prognosis in prostate carcinomas and with shorter relapse-free survival in colon carcinoma. The previously described interaction with translation complexes and its direct binding to ALDH1A1 and CCNG2 promoters found here reveal the PTOV1 capacity to modulate the expression of critical genes at multiple levels in aggressive cancers. Remarkably, the AT-hook motifs in PTOV1 open possibilities for selective targeting its nuclear and/or cytoplasmic activities.

Prostate Tumor Overexpressed-1 (PTOV1) promotes docetaxel-resistance and survival of castration resistant prostate cancer cells.

Metastatic prostate cancer is presently incurable. The oncogenic protein PTOV1, first described in prostate cancer, was reported as overexpressed and significantly correlated with poor survival in numerous tumors. Here, we investigated the role of PTOV1 in prostate cancer survival to docetaxel and self-renewal ability. Transduction of PTOV1 in docetaxel-sensitive Du145 and PC3 cells significantly increased cell survival after docetaxel exposure and induced docetaxel-resistance genes expression (ABCB1, CCNG2 and TUBB2B). In addition, PTOV1 induced prostatospheres formation and self-renewal genes expression (ALDH1A1, LIN28A, MYC and NANOG). In contrast, Du145 and PC3 cells knockdown for PTOV1 significantly accumulated in the G2/M phase, presented a concomitant increased subG1 peak, and cell death by apoptosis. These effects were enhanced in docetaxel-resistant cells. Analyses of tumor datasets show that PTOV1 expression significantly correlated with prostate tumor grade, drug resistance (CCNG2) and self-renewal (ALDH1A1, MYC) markers. These genes are concurrently overexpressed in most metastatic lesions. Metastases also show PTOV1 genomic amplification in significant co-occurrence with docetaxel-resistance and self-renewal genes. Our findings identify PTOV1 as a promoter of docetaxel-resistance and self-renewal characteristics for castration resistant prostate cancer. The concomitant increased expression of PTOV1, ALDH1A1 and CCNG2 in primary tumors, may predict metastasis and bad prognosis.

Equine bone marrow mesenchymal or amniotic epithelial stem cells as feeder in a model for the in vitro culture of bovine embryos.

Various studies have shown that the in vitro culture environment is one of the key determinants of the blastocyst output. In the present study we investigated the effects of co-culturing bovine embryos with equine bone marrow mesenchymal stem cells (BM-MSCs) or equine amniotic epithelial stem cells (AE-SCs) on in vitro blastocysts development. BM specimens were obtained aseptically from sternal aspirates of horses under local anaesthesia and the isolated cells were resuspended in Dulbecco Modified Earle's Medium supplemented with 10 ng/ml of basic fibroblast growth factor (bFGF). Amniotic membranes were obtained from fresh placentas and, to release the AE cells, amniotic fragments were incubated with 0.05% trypsin for 45 min. Separated AE cells were plated in standard culture medium containing 10 ng/ml epidermal growth factor (EGF). Seven hundred and five cumulus-oocyte complexes were used and, after IVM and IVF, cumulus-free presumptive zygotes were randomly transferred into one of three co-culture systems in which they were cultured up to day 7: (1) co-culture with cumulus cells (control); (2) co-culture with BM-MSCs; and (3) co-culture with AE-SCs. Statistical analyses were performed by ANOVA. Blastocyst developmental rates were significantly different (p < 0.001) between control, AE-SCs and BM-MSCs (respectively 35.45, 41.84 and 30.09%). In conclusion, the AE-SC monolayer create a more suitable microenvironment necessary for inducing local cell activation and proliferation of the growing embryos in comparison with BM-MSCs and cumulus cells. It can be suggested that these cells secrete biologically active substances, including signalling molecules and growth factors of epithelial nature, different to those of the BM cells of mesenchymal origin.