New concepts and challenges in the clinical translation of cancer preventive therapies: the role of pharmacodynamic biomarkers.

Implementation of therapeutic cancer prevention strategies has enormous potential for reducing cancer incidence and related mortality. Trials of drugs including tamoxifen and aspirin have led the way in demonstrating proof-of-principle that prevention of breast and colorectal cancer is feasible. Many other compounds ranging from drugs in widespread use for various indications, including metformin, bisphosphonates, and vitamin D, to dietary agents such as the phytochemicals resveratrol and curcumin, show preventive activity against several cancers in preclinical models. Notwithstanding the wealth of opportunities, major challenges have hindered the development process and only a handful of therapies are currently approved for cancer risk reduction. One of the major obstacles to successful clinical translation of promising preventive agents is a lack of pharmacodynamic biomarkers to provide an early read out of biological activity in humans and for optimising doses to take into large scale randomised clinical trials. A further confounding factor is a lack of consideration of clinical pharmacokinetics in the design of preclinical experiments, meaning results are frequently reported from studies that use irrelevant or unachievable concentrations. This article focuses on recent findings from investigations with dietary-derived agents to illustrate how a thorough understanding of the mechanisms of action, using models that mimic the clinical scenario, together with the development of compound-specific accompanying pharmacodynamic biomarkers could accelerate the developmental pipeline for preventive agents and maximise the chances of success in future clinical trials. Moreover, the concept of a bell-shaped dose-response curve for therapeutic cancer prevention is discussed, along with the need to rethink the traditional 'more is better' approach for dose selection

Metabolic pathways regulated by TAp73 in response to oxidative stress

Reactive oxygen species are involved in both physiological and pathological processes including neurodegeneration and cancer. Therefore, cells have developed scavenging mechanisms to maintain redox homeostasis under control. Tumor suppressor genes play a critical role in the regulation of antioxidant genes. Here, we investigated whether the tumor suppressor gene TAp73 is involved in the regulation of metabolic adaptations triggered in response to oxidative stress. H2O2 treatment resulted in numerous biochemical changes in both control and TAp73 knockout (TAp73-/-) mouse embryonic fibroblasts, however the extent of these changes was more pronounced in TAp73-/- cells when compared to control cells. In particular, loss of TAp73 led to alterations in glucose, nucleotide and amino acid metabolism. In addition, H2O2 treatment resulted in increased pentose phosphate pathway (PPP) activity in null mouse embryonic fibroblasts. Overall, our results suggest that in the absence of TAp73, H2O2 treatment results in an enhanced oxidative environment, and at the same time in an increased pro-anabolic phenotype. In conclusion, the metabolic profile observed reinforces the role of TAp73 as tumor suppressor and indicates that TAp73 exerts this function, at least partially, by regulation of cellular metabolism

TAp73 promotes anti-senescence-anabolism not proliferation

TAp73, a member of the p53 family, has been traditionally considered a tumor suppressor gene, but a recent report has claimed that it can promote cellular proliferation. This assumption is based on biochemical evidence of activation of anabolic metabolism, with enhanced pentose phosphate shunt (PPP) and nucleotide biosynthesis. Here, while we confirm that TAp73 expression enhances anabolism, we also substantiate its role in inhibiting proliferation and promoting cell death. Hence, we would like to propose an alternative interpretation of the accumulating data linking p73 to cellular metabolism: we suggest that TAp73 promotes anabolism to counteract cellular senescence rather than to support proliferation

Role of p63 and p73 isoforms on the cell death in patients with hepatocellular carcinoma submitted to orthotopic liver transplantation

BACKGROUND & AIMS: Patients with hepatocellular carcinoma (HCC) submitted to orthotopic liver transplantation (OLT) have a variable 5-year survival rate limited mostly by tumor recurrence. The etiology, age, sex, alcohol, Child-Pugh, and the immunesuppressor have been associated with tumour recurrence. The expression of ΔNp73 is related to the reduced survival of patients with HCC. The study evaluated the expression of p63 and p73 isoforms and cell death receptors, and their relation to tumour recurrence and survival. The results were in vitro validated in HCC cell lines. METHODS: HCC sections from patients submitted to OLT were used. The in vitro study was done in differentiated hepatitis B virus (HBV)-expressing Hep3B and control HepG2 cells. The expression of cell death receptors and cFLIPS/L, caspase-8 and -3 activities, and cell proliferation were determined in control and p63 and p73 overexpressing HCC cells. RESULTS: The reduced tumor expression of cell death receptors and TAp63 and TAp73, and increased ΔNp63 and ΔNp73 expression were associated with tumor recurrence and reduced survival. The in vitro study demonstrated that HBV-expressing Hep3B vs HepG2 cells showed reduced expression of p63 and p73, cell death receptors and caspase activation, and increased cFLIPL/cFLIPS ratio. The overexpression of TAp63 and TAp73 exerted a more potent pro-apoptotic and anti-proliferative effects in Hep3B than HepG2-transfected cells which was related to cFLIPL upregulation. CONCLUSIONS: The reduction of TAp63 and TAp73 isoforms, rather than alteration of ΔN isoform expression, exerted a significant functional repercussion on cell death and proliferation in HBV-expressing HepB cells

cFLIP_L and cFLIP_S expression in transfected HepG2 and Hep3B cells with TAp63α, ΔNp63α, TAp73α and ΔNp73α overexpressing plasmids.

<p>The protein expression of cFLIP was assessed by western-blot analysis following the procedure described in Material and Methods. Data are expressed as mean ± SEM either from the densitometry analysis referred to the value of stain-free procedure. The asterisks indicate statistical significance (**p≤0.01 or ***p≤0.001) compared with their corresponding empty vector transfected cell line. The groups denoted by different letters (a, b, c or d) are significantly different among them (p≤0.05).</p

TNF-R1 (A), CD95 (B) and TRAIL-R1 (C) expression, caspase-8 (D) and caspase-3 (E) activities and cell proliferation (F) in transfected HepG2 and Hep3B cells with TAp63α, ΔNp63α, TAp73α and ΔNp73α overexpressing plasmids.

<p>The parameters were assessed 48 hours after cell transfections. The protein expression of cell death receptors was assessed by western-blot analysis following the procedure described in Material and Methods. Caspase-8 and -3 activities were determined using commercial assays described in Material and Methods. Cell proliferation was determined by BrdU incorporation administered 2 hours before cell collection following the procedure described in Material and Methods. Data are expressed as mean ± SEM either from the densitometry analysis of blots referred to the corresponding loading control (stain-free procedure) or to caspase activity referred to a calibration curve developed using recombinant purified enzyme. The asterisks indicate statistical significance (*p≤0.05, **p≤0.01 or ***p≤0.001) compared with their corresponding empty vector transfected cell line. The groups denoted by different letters (a, b, c or d) are significantly different among them (p≤0.05).</p

Values of Pearson correlation coefficient (r) and the significance of the correlation (<i>p</i>) between the expression of p63 and p73 isoforms, and cell death receptors in studied population.

<p>Values of Pearson correlation coefficient (r) and the significance of the correlation (<i>p</i>) between the expression of p63 and p73 isoforms, and cell death receptors in studied population.</p

Distribution of all pairwise variables and the corresponding linear equations in the three-studied populations (alive, dead by HCC recurrence and dead by other causes).

<p>The values of R² lineal of the linear equation were superior between ΔN isoforms and cell death receptors (Fig 3D-3F and 3K-3M) that those between ΔN isoforms and cell death receptors (Fig 3A-3C and 3H-3J).</p

TNF-R1 (A), CD95 (B) and TRAIL-R1 (C) expression obtained from HepG2 and Hep3B.

<p>The protein expression of cell death receptors was assessed by western-blot analysis following the procedure described in Material and Methods. Data are expressed as mean ± SEM of the densitometry analysis referred to the corresponding loading control (β-actin). The asterisks indicate statistical significance compared with their corresponding group (HepG2) (***p≤0.001).</p

TAp63, ΔNp63, TAp73 and ΔNp73 expression in well-differentiated (A) and moderately-poorly differentiated HCC (B) in patients subject to OLT, and their correlation to survival or death as a consequence of tumor recurrence or other causes such as pneumonia, fungal sepsis, heart complications and septic shock (C).

<p>The protein expression of p53 gene family members was assessed by immunohistochemistry following the procedure described in Material and Methods. Data are expressed as mean ± SEM of the densitometry analysis. The asterisks indicate statistical significance (*p≤0.05, **p≤0.01 or ***p≤0.001) compared with their corresponding control. The images are representative of the corresponding group. Image magnification x10.</p