Disruption of TP63-miR-27a* Feedback Loop by Mutant TP53 in Head and Neck Cancer.

BACKGROUND: Alterations in the epidermal growth factor receptor and PI3K pathways in head and neck squamous cell carcinomas (HNSCCs) are frequent events that promote tumor progression. Ectopic expression of the epidermal growth factor receptor-targeting microRNA (miR), miR-27a* (miR-27a-5p), inhibits tumor growth. We sought to identify mechanisms mediating repression of miR-27a* in HNSCC, which have not been previously identified. METHODS: We quantified miR-27a* in 47 oral cavity squamous cell carcinoma patient samples along with analysis of miR-27a* in 73 oropharyngeal and 66 human papillomavirus-positive (HPV+) samples from The Cancer Genome Atlas. In vivo and in vitro TP53 models engineered to express mutant TP53, along with promoter analysis using chromatin immunoprecipitation and luciferase assays, were used to identify the role of TP53 and TP63 in miR-27a* transcription. An HNSCC cell line engineered to conditionally express miR-27a* was used in vitro to determine effects of miR-27a* on target genes and tumor cells. RESULTS: miR-27a* expression was repressed in 47 oral cavity tumor samples vs matched normal tissue (mean log2 difference = -0.023, 95% confidence interval = -0.044 to -0.002; two-sided paired t test, P = .03), and low miR-27a* levels were associated with poor survival in HPV+ and oropharyngeal HNSCC samples. Binding of ΔNp63α to the promoter led to an upregulation of miR-27a*. In vitro and in vivo findings showed that mutant TP53 represses the miR-27a* promoter, downregulating miR-27a* levels. ΔNp63α and nucleoporin 62, a protein involved in ΔNP63α transport, were validated as novel targets of miR-27a*. CONCLUSION: Our results characterize a negative feedback loop between TP63 and miR-27a*. Genetic alterations in TP53, a frequent event in HNSCC, disrupt this regulatory loop by repressing miR-27a* expression, promoting tumor survival.

PI3 Kinase inhibition on TRAIL-induced apoptosis correlates with androgen-sensitivity and p21 expression in prostate cancer cells.

TNF-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in many types of cancer cells. TRAIL is considered a therapeutic target, therefore, it was of interest to examine molecular mechanisms that may modulate sensitivity to TRAIL signaling in prostate cancer cells. LNCaP cells were found to be relatively resistant to TRAIL induced cell death while PC3 cells were sensitive. PI3-kinase (PI3 K) inhibitors were able to render LNCaP cells sensitive to TRAIL but conferred resistance to PC3 cells. PI3 K inhibitors were associated with an increase in p21(waf1, cip1) expression in PC3 cells where as p21 decreases in LNCaP cells suggesting that p21 may impart TRAIL resistance. Since androgen receptor (AR) signaling can be modulated by AKT, and p21 is an AR responsive gene, the impact of PI3 K inhibition on TRAIL sensitivity was evaluated in AR transfected PC3 cells (PC3AR). The expression of AR was significantly downregulated by PI3 K inhibition in LNCaP cells, which have an intact AR signaling axis. PC3AR cells expressed higher levels of p21 protein and were relatively resistant to TRAIL compared to control cells. Finally, using adenoviral p21 gene transfer we directly demonstrated that p21 can confer resistance to TRAIL-induced cell death. These results suggest that TRAIL resistance is not regulated simply by a PI3 K/AKT survival pathway associated with inactivating PTEN mutations but may also be modulated by downstream AR responsive targets such as p21. These findings may have significant clinical implications for the utility of TRAIL in the management of prostate cancer.

The p53 tumor suppressor network in cancer and the therapeutic modulation of cell death.

The molecular subversion of cell death is acknowledged as a principal contributor to the development and progression of cancer. The p53 tumor suppressor protein is among the most commonly altered proteins in human cancer. The p53 protein mediates critical functions within cells including the response to genotoxic stress, differentiation, senescence, and cell death. Loss of p53 function can result in enhanced rates of cell proliferation, resistance to cell death stimuli, genomic instability, and metastasis. The community of cancer scientists is now in possession of a vast repository of information regarding the frequency, specific mechanisms, and clinical context of cell death deregulation in cancer. This information has enabled the design of therapeutic agents to target proteins, including p53. The feasibility and impact of targeting cell death signaling proteins has been established in preclinical models of human cancer. The appropriate application of these targeted agents is now being established in clinical trials.

Biomarker expression patterns that correlate with high grade features in treatment naive, organ-confined prostate cancer.

BACKGROUND: The early detection of prostate cancer has resulted in an increase in the number of patients with localized prostate cancer and has paralleled the reported reduction in prostate cancer mortality. The increased rate of detection of patients with localized prostate cancer may also increase the risk of potentially morbid therapy in a patient with indolent cancer. Defining the biomarker correlates of prostate cancer virulence will facilitate the appropriate application and development of therapy for patients with early disease. METHODS: A 255 core prostate cancer tissue microarray (TMA) from 47 prostatectomy specimens with organ confined tumor was constructed. Prostate cancer foci of transition and peripheral zone origin were represented on the TMA. Further, replicate cores of the two Gleason grades comprising the Gleason score, representative of Gleason scores 5-9, were arrayed from each prostatectomy specimen. Standard immunohistochemical techniques were used to assess expression of nine, cell death and cell cycle regulatory proteins implicated in the pathogenesis of prostate cancer (bax, bcl-2, bcl-xL, bin1, CD95, mdm2, p21, p53, and NFkappaB). RESULTS: The Spearman correlation coefficient revealed a strong correlation of bax, bin1, FAS, p65 and p21 expression with Gleason grade. Spearman correlation coefficients showed that expression of, bax and bin1, bax and MDM2, Bax and p21, and bax and p65 NFkappaB was highly associated. Other significant associations were identified between bin1 and p21, bin1 and MDM2, bin1 and p65 NFkappaB and between p21 and p65 NFkappaB. A model for predicting the biological potential of Gleason score 7 prostate cancer using multivariable logistic regression methods was developed. The findings also indicate that the profile of specific markers for Gleason grade 3 prostate cancer correlates with the overall context of the Gleason score. CONCLUSION: These data support the view that important molecular differences exist among and between the Gleason scores. Furthermore, there is significant molecular heterogeneity among prostatectomy specimens containing Gleason grade 3 cancer. This observation may have broader implications regarding the determination of risk among patients with prostate cancer that is currently considered to be of either good prognosis or unclear prognosis, i.e. Gleason score 7 tumors.

The sonic hedgehog signaling network in development and neoplasia.

The sonic hedgehog (SHH) pathway was first defined genetically in fruit flies. Subsequently, the SHH network has been shown to be critical for normal mammalian development, by mediating interactions between stromal and epithelial cells. Recent evidence suggests that, deregulation of SHH signaling is important in the pathogenesis of cancer. Further, some observations suggest that a SHH paracrine mechanism mediating tumor-mesenchymal interactions may contribute to the metastatic capacity of cancer. Preclinical studies demonstrate that tumor cells in which SHH is deregulated are dependent on signaling through this pathway for the maintenance of proliferation and viability. SHH antagonists have been identified and show promise in inhibiting tumor growth in preclinical studies. The utility of these agents in the management of cancer patients awaits the outcome of ongoing and future clinical trials.

Cooperation of Ha-ras and Bcl-2 during multistep skin carcinogenesis.

Nonmelanoma skin cancer (NMSC) is the most frequently diagnosed cancer in the United States. Deregulation of bcl-2 and ras family members is commonly observed in NMSC. It has been previously demonstrated that simultaneous bcl-2 and Ha-ras gene expression in keratinocytes results in disordered differentiation and resistance to cell death induced by ultraviolet (UV) radiation. It was, therefore, interest to assess the extent of cooperation between bcl-2 and Ha-ras during multistep skin carcinogenesis. The keratin 1 promoter was used to generate HK1.ras and HK1.bcl-2 transgenic mice, which were subsequently crossed to generate HK1.ras/bcl-2 double transgenic mice. The apoptotic index (AI) following UV-irradiation was significantly lower in HK1.bcl-2 and HKI.ras/bcl-2 epidermis compared to control littermates. Interestingly, the AI of HK1.ras/bcl-2 mice was significantly lower than even HK1.bcl-2 mice following UV-irradiation. To investigate the interaction of these oncogenes in skin tumorigenesis, a two-stage chemical carcinogenesis protocol was used to induce tumors. The individual contributions of Ha-ras and bcl-2 to papilloma latency, incidence, and growth rate in HK1.ras/bcl-2 double transgenic mice was marginally additive. Papillomas arising in HK1.ras transgenic mice exhibited the highest rate of apoptosis whereas papillomas arising in the HK1.ras/bcl-2 double transgenic mice exhibited rates of apoptosis that were significantly lower than papillomas arising in either control littermate or HK1.ras mice. Constitutive expression of either Ha-ras or bcl-2 exhibited similar rates of malignant tumor progression and they were not significantly different than control littermates. Importantly, when these two oncoproteins were coexpressed, a significant, and synergistic, increase in malignant transformation was observed.

Sonic hedgehog induces epidermal growth factor dependent matrix infiltration in HaCaT keratinocytes.

The deregulation of the sonic hedgehog (shh) signaling pathway in epidermal keratinocytes is a primary event leading to the formation of basal cell carcinoma (BCC). The mechanisms by which this pathway exerts this effect remain largely undefined. We demonstrate that overexpression of shh in HaCaT keratinocytes grown in organotypic cultures induced a basal cell phenotype, as evidenced by their morphology, trans-epithelial staining of cytokeratin 14, and suprabasalar proliferation. Shh also induced keratinocyte infiltration into the underlying collagen matrix. Constitutive shh expression was associated with increased phosphorylation of the epidermal growth factor receptor (EGFR) as well as jnk and raf. Additionally, levels of c-jun and matrix metalloproteinase-9 (MMP-9) protein were elevated in shh-expressing cells. Inhibition of EGFR activity with either the tyrphostin, AG1478, or blocking receptor-ligand interaction with the monoclonal antibody, C-225, blocked matrix infiltration. In contrast, exogenously supplied EGF significantly augmented the invasiveness of the HaCaT cells. These observations provide insight into the impact of deregulated shh on epidermal homeostasis. The findings further suggest that an intact EGF signaling axis cooperates with shh and is a critical mediator of matrix invasion in a tumor type characterized by disrupted shh.

Transcriptional regulation of bcl-2 mediated by the sonic hedgehog signaling pathway through gli-1.

Basal cell carcinomas (BCCs) express high levels of the antiapoptotic proto-oncogene, bcl-2, and we have shown that bcl-2 contributes to the malignant phenotype in a transgenic mouse model. The basis of bcl-2 transcriptional regulation in keratinocytes is unknown. The sonic hedgehog (SHH) signaling pathway is frequently altered in BCCs. Mediators of shh signaling include the downstream transactivator, gli-1, and transrepressor, gli-3. Seven candidate gli binding sites were identified in the bcl-2 promoter. Cotransfection of increasing amounts of gli-1 in keratinoycytes resulted in a corresponding dose-dependent increase in bcl-2 promoter luciferase activity. Gli-1 was also able to up-regulate endogenous bcl-2. Gli-3 cotransfection resulted in no significant changes in bcl-2 promoter activity compared with control. Gli-3 has been demonstrated to be proteolytically processed into an N-terminal repressive form that can inhibit downstream transactivation by gli-1. Gli-3 mutants possessing only the N-terminal region or the C-terminal region were made and used in luciferase assays. The N terminus of gli-3 inhibited gli-1 transactivation of the bcl-2 promoter. Gel shift analysis and luciferase assays demonstrated that gli binding site 4 (-428 to -420), is important for gli transcriptional regulation. Skin samples from transgenic mice expressing an RU486 gli-1 transgene exhibited significantly higher levels of endogenous bcl-2 protein in epidermal keratinocytes as assessed by immunoblotting and immunohistochemistry. Together, these findings provide consistent evidence that gli proteins can transcriptionally regulate the bcl-2 promoter and that gli-3 can inhibit transactivation by gli-1. These studies further suggest that one consequence of the deregulation of shh signaling in BCC is the up-regulation of bcl-2.