IntroductionNuclear accumulation of β-catenin is important for cancer development and it is found to overlap with p68 (DDX5) immunoreactivity in most breast cancers, as indicated by both clinical investigations and studies in cell lines. In this study, we aim to investigate the regulation of p68 gene expression through β-catenin/transcription factor 4 (TCF4) signaling in breast cancer.MethodsFormalin-fixed paraffin-embedded sections derived from normal human breast and breast cancer samples were used for immunohistochemical analysis. Protein and mRNA expressions were determined by immunoblotting and quantitative RT-PCR respectively. Promoter activity of p68 was checked using luciferase assay. Occupancy of several factors on the p68 promoter was evaluated using chromatin immunoprecipitation. Finally, a syngeneic mouse model of breast cancer was used to assess physiological significance.ResultsWe demonstrated that β-catenin can directly induce transcription of p68 promoter or indirectly through regulation of c-Myc in both human and mouse breast cancer cells. Moreover, by chromatin immunoprecipitation assay, we have found that both β-catenin and TCF4 occupy the endogenous p68 promoter, which is further enhanced by Wnt signaling. Furthermore, we have also established a positive feedback regulation for the expression of TCF4 by p68. To the best of our knowledge, this is the first report on β-catenin/TCF4-mediated p68 gene regulation, which plays an important role in epithelial to mesenchymal transition, as shown in vitro in breast cancer cell lines and in vivo in an animal breast tumour model.ConclusionsOur findings indicate that Wnt/β-catenin signaling plays an important role in breast cancer progression through p68 upregulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0496-5) contains supplementary material, which is available to authorized users.

PTEN mutation is a frequent feature across a plethora of human cancers, the hot-spot being its C-terminus (PTEN-CT) regulatory domain resulting in a much diminished protein expression. In this study, the presence of C-terminus mutations was confirmed through sequencing of different human tumor samples. The kinase CKII-mediated phosphorylation of PTEN at these sites makes it a loopy structure competing with the E3 ligases for binding to its lipid anchoring C2 domain. Accordingly, it was found that PTEN-CT expressing stable cell lines could inhibit tumorigenesis in syngenic breast tumor models. Therefore, we designed a novel exosome-mediated delivery of the intrinsic PTEN domain, PTEN-CT into different cancer cells and observed reduced proliferation, migration, and colony forming ability. The delivery of exosome containing PTEN-CT to breast tumor mice model was found to result in significant regression in tumor size with the tumor sections showing increased apoptosis. Here, we also report for the first time an active PTEN when its C2 domain is bound by PTEN-CT, probably rendering its anti-tumorigenic activities through the protein phosphatase activity. Therefore, therapeutic interventions that focus on PTEN E3 ligase inhibition through exosome-mediated PTEN-CT delivery can be a probable route in treating cancers with low PTEN expression.

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Increased abundance of proto-oncogene AKT and reduced expression of tumor suppressor Forkhead box O3 (FOXO3a), the downstream target of AKT, is frequent in carcinogenesis. Mechanistic insights of AKT gene regulation are limited. DEAD box RNA helicase p68 is overexpressed in various cancers and acts as a transcriptional co-activator of several transcription factors, including β-catenin. Here, we report a novel mechanism of p68-mediated transcriptional activation of AKT, and its ensuing effect on FOXO3a, in colon carcinogenesis. Interestingly, we found that the expression of p68 and AKT exhibits strong positive correlation in normal and colon carcinoma patient samples. In addition, p68 increased both AKT messenger RNA (mRNA) and protein, enhanced AKT promoter activity in multiple colon cancer cell lines. Conversely, p68 knockdown led to reduced AKT mRNA and protein, diminished AKT promoter activity. Here, we demonstrated that p68 occupies AKT promoter with β-catenin as well as nuclear factor-κB (NF-κB)and cooperates with these in potentiating AKT transcription. Furthermore, p68 and FOXO3a expression followed inverse correlation in the same set of colon carcinoma samples. We observed that p68 significantly reduced FOXO3a protein level in an AKT-dependent manner. Studies in primary tumors and metastatic lung nodules generated in mice colorectal allograft model, using syngeneic cells stably expressing p68, corroborated our in vitro findings. Hence, a new mechanism of oncogenesis is attributed to p68 by upregulation of AKT and consequent nuclear exclusion and degradation of tumor suppressor FOXO3a.

Despite recent advances in medicine, 30–40% of patients with breast cancer show recurrence underscoring the need for improved effective therapy. In this study, by in vitro screening we have selected a novel synthetic indole derivative 2,2'-diphenyl-3,3'-diindolylmethane (DPDIM) as a potential anti- breast cancer agent. DPDIM induces apoptosis both in vitro in breast cancer cells MCF7, MDA-MB 231 and MDA-MB 468 and in vivo in 7,12-dimethylbenz[α]anthracene (DMBA) induced Sprague-Dawley (SD) rat mammary tumor. Our in vitro studies show that DPDIM exerts apoptotic effect by negatively regulating the activity of EGFR and its downstream molecules like STAT3, AKT and ERK1/2 which are involved in the proliferation and survival of these cancer cells. In silico predictions also suggest that DPDIM may bind to EGFR at its ATP binding site. DPDIM furthermore inhibits EGF induced increased cell viability. We have also shown decreased expression of pro-survival factor Bcl-XL as well as increase in the level of pro-apoptotic proteins like Bax, Bad, Bim in DPDIM treated cells in vitro and in vivo. Our results further indicate that the DPDIM induced apoptosis is mediated through mitochondrial apoptotic pathway involving the caspase-cascade. To the best of our knowledge this is the first report of DPDIM for its anticancer activity. Altogether this report suggests that DPDIM could be an effective therapeutic agent for breast cancer.

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In mulberry (Morus alba L.) plants NaCI stress imposed through roots by irrigation during growth period decreased the net photosynthetic rate (NPR), physiological water use efficiency (pWUE), which ultimately reflected on the reduction of growth parameters and leaf yield. Foliar spray of kinetin and spermidine ~both at 1 raM) on salinized plants reduced the detrimental effects of saline stress. Kinetin and spermidine sprayed plants increased the total chlorophyll, protein content, as well as leaf yield, but reduced the sugar and proline contents as compared to NaCI treated plants. Kinetin was more effective than spermidine in increasing NPR, pWUE and leaf yield both in nonsalinized and salinized condition.

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