While p73 overexpression has been associated with increased apoptosis in cancer tissues, p73 overexpressing tumors appear to be of high grade malignancy. Why this putative tumor suppressor is overexpressed in cancer cells and what the function of overexpressed p73 is in breast cancers are critical questions to be addressed. By investigating the effect of p53 inactivation on p73 expression, we found that both protein and mRNA levels of TAp73 were increased in MCF-7/p53siRNA cells, MCF-7/p53mt135 cells and HCT-116/p53−/− cells, as compared to wild type control, suggesting that p53 inactivation by various forms upregulates p73. We showed that p53 knockdown induced p73 was mainly regulated at the transcriptional level. However, although p53 has a putative binding site in the TAp73 promoter, deletion of this binding site did not affect p53 knockdown mediated activation of TAp73 promoter. Chromatin immuno-precipitation (ChIP) data demonstrated that loss of p53 results in enhanced occupancy of E2F-1 in the TAp73 promoter. The responsive sequence of p53 inactivation mediated p73 upregulation was mapped to the proximal promoter region of the TAp73 gene. To test the role of E2F-1 in p53 inactivation mediated regulation of p73 transcription, we found that p53 knockdown enhanced E2F-1 dependent p73 transcription, and mutations in E2F-1 binding sites in the TAp73 promoter abrogated p53 knockdown mediated activation of TAp73 promoter. Moreover, we demonstrated that p21 is a mediator of p53-E2F crosstalk in the regulation of p73 transcription. We concluded that p53 knockdown/inactivation may upregulate TAp73 expression through E2F-1 mediated transcriptional regulation. p53 inactivation mediated upregulation of p73 suggests an intrinsic rescuing mechanism in response to p53 mutation/inactivation. These findings support further analysis of the correlation between p53 status and p73 expression and its prognostic/predictive significance in human cancers.

Environmental factors, including 7,12‑dimethylbenz[a]anthracene (DMBA) exposure, and genetic predisposition, including ErbB2 overexpression/amplification, have been demonstrated to increase breast cancer susceptibility. Although DMBA‑ and ErbB2‑mediated breast cancers are well‑studied in their respective models, key interactions between environmental and genetic factors on breast cancer risk remain unclear. Therefore, the present study aimed to investigate the effect of DMBA exposure on ErbB2‑mediated mammary tumorigenesis. MMTV‑ErbB2 transgenic mice exposed to DMBA (1 mg) via weekly oral gavage for 6 weeks exhibited significantly enhanced mammary tumor development, as indicated by reduced tumor latency and increased tumor multiplicity compared with control mice. Whole mount analysis of premalignant mammary tissues from 15‑week‑old mice revealed increased ductal elongation and proliferative index in DMBA‑exposed mice. Molecular analyses of premalignant mammary tissues further indicated that DMBA exposure enhanced epidermal growth factor receptor (EGFR)/ErbB2 and estrogen receptor (ER) signaling, which was associated with increased mRNA levels of EGFR/ErbB2 family members and ER‑targeted genes. Furthermore, analysis of tumor karyotypes revealed that DMBA‑exposed tumors displayed more chromosomal alterations compared with control tumors, implicating DMBA‑induced chromosomal instability in tumor promotion in this model. Together, the data suggested that DMBA‑induced deregulation of EGFR/ErbB2‑ER pathways plays a critical role in the enhanced chromosomal instability and promotion of ErbB2‑mediated mammary tumorigenesis. The study highlighted gene‑environment interactions that may increase risk of breast cancer, which is a critical clinical issue.

Caloric intake influences the onset of many diseases, including cancer. In particular, caloric restriction (CR) has been reported to suppress mammary tumorigenesis in various models. However, the underlying cancer preventive mechanisms have not been fully explored. To this end, we aimed to characterize the anticancer mechanisms of CR using MMTV-ErbB2 transgenic mice, a well-established spontaneous ErbB2-overexpressing mammary tumor model, by focusing on cellular and molecular changes in premalignant tissues. In MMTV-ErbB2 mice with 30% CR beginning at 8 weeks of age, mammary tumor development was dramatically inhibited, as exhibited by reduced tumor incidence and increased tumor latency. Morphogenic mammary gland analyses in 15- and 20-week-old mice indicated that CR significantly decreased mammary epithelial cell (MEC) density and proliferative index. To understand the underlying mechanisms, we analyzed the effects of CR on mammary stem/progenitor cells. Results from fluorescence-activated cell sorting analyses showed that CR modified mammary tissue hierarchy dynamics, as evidenced by decreased luminal cells (CD24highCD49flow), putative mammary reconstituting unit subpopulation (CD24highCD49fhigh) and luminal progenitor cells (CD61highCD49fhigh). Mammosphere and colony-forming cell assays demonstrated that CR significantly inhibited mammary stem cell self-renewal and progenitor cell numbers. Molecular analyses indicated that CR concurrently inhibited estrogen receptor (ER) and ErbB2 signaling. These molecular changes were accompanied by decreased mRNA levels of ER-targeted genes and epidermal growth factor receptor/ErbB2 family members and ligands, suggesting ER-ErbB2 signaling cross-talk. Collectively, our data demonstrate that CR significantly impacts ER and ErbB2 signaling, which induces profound changes in MEC reprogramming, and mammary stem/progenitor cell inhibition is a critical mechanism of CR-mediated breast cancer prevention.

The complexity of rheumatoid arthritis (RA) pathogenesis makes combined blockade of key pathogenic factors an attractive therapeutic strategy. We have previously reported a novel recombinant adeno-associated virus (AAV) vector, AAV.TFCF, which mediates separate coexpression of TNFα antagonist TNFR-Fc and T cell antagonist CTLA4-FasL both in vitro and in vivo (the injected joints). The purpose of this study was to examine the efficacy of TNFR-Fc/CTLA4-FasL combination therapy mediated by AAV.TFCF in experimental model of RA. Adjuvant-induced arthritis (AIA) was induced in Lewis rats, and the recombinant AAV.TFCF was injected into rat ankle joints. AAV vector encoding CTLA4-FasL (AAV.CTFA) or TNFR-Fc (AAV.TRFC) was used as the monotherapy control, and an AAV vector mediating the expression of enhanced green fluorescent protein (AAV.EGFP) was used as the negative control. The combination treatment mediated by AAV.TFCF demonstrated a more effective suppression of AIA compared with those monotherapy controls, as reflected in the clinical and histological observations. The synergistic anti-inflammatory effect of TNFR-Fc combining with CTLA4-FasL was proved to be associated with the greater reductions of inflammatory CD4+ T cell infiltration and proinflammatory cytokine TNFα level in the arthritic joints. In addition, the combination therapy was found to be able to increase the frequency of CD4 + CD25 + FoxP3+ regulatory T cell population in rat draining lymph nodes and suppress splenic inflammatory responses. These results suggest that combination treatment with TNFR-Fc and CTLA4-FasL may achieve superior efficacy in suppressing RA, and using this novel recombinant AAV.TFCF to obtain the combined counteraction of both pathogenic T cells and the key proinflammatory cytokine TNFα may provide a more effective and desirable strategy for treatment of RA.

We generated replication-defective adenovirus Ad-p53AIP1 and studied its anti-tumor efficacy both in vitro and in vivo. We demonstrated that Ad-p53AIP1 infection elicited high levels of p53AIP1 expression in cancer cells. We also found that Ad-p53AIP1 expression induced marked apoptosis and cell cycle arrest in HepG2 cells. Moreover, Ad-p53AIP1 infection significantly inhibited the tumorigenesis of 4T1 mouse mammary cancer cells in vivo. In particular, we discovered that p53AIP1 overexpression up-regulated the protein levels of p53 in HepG2 cells, which was accompanied by down-regulation of MDM2 mRNA and protein, suggesting an interaction between MDM2 and p53 in p53AIP1-induced apoptosis and cell cycle arrest. Our data demonstrated the feasibility of Ad-p53AIP1-mediated cancer gene therapy. p53AIP1-induced up-regulation of p53 protein through MDM2 suggests that p53AIP1 gene therapy may be more advantageous in tumors expressing high levels of oncoprotein MDM2 or having a mutation in MDM2 inhibitor p16INK4.