Faina Vikhanskaya

c-Jun promotes cellular survival by suppression of PTEN

Activation of c-Jun, a component of the AP-1 family of transcription factors, leads to either promotion or prevention of apoptosis. However, the molecular determinants of c-Jun-mediated cell survival are still unclear. We show here that inducible expression of c-Jun promotes cellular survival by negatively regulating the expression of the tumor-suppressor PTEN, resulting in the concomitant activation of the Akt survival pathway. Consistently, c-jun−/− fibroblasts, which are sensitive to nutrient deprivation, and human cell lines in which c-Jun expression is silenced, express elevated levels of PTEN. siRNA-mediated silencing of PTEN resulted in the reduction of cell-death owing to c-Jun deficiency. c-Jun was found to suppress PTEN expression by binding to a variant AP-1 site found in the 5′ upstream sequences of PTEN promoter. Finally, an inverse correlation between c-Jun and PTEN levels was apparent in a panel of human tumor cell lines, independent of their p53 status. Together, the data demonstrate that c-Jun contributes to the promotion of cellular survival by regulating the expression of PTEN.

p73 supports cellular growth through c-Jun-dependent AP-1 transactivation

The cause or consequence of overexpression of p73 (refs 1, 2), the structural and functional homologue of the tumour-suppressor gene product p53 (refs 3, 4), in human cancers is poorly understood. Here, we report a role for p73 in supporting cellular growth through the upregulation of AP-1 transcriptional activity. p73 suppresses growth when overexpressed alone, but synergises with the proto-oncogene c-Jun to promote cellular survival. Conversely, silencing of p73 expression compromises cellular proliferation. Molecular analysis revealed that expression of the AP-1 target-gene product cyclinD1 (ref. 5) is reduced concomitant with p73, but not p53, silencing. Moreover, cyclinD1 was induced by p73 expression in a c-Jun-dependent manner, and was required for p73-mediated cell survival. Furthermore, c-Jun-dependent AP-1 transcriptional activity was augmented by p73 and, consistently, induction of endogenous AP-1 target genes was compromised in the absence of p73. Chromatin immunoprecipitation and electrophoretic mobility shift analysis indicated that p73 enhanced the binding of phosphorylated c-Jun and Fra-1, another AP-1 family member, to AP-1 consensus DNA sequences, by regulating c-Jun phosphorylation and Fra-1 expression. Collectively, our data demonstrates a novel and unexpected role of p73 in augmenting AP-1 transcriptional activity through which it supports cellular growth.

p73 Overexpression increases VEGF and reduces thrombospondin-1 production: implications for tumor angiogenesis.

Tumor neovascularization is controlled by a balance between positive and negative effectors, whose production can be regulated by oncogenes and tumor suppressor genes. The aim of this study was to investigate whether the angiogenic potential of tumors could also be controlled by p73, a gene homologous to the tumor suppressor p53, whose involvement in tumor angiogenesis is known. We have studied the production of proangiogenic (VEGF, FGF-2, PIGF and PDGF) and antiangiogenic (TSP-1) factors in two p73 overexpressing clones obtained from the human ovarian carcinoma cells A2780. TSP-1 was downregulated in both clones compared to mock transfected cells, both at mRNA and protein level. Conversely, both clones showed an increased production of VEGF mRNA and protein. For both TSP-1 and VEGF, regulation of expression was partially due to modulation of the promoter activity, and was dependent on p53 status. Production of the other angiogenic factors FGF-2, PIGF and PDGF-B was also increased in p73 overexpressing clones. The two clones were more angiogenic than parental cells, as shown in vitro by their increased chemotactic activity for endothelial cells, and in vivo by the generation of more vascularized tumors. These findings suggest a potential role of p73 in tumor angiogenesis.

Evaluation of the combined effect of p53 codon 72 polymorphism and hotspot mutations in response to anticancer drugs

Mutations in p53 are common events during carcinogenesis and have been suggested to affect sensitivity to chemotherapy. Recently, the common polymorphism at codon 72, resulting in either an arginine (72R) or proline (72P) residue, was shown to differentially affect the response to anticancer drugs. Here, we have generated isogenic lung cancer cell lines to evaluate the effect of six p53 hotspot mutations (R175H, G245S, R248W, R249S, R273H, and R282W) in conjunction with the codon 72 polymorphism, for their response to a variety of anticancer drugs, either alone or in combination. The data indicate that 72R mutations do not confer general resistance to cisplatin, etoposide, gemcitabine, vinblastine, and taxol. For doxorubicin, cells expressing 249-72R were more resistant than the 249-72P cells. Combined treatment with cisplatin + etoposide resulted in an additive effect in cells expressing most 72R and 72P mutations, except for the 175-72R cells which were refractory to combined treatment. However, combined treatment with cisplatin + gemcitabine resulted in the absence of an additive effect in cells expressing the 273-72R and 282-72R mutants, unlike their 72P counterparts. Nonetheless, all p53 mutants (72R or 72P) equally inhibited p73-mediated transcriptional activity in lung cancer cells, suggesting that the selective resistance conferred by some 72R mutants to certain drugs is probably due to other p73-independent effects of these mutants. Together, the data show that the status of codon 72 polymorphism and p53 mutations can be used as a means for prediction of treatment response, although variables for each cancer type requires detailed evaluation.

Characterization of the 5'flanking region of the human Chk1 gene: identification of E2F1 functional sites.

Chk1 kinase is a key signal transducer in the checkpoint pathways responding to DNA damage. Chk1 has been shown to be transcriptionally down-regulated in response to cis-dichloro-diamino platinum treatment in a p53-dependent manner. In this paper we isolated and characterised the 5’ flanking region of the chk1 gene. We demonstrated that the isolated region has promoter activity when cloned upstream to a reporter luciferase gene and we could define the minimal promoter region. The modulation of the transcriptional activity of the cloned chk1 promoter region by different transcription factors was investigated by co-transfection experiments and by using different isogenic systems. It was shown that p53 is indeed able to down-regulate the promoter activity of the cloned region, supporting the published observation of a p53-dependent decrease in chk1 protein levels after DNA damage. A number of E2F binding sites were detected in the genomic sequence and E2F1 is able to induce chk1 promoter activity in co-transfection experiments. This induction is abolished when a mutated form of E2F1, not able to bind DNA, was used. SAOS-2 cells over-expressing E2F1 sustained an increased chk1 promoter activity than parental SAOS-2 cells, whereas a tetracycline-induced expression of Rb protein, leading to a sequestering of E2F factors, correlated with a decreased chk1 promoter activity. We discuss our results in the context of chk1 involvment in DNA damage checkpoint pathways.

Human papillomavirus type 16 E6‐enhanced susceptibility to apoptosis induced by TNF in A2780 human ovarian cancer cell line

In our study, we show that expression of HPV‐16 E6 sensitizes TNF‐induced cytotoxicity of human ovarian cancer cell line A2780. This effect is not related to a different number of TNF receptors present on cell membrane. The major induction of massive apoptosis induced by TNF is not p53‐ and p21waf‐1‐dependent but it is principally related to NF‐κB inhibition in A2780/E6 cells. Consistently to NF‐κB inhibition a rapidly release of cytochrome c and severe induction of DNA fragmentation are seen in A2780/E6 cells. Also in human colon cancer cell line HCT‐116/E6 the expression of HPV‐16 E6 enhances TNF‐cytotoxicity. This effect is not present in the HCT‐116/mu‐p53 clone (transfected with a dominant‐negative mutated p53 transgene). Thus, taken together all these observations suggest that HPV‐16 E6 sensitizes A2780 and HCT‐116 cells to TNF; this effect is not p53‐dependent, but it is essentially mediated through an inhibition in activating NF‐κB activities.

Mechanism of resistance to cisplatin in a human ovarian‐carcinoma cell line selected for resistance to doxorubicin: Possible role of p53

A possible novel mechanism of cross‐resistance to cisplatin (CDDP) in the doxorubicin‐resistant ovarian‐cancer cell line A2780‐DX3, which displays atypical multidrug resistance, is presented. A2780‐DX3 is found to be more resistant than the parental line A2780 in terms of CDDP‐induced cytotoxicity and apoptosis. Resistance is not related to the amount of cross‐links. Topoisomerase‐II (topII) protein levels were similar in both cell lines, with lower cleavage activity in A2780‐DX3 cells. The parental and the doxorubicin‐resistant cells expressed the same level of c‐erb2, which could be implicated in CDDP resistance. bcl2 was almost undetectable in both cell lines. At the same time, we found strong induction of p53, waf‐1 and bax protein levels after CDDP treatment in the A2780, but not in the A2780‐DX3, cell line. Treatment of both cell lines with mitomycin C (MMC), which acts with a mechanism different from CDDP, caused equal accumulation of p53 and induction of bax. We found that A2780‐DX3 cells exhibit altered cellular localization of p53 protein in comparison with A2780. A significant proportion of p53 in A2780‐DX3 cells was found in the cytoplasmic compartment, and CDDP treatment induced a functional p53 protein in the nucleus of A2780 much more strongly than in A2780‐DX3, which coincides with an increase of transcriptional activity of p53 in treated A2780 cells. We propose that the cross‐resistance to CDDP in the A2780‐DX3 cell line may be due to inactivation of a CDDP‐dependent p53‐accumulation pathway. Int. J. Cancer 72:155–159, 1997.

HtrA2 enhances the apoptotic functions of p73 on bax.

Regulation of the p73 gene is complex due to the presence of two promoters and the very complex mRNA maturation in both the N-terminal and C-terminal parts of the protein. We have found an additional regulation mechanism for the p73-α form that occurs through proteolytic cleavage connected to the activity of the serine protease HtrA2. Following apoptotic stimuli, HtrA2 accumulates in the nucleus and cleaves p73α in the C-terminal portion, enabling the protein to increase its transactivation activity on the apoptotic gene bax but not on the cell-cycle regulator gene p21. In the presence of HtrA2, p73 is more prone to cause caspase activation and nuclei fragmentation: p73 needs HtrA2 to activate and enhance its apoptotic functions. This new relation between p73 and HtrA2 may help to understand the different behavior of the p73 protein in cell physiology and in the responses of cancer cells to chemotherapy.

p53Transfectants in Ovarian Cancer

The tumor suppressor gene tp53 is mutated, deleted, or rearranged in more than 50% of human tumors (1). Wild-type (wt) tp53 stops growth and/or induces apoptosis in most transformed cells into which it is introduced, thus restricting research of such cells. One means of studying the effects of both wt and mutant tp53 is to generate cells in which tp53 activity can be experimentally manipulated using inducible transcriptional control elements to drive wt tp53 expression (2-4). Alternatively, temperature-sensitive (ts) tp53 mutants may be used. Such mutants were first analyzed by Oren (5) and possess wt tp53 activity at 32°C, but behave like other mutants tp53 molecules at 37°C.