Intact P53 Research Articles

Abstract 4730: Inhibition of the p53-HDMX interaction sensitizes melanoma to chemotherapy

Abstract Melanoma is the leading cause of death from skin disease worldwide. It is an aggressive cancer that is often resistant to conventional chemotherapy and radiation-based therapy. The combination of a low survival rate in metastatic disease and lack of successful treatment options underscores the need to elucidate novel methods to target melanoma. More than 95% of human melanomas retain wild type p53, the protein responsible for maintaining genomic integrity; and HDMX, a negative regulator of p53 that neutralizes p53, has recently been found to be overexpressed in 65% of human cutaneous melanoma. These features suggest that targeting the p53-HDMX interaction in melanoma could not only restore the tumor suppressor function of p53, but also sensitize melanoma cells to treatment with conventional chemotherapeutic agents which rely on an intact p53 response. We have previously demonstrated that a hydrocarbon-stapled α-helix based on the transactivation domain of p53 (SAH-p53-8) efficiently disrupts p53-HDM2 and p53-HDMX complexes allowing for p53 to induce apoptotic cell death. Here, we show that SAH-p53-8 treatment is cytotoxic to metastatic melanoma cells by a p53-dependent mechanism. Additionally, SAH-p53-8 sensitizes melanoma to treatment with either cisplatin or the HDM2-specific inhibitor Nutlin-3. Importantly, the inhibition of the p53-HDMX complex arrests the growth of human metastatic melanoma in vivo. The restoration of functional p53 activity provides a basis for a previously unrecognized targeted therapy for metastatic melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4730. doi:1538-7445.AM2012-4730

Abstract 2784: Nutlin-3 induces cell cycle arrest in p53 wild-type ovarian carcinomas via upregulation of p21

Abstract Despite the fact that ovarian epithelial ovarian cancer is a molecularly and histologically heterogeneous disease, standard treatment is the same for all subtypes. Most high-grade serous ovarian carcinomas (OCs) are responsive to initial platinum/taxane chemotherapy, but low-grade serous OCs and clear cell OCs are relatively chemoresistant with limited treatment options upon recurrence, and most patients succumb to the disease. It is therefore of interest to exploit the molecular differences between high-grade serous OCs and other histological types in order to develop novel therapeutics. Over 90% of high-grade serous OCs harbor p53 mutations, whereas low-grade serous OCs are p53 wild-type but more frequently contain KRAS and BRAF mutations. Similarly, ovarian clear cell carcinomas are p53 wild-type but more frequently contain PI3K mutations. Nutlin-3 functions via inhibition of MDM2, thereby activating wild-type p53 and inducing apoptosis. We therefore sought to validate nutlin-3 as a potential novel therapeutic compound for p53 wild-type ovarian carcinomas. Two low-grade serous OC cell lines (HOC-7, MPSC-1), two clear cell OC cell lines (TOV21G, OVAS), and one high-grade serous OC p53-null cell line (SKOV3) were treated with varying concentrations of nutlin-3 (0-70 µm) for 72 hours, and cellular proliferation assays were performed. The three serous OC cell lines were then treated with nutlin-3 (5 µm), and quantitative reaction real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) were employed to quantify MDM2, p53, and p21 expression at 24, 48, and 72 hours after treatment. The MPSC-1 cell line was then sequenced for p53 mutations in exons 5, 6, 7, and 8. Nutlin-3 inhibited cellular proliferation in a dose-dependent manner in the clear cell and low-grade serous cell lines, but not the p53-null high-grade serous cell line, as expected. Both PI3K-mutant clear cell ovarian cancer cell lines exhibited decreased growth in response to nutlin-3, but to a lesser extent than the low-grade serous cell lines. HOC-7, which bears a KRAS mutation, was more sensitive to inhibition than BRAF-mutated MPSC-1. Sequencing for p53 mutation in MPSC-1 surprisingly revealed a heterozygous p53 40 bp deletion in exon 5, which accounted for the reduced sensitivity of MPSC-1 to nutlin-3. On qRT-PCR and WB, levels of p53 were slightly decreased after nutlin-3 treatment and were absent in SKOV3. MDM2 and p21 were initially upregulated, but returned to near-baseline levels after 72 hours. Nutlin-3 is a novel potential therapeutic agent for low-grade serous ovarian carcinoma, and is dependent on an intact p53 pathway. It appears to function via upregulation of p21 with subsequent cell cycle arrest. Further studies characterizing response to nutlin-3 according to tumor mutation status are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2784. doi:1538-7445.AM2012-2784

Abstract 2876: Farnesyltransferase inhibitor induces p53-dependent cellular senescence in colon cancer cells

Abstract Farnesyltransferase inhibitors (FTIs), small molecules that inhibit prenylation of multiple proteins such as Ras and Rho, have clinical promise for cancer. FTIs block malignant growth and promote cell death in a wide variety of tumor types in vitro and in vivo, but the mechanisms underlying the antitumor activity of FTIs are not completely understood. We have observed that colon cancer cell line OS187 responds to the FTI tipifarnib with a senescent phenotype, demonstrated as altered cell morphology, elevated senescence-associated β-galactosidase activity, upregulated protein and mRNA levels of senescence markers, and long-term cell cycle arrest. Our previous results also indicated that cells retaining intact p53 are more sensitive to tipifarnib than are cells with absent/inactive p53, suggesting a possible role for p53 in the senescent response to tipifarnib. To test this hypothesis, we used shRNA to reduce p53 expression in OS187 cells. Loss of p53 reversed the swollen and enlarged senescent-like cellular phenotype induced by tipifarnib. In addition, p53 knockdown cells showed less senescence-associated β-galactosidase activity than did scrambled control cells after 7-day treatment with tipifarnib. Cell viability assay suggested that p53 knockdown partially rescued the cytotoxic effects of tipifarnib at low concentrations. Western blot also demonstrated that cell cycle regulatory proteins such as p27, p21, and p15 were significantly upregulated in scrambled control cells while only a slight increase of these proteins were observed in p53 knockdown cells after tipifarnib treatment. Flow cytometry analysis showed that cells with decreased p53 level had a reduced percentage of cells in sub G1 phase when treated with a high concentration of tipifarnib, and fewer cells arresting in G2/M phase when treated with low concentration of tipifarnib compared to the scrambled control cells. This observation suggested that p53 may involve in different pathways depending on the strength of the stress signal imposed by different concentration of tipifarnib. In conclusion, these results confirm that p53 is important for inducing senescence when farnesylation is inhibited in OS187 cells. However, since p53 knockdown has very limited impact on the anti-proliferation effects of tipifarnib at high concentrations, other pathways are expected to play a role in mediating cell susceptibility to tipifarnib. Future experiments will focus on investigating the p53-independent pathways that are responsible for tipifarnib-induced senescence and apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2876. doi:1538-7445.AM2012-2876

Negative Regulation-Resistant p53 Variant Enhances Oncolytic Adenoviral Gene Therapy

Intact p53 function is essential for responsiveness to cancer therapy. However, p53 activity is attenuated by the proto-oncoprotein Mdm2, the adenovirus protein E1B 55kD, and the p53 C-terminal domain. To confer resistance to Mdm2, E1B 55kD, and C-terminal negative regulation, we generated a p53 variant (p53VPΔ30) by deleting the N-terminal and C-terminal regions of wild-type p53 and inserting the transcriptional activation domain of herpes simplex virus VP16 protein. The oncolytic adenovirus vector Ad-mΔ19 expressing p53VPΔ30 (Ad-mΔ19/p53VPΔ30) showed greater cytotoxicity than Ad-mΔ19 expressing wild-type p53 or other p53 variants in human cancer cell lines. We found that Ad-mΔ19/p53VPΔ30 induced apoptosis through accumulation of p53VPΔ30, regardless of endogenous p53 and Mdm2 status. Moreover, Ad-mΔ19/p53VPΔ30 showed a greater antitumor effect and increased survival rates of mice with U343 brain cancer xenografts that expressed wild-type p53 and high Mdm2 levels. To our knowledge, this is the first study reporting a p53 variant modified at the N terminus and C terminus that shows resistance to degradation by Mdm2 and E1B 55kD, as well as negative regulation by the p53 C terminus, without decreased trans-activation activity. Taken together, these results indicate that Ad-mΔ19/p53VPΔ30 shows potential for improving p53-mediated cancer gene therapy.

P53 Rescue through HDM2 Antagonism Suppresses Melanoma Growth and Potentiates MEK Inhibition

Oncogenesis reflects an orchestrated interaction between misguided growth signals. Although much effort has been launched to pharmacologically disable activated oncogenes, one sidelined approach is the restoration of tumor suppressive signals. As TP53 is often structurally preserved, but functionally crippled, by CDKN2A/ARF loss in melanoma, rescue of p53 function represents an attractive point of vulnerability in melanoma. In this study, we showed that both p53 protein and activity levels in melanoma cells were strongly induced by nutlin-3, a canonical HDM2 antagonist. Among a test panel of 51 cell lines, there was a marked reduction in melanoma viability that was directly linked to TP53 status. Moreover, we also found that the melanoma growth suppression mediated by mitogen-activated protein kinase/extracellular signal-regulated kinase inhibition was potentiated by HDM2 antagonism. These results provide fundamental insights into the intact p53 circuitry, which can be restored through small molecule inhibitors and potentially deployed for therapeutic gain.

Adenoviral Vector Driven by a Minimal Rad51 Promoter Is Selective for p53-Deficient Tumor Cells

BackgroundThe full length Rad51 promoter is highly active in cancer cells but not in normal cells. We therefore set out to assess whether we could confer this tumor-selectivity to an adenovirus vector.Methodology/Principal FindingsExpression of an adenovirally-vectored luciferase reporter gene from the Rad51 promoter was up to 50 fold higher in cancer cells than in normal cells. Further evaluations of a panel of truncated promoter mutants identified a 447 bp minimal core promoter element that retained the full tumor selectivity and transcriptional activity of the original promoter, in the context of an adenovirus vector. This core Rad51 promoter was highly active in cancer cells that lack functional p53, but less active in normal cells and in cancer cell lines with intact p53 function. Exogenous expression of p53 in a p53 null cell line strongly suppressed activity of the Rad51 core promoter, underscoring the selectivity of this promoter for p53-deficient cells. Follow-up experiments showed that the p53-dependent suppression of the Rad51 core promoter was mediated via an indirect, p300 coactivator dependent mechanism. Finally, transduction of target cells with an adenovirus vector encoding the thymidine kinase gene under transcriptional control of the Rad51 core promoter resulted in efficient killing of p53 defective cancer cells, but not of normal cells, upon addition of ganciclovir.Conclusions/SignificanceOverall, these experiments demonstrated that a small core domain of the Rad51 promoter can be used to target selective transgene expression from adenoviral vectors to tumor cells lacking functional p53.

Zinc supplementation augments in vivo antitumor effect of chemotherapy by restoring p53 function

Activated p53 is necessary for tumor suppression. Homeodomain-interacting protein kinase-2 (HIPK2) is a positive regulator of functional p53. HIPK2 modulates wild-type p53 activity toward proapoptotic transcription and tumor suppression by the phosphorylation of serine 46. Knock-down of HIPK2 interferes with tumor suppression and sensitivity to chemotherapy. Combined administration of adriamycin and zinc restores activity of misfolded p53 and enables the induction of its proapoptotic and tumor suppressor functions in vitro and in vivo. We therefore looked for a cancer model where HIPK2 expression is low. MMTV-neu transgenic mice overexpressing HER2/neu, develop mammary tumors at puberty with a long latency, showing very low expression of HIPK2. Here we show that whereas these tumors are resistant to adriamycin treatment, a combination of adriamycin and zinc suppresses tumor growth in vivo in these mice, an effect evidenced by the histological features of the mammary tumors. The combined treatment of adriamycin and zinc also restores wild-type p53 conformation and induces proapoptotic transcription activity. These findings may open up new possibilities for the treatment of human cancers via the combination of zinc with chemotherapeutic agents, for a selected group of patients expressing low levels of HIPK2, with an intact p53. In addition, HIPK2 may serve as a new biomarker for tumor aggressiveness.

Abstract PR9: PICT1/GLTSCR2 is a critical nucleolar binding partner of RPL11 that regulates the MDM2-p53 pathway and tumor growth

Abstract The tumor-suppressive MDM2-p53 intracellular signaling pathway is activated by cellular stress. Such stress includes DNA damage, which triggers the ATM-Chk2 and ATR-Chk1 cascades; infection or oncogene activation, which induce the p19ARF pathway; and nucleolar stress, which initiates a cascade mediated by ribosomal proteins (RPs), particularly RPL5, RPL11, RPL23, and RPS7. These RPs are usually located in the nucleolus but are rapidly released into the nucleoplasm upon nucleolar stress, activating the MDM2-p53 pathway. However, it is unknown whether there are direct connections between p53-activating RP genes and cancer development in vivo, and which mechanism these RPs employ to translocate from the nucleolus to the nucleoplasm in response to stress. PICT1/GLTSCR2 was originally identified as a candidate tumor suppressor gene located at human chromosome 19q13.32, the site of deletions in human gliomas. Subsequent in vitro studies have demonstrated that PICT1 regulates the stability of PTEN, and that low PICT1 expression caused growth advantage. Based on these findings, PICT1 has been deemed a tumor suppressor. Other evidence, however, suggests that PICT1 may not always dampen cancer progression. In oligodendroglial tumors, loss of heterozygosity (LOH) at chromosome 19q13 is associated with longer disease-free survival after chemotherapy. These contradictory observations indicate that one or more genes mapped to chromosome 19q may contribute to brain cancer development. We speculated that PICT1 might be an important chromosome 19q–mapped gene that regulates tumor progression. To clarify PICT1 function, we generated PICT1-deficient mice and ES cells and carried out extensive biochemical and molecular analyses. Our results show that PICT1 is a nucleolar protein essential for murine embryogenesis and ES cell survival. Even without DNA damage, PICT1 loss results in p53-dependent G1 arrest and apoptosis in vitro and in vivo. Strikingly, PICT1-deficient cells accumulate p53 due to impaired MDM2 function. We further show that PICT1 binds to RPL11, and that RPL11 is released from nucleoli in the absence of PICT1. In PICT1-deficient cells, increased binding of RPL11 to MDM2 blocks MDM2-mediated ubiquitination of p53, leading to p53 accumulation and repression of cell proliferation. In cases of human colon and esophageal cancer, patients with low PICT1 expression have better prognoses. Similarly, when shRNA is used to deplete PICT1 in various tumor cell lines with intact p53 signaling, the cells grow more slowly and accumulate p53. These data suggest that PICT1 is a novel key regulator of the MDM2-p53 pathway and promotes tumor progression by retaining RPL11 in the nucleolus. Thus, PICT1 is an important cancer-related gene that, at the very least, does not behave like a typical tumor suppressor and may instead have oncogenic effects. Studies on identification of factors which influence PICT1 expression or stability, or interfering with PICT1-RPL11 binding may lead to new cancer therapeutics, especially for individuals with intact-p53 signaling. This abstract is also presented as Poster B30. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr PR9.

Regulation of the MDM2-P53 pathway and tumor growth by PICT1 via nucleolar RPL11

PICT1 (also known as GLTSCR2) is considered a tumor suppressor because it stabilizes phosphatase and tensin homolog (PTEN), but individuals with oligodendrogliomas lacking chromosome 19q13, where PICT1 is located, have better prognoses than other oligodendroglioma patients. To clarify the function of PICT1, we generated Pict1-deficient mice and embryonic stem (ES) cells. Pict1 is a nucleolar protein essential for embryogenesis and ES cell survival. Even without DNA damage, Pict1 loss led to p53-dependent arrest of cell cycle phase G(1) and apoptosis. Pict1-deficient cells accumulated p53, owing to impaired Mdm2 function. Pict1 binds Rpl11, and Rpl11 is released from nucleoli in the absence of Pict1. In Pict1-deficient cells, increased binding of Rpl11 to Mdm2 blocks Mdm2-mediated ubiquitination of p53. In human cancer, individuals whose tumors express less PICT1 have better prognoses. When PICT1 is depleted in tumor cells with intact P53 signaling, the cells grow more slowly and accumulate P53. Thus, PICT1 is a potent regulator of the MDM2-P53 pathway and promotes tumor progression by retaining RPL11 in the nucleolus.

Cardiomyocyte Specific Ablation of p53 Is Not Sufficient to Block Doxorubicin Induced Cardiac Fibrosis and Associated Cytoskeletal Changes

Doxorubicin (Dox) is an anthracycline used to effectively treat several forms of cancer. Unfortunately, the use of Dox is limited due to its association with cardiovascular complications which are manifested as acute and chronic cardiotoxicity. The pathophysiological mechanism of Dox induced cardiotoxicity appears to involve increased expression of the tumor suppressor protein p53 in cardiomyocytes, followed by cellular apoptosis. It is not known whether downregulation of p53 expression in cardiomyocytes would result in decreased rates of myocardial fibrosis which occurs in response to cardiomyocyte loss. Further, it is not known whether Dox can induce perivascular necrosis and associated fibrosis in the heart. In this study we measured the effects of acute Dox treatment on myocardial and perivascular apoptosis and fibrosis in a conditional knockout (CKO) mouse model system which harbours inactive p53 alleles specifically in cardiomyocytes. CKO mice treated with a single dose of Dox (20 mg/kg), did not display lower levels of myocardial apoptosis or reactive oxygen and nitrogen species (ROS/RNS) compared to control mice with intact p53 alleles. Interestingly, CKO mice also displayed higher levels of interstitial and perivascular fibrosis compared to controls 3 or 7 days after Dox treatment. Additionally, the decrease in levels of the microtubule protein α-tubulin, which occurs in response to Dox treatment, was not prevented in CKO mice. Overall, these results indicate that selective loss of p53 in cardiomyocytes is not sufficient to prevent Dox induced myocardial ROS/RNS generation, apoptosis, interstitial fibrosis and perivascular fibrosis. Further, these results support a role for p53 independent apoptotic pathways leading to Dox induced myocardial damage and highlight the importance of vascular lesions in Dox induced cardiotoxicity.

Expression of Fbxo7 in Haematopoietic Progenitor Cells Cooperates with p53 Loss to Promote Lymphomagenesis

Fbxo7 is an unusual F box protein that augments D-type cyclin complex formation with Cdk6, but not Cdk4 or Cdk2, and its over-expression has been demonstrated to transform immortalised fibroblasts in a Cdk6-dependent manner. Here we present new evidence in vitro and in vivo on the oncogenic potential of this regulatory protein in primary haematopoietic stem and progenitor cells (HSPCs). Increasing Fbxo7 expression in HSPCs suppressed their colony forming ability in vitro, specifically decreasing CD11b (Mac1) expression, and these effects were dependent on an intact p53 pathway. Furthermore, increased Fbxo7 levels enhanced the proliferative capacity of p53 null HSPCs when they were grown in reduced concentrations of stem cell factor. Finally, irradiated mice reconstituted with p53 null, but not wild-type, HSPCs expressing Fbxo7 showed a statistically significant increase in the incidence of T cell lymphoma in vivo. These data argue that Fbxo7 negatively regulates the proliferation and differentiation of HSPCs in a p53-dependent manner, and that in the absence of p53, Fbxo7 expression can promote T cell lymphomagenesis.

Abstract 2478: Silencing Aurora Kinase A radio-sensitizes glioma cells

Abstract Purpose: It has been reported that Aurora Kinase A (AurKA) is overexpressed in many cancer types, including breast, ovarian, pancreatic, head and neck, esophageal, renal, and lung. Upregulation of AurKA can lead to oncogenic transformation and aneuploidy. AurKA is a member of the serine/threonine family of protein kinases that is localized to the mitotic spindle during cell division. AurKA is involved in spindle assembly and regulated by phosphorylation-dependent proteosomal degradation. Cells that overexpress AurKA surpass the G2/M checkpoint prematurely, resulting in chromosomal abnormalities. Silencing AurKA will prevent premature progression through the G2/M checkpoint, allowing for cells to be targeted in the radiosensitive G2/M phase. Materials and Methods: We first looked at the expression of AurKA in established (ATCC) and primary glioblastoma (GBM) cell lines. Stable silencing of AurKA was done via a lentiviral transfection with shRNA in U87, LN18, and LN229 cell lines. We performed clonogenic survival and MTS assays to measure the cell death after radiation of these AurKA knockdown (AurKA KD) cells versus cells transfected with non-targeting (NT) shRNA. Western blot was performed using these same cells post-radiation treatment to investigate the expression of proteins in both apoptotic and pro-survival pathways. To investigate the effect of AurKA KD on the formation of polyploidy cells and cell cycle regulation. Cell cycle analysis on AurKA KD and NT cells was conducted using flow cytometry. AurKA is also known to phosphorylate tumor suppressor protein p53 at Ser315, which leads to degradation of p53 via ubiquitination by Mdm2. We carried out experiments where AurKA KD cells, with either intact or mutant p53, were treated with a p53 inhibitor Pifithrin α and subsequently measured apoptosis using Annexin V and Western blot. Initial sequencing results of AurKA in our GBM cell lines did not lead to any conclusive results. Furthermore, we assessed the copy number variation of AurKA to correlate with radiosensitivity of our GBM cell lines. Results and Conclusions: Radiation treatment increases AurKA expression in our established and primary GBM cell lines. Next we silenced AurKA using lentiviral mediated shRNA transduction. AurKA KD cells exhibit greater radiosensitivity and reduced proliferation when compared to control cells, as shown by the clonogenic survival and MTS assays, respectively. Increased expression of apoptotic proteins, such as cleaved PARP and cleaved caspase-3, is seen in AurKA KD cells after treatment with radiation when compared to controls. Moreover, silencing AurKA prevented the accumulation of polyploid cells after radiation treatment. Our data suggest that AurKA could be a promising therapeutic target for radiosensitizing of GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2478. doi:10.1158/1538-7445.AM2011-2478

Abstract 2640: Inhibition of farnesylation induces senescence and apoptosis in osteosarcoma

Abstract Farnesyltransferase inhibitors (FTIs), small molecules that inhibit prenylation of multiple proteins such as Ras and Rho, have clinical promise for cancer. FTIs block malignant growth and promote cell death in a wide variety of tumor types in vitro and in vivo, but the mechanisms underlying the antitumor activity of FTIs are not completely understood. We have observed that osteosarcoma cell lines respond to the FTI tipifarnib with growth arrest, cellular enlargement, and flattened morphology, all changes consistent with the senescent phenotype (Hall et al., 2010). These observations led us to evaluate the ability of FTIs to induce senescence in osteosarcoma. Further, it was clear that cells retaining intact p53 responded at much lower concentrations of FTIs than did cells with absent/inactive p53, suggesting a possible role for p53 in this response. We measured endogenous β-galactosidase activity by in situ chromatography and quantified expression levels of common senescence markers including p53, p21Cip1, p16INK4a and DEC1 by densitometry analysis of western blot and qPCR in two human osteosarcoma cell lines, OS187 and Saos2. Cell growth was measured by direct counting of nuclei, and cell cycle was analyzed by PI staining and flow cytometry. Apoptosis was assessed by PARP cleavage, annexin V staining and flow cytometry. Variable responses to tipifarnib correlated with p53 status. In p53 proficient OS187 cells, 24h exposure to very low concentration of tipifarnib (10−9 M) induced upregulation of DEC1 and a 45-fold increase in p21Cip1 protein, accompanied by increased expression and phosphorylation of p53. This event also correlated with transient reduction of cyclin A, B1, and E proteins in a time-dependent manner, leading to cell cycle arrest which is comparable to tipifarnib-treated 293T cells. OS187 cells also exhibited elevated senescence-associated β-galactosidase activity in response to low-dose tipifarnib treatment after 24h. Meanwhile, Saos2 cells (p53-null) gave no response at 10−9 M tipifarnib. At higher concentrations (10−7 M), modest induction of p21Cip1 (10-fold increase) and p16INK4a (2-fold increase) proteins and mild inhibition of cell cycle progression were observed. Augmented senescence-associated β-galactosidase activity became detectable after Saos2 cells were exposed to high concentration of tipifarnib (10−6 M) for at least 48h. In addition to senescence, tipifarnib increased PARP cleavage and annexin V staining in OS187 cells, indicating that apoptotic cell death may also play a role in the anti-tumor activity of FTIs. These findings suggest that tipifarnib could efficiently promote senescence through a p53-dependent pathway at low concentrations, and via p53 independent pathways at higher concentrations. Induction of both cellular senescence and apoptosis may contribute to the growth inhibition induced by FTIs in osteosarcoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2640. doi:10.1158/1538-7445.AM2011-2640

Escape from p21-mediated Oncogene-induced Senescence Leads to Cell Dedifferentiation and Dependence on Anti-apoptotic Bcl-xL and MCL1 Proteins

Oncogene-induced senescence (OIS) is a tumor suppressor response that induces permanent cell cycle arrest in response to oncogenic signaling. Through the combined activation of the p53-p21 and p16-Rb suppressor pathways, OIS leads to the transcriptional repression of proliferative genes. Although this protective mechanism has been essentially described in primary cells, we surprisingly observed in this study that the OIS program is conserved in established colorectal cell lines. In response to the RAS oncogene and despite the inactivation of p53 and p16(INK4), HT29 cells enter senescence, up-regulate p21(WAF1), and induce senescence-associated heterochromatin foci formation. The same effect was observed in response to B-RAF(v600E) in LS174T cells. We also observed that p21(WAF1) prevents the expression of the CDC25A and PLK1 genes to induce cell cycle arrest. Using ChIP and luciferase experiments, we have observed that p21(WAF1) binds to the PLK1 promoter to induce its down-regulation during OIS induction. Following 4-5 weeks, several clones were able to resume proliferation and escape this tumor suppressor pathway. Tumor progression was associated with p21(WAF1) down-regulation and CDC25A and PLK1 reexpression. In addition, OIS and p21(WAF1) escape was associated with an increase in DNA damage, an induction of the epithelial-mesenchymal transition program, and an increase in the proportion of cells expressing the CD24(low)/CD44(high) phenotype. Results also indicate that malignant cells having escaped OIS rely on survival pathways induced by Bcl-xL/MCL1 signaling. In light of these observations, it appears that the transcriptional functions of p21(WAF1) are active during OIS and that the inactivation of this protein is associated with cell dedifferentiation and enhanced survival.

The RP-Mdm2-p53 Pathway and Tumorigenesis

The dynamic processes of cell growth and division are under constant surveillance. As one of the primary "gatekeepers" of the cell, the p53 tumor suppressor plays a major role in sensing and responding to a variety of stressors to maintain cellular homeostasis. Recent studies have shown that inhibition of ribosomal biogenesis can activate p53 through ribosomal protein (RP)-mediated suppression of Mdm2 E3 ligase activity. Mutations in Mdm2 that disrupt RP binding have been detected in human cancers; however, the physiological significance of the RP-Mdm2 interaction is not completely understood. We generated mice carrying a single cysteine-to-phenylalanine substitution in the central zinc finger of Mdm2 (Mdm2C305F) that disrupts Mdm2's binding to RPL11 and RPL5. Despite being developmentally normal and maintaining an intact p53 response to DNA damage, the Mdm2C305F mice demonstrate a diminished p53 response to perturbations in ribosomal biogenesis, providing the first in vivo evidence for an RP-Mdm2-p53 signaling pathway. Here we review some recent studies about RP-Mdm2-p53 signaling and speculate on the relevance of this pathway to human cancer.

Reconstitution of human MCF‐7 breast cancer cells with caspase‐3 does not sensitize them to action of CDK inhibitors

Human MCF-7 breast cancer cells are resistant to pro-apoptotic stimuli due to caspase-3 inactivation. On the other hand, they should be sensitive to agents like selective pharmacological inhibitors of cyclin-dependent kinases (CDKs) that (re)activate p53 tumor suppressor protein because they harbor intact p53 pathways. In this study we examined whether reconstitution of caspase-3 in MCF-7 cells sensitizes them to inhibitors of CDKs, by analyzing the effects of roscovitine (ROSC) and olomoucine (OLO), two closely related selective pharmacological CDK inhibitors, on both mother MCF-7 cells and a secondary mutant line, MCF-7.3.28 that stably expresses human caspase-3. The results show that ROSC is, as expected, much more potent than OLO. Surprisingly; however, ROSC and OLO reduced proliferation of parental MCF-7 cells more strongly than caspase-3-proficient counterparts. Both inhibitors arrest human breast cancer cells at the G(2)-phase of the cell cycle. Analysis of cell-cycle regulators by immunoblotting revealed that ROSC strongly induces p53 protein activity by inducing its phosphorylation at Ser46 in the MCF-7 cells lacking caspase-3, but not in caspase-3-proficient cells. Furthermore, reconstitution of caspase-3 in MCF-7 cells neither elevates the mitochondrial apoptosis rate nor significantly increases caspases activity upon ROSC treatment. However, the stabilization of p53 in response to DNA damaging agents is the same in both caspase negative and positive MCF-7 cells. Cytotoxic agents induce caspase-3-dependent apoptosis in caspase-3-proficient cells. These results indicate that reconstitution of MCF-7 cancer cells with caspase-3 sensitize them to the action of DNA damaging agents but not to ATP-like pharmacological inhibitors of CDKs.

The Hsp90 Inhibitor SNX7081 Restores the Fludarabine Sensitivity of Chronic Lymphocytic Leukemia (CLL) Cells Harbouring Mutations of ATM or TP53

AbstractAbstract 2473 Introduction:Resistance to fludarabine-based treatment represents a challenge in the clinical management of Chronic Lymphocytic Leukemia (CLL). Despite the unprecedented response rates seen with the fludarabine (F), cyclophosphamide (C), Rituximab (R) regimen novel treatment strategies are required that do not rely on an intact p53 signaling pathway. We recently described the activity of a novel, synthetic inhibitor of the molecular chaperone, heat-shock protein 90 (Hsp90) named SNX7081 (Serenex, now Pfizer) against CLL cells in vitro (Best et al., 2010 BJH). Here we explored the effect of this inhibitor on the fludarabine sensitivity of 3 haematological cell lines and 12 patient samples with mutations of ATM or TP53. Methods:Mononuclear cells were isolated by density centrifugation from CLL patients following informed consent. The 13 patient samples selected for study were determined to have mutations of either ATM or TP53 using a functional assay described in detail elsewhere (Best et al., 2008). The Mec1 (CLL), Mec2 (CLL) and U266 (B-ALL) cell lines were maintained under standard conditions in RPMI-1640 with 2mM L-glut and 1% pen/strep. Sensitivity to fludarabine, with and without SNX7081, was assessed using the MTT (3–4, 5-dimethylthiazol-2,5-diphenyl tetrazolium bromide) assay. Synergy between the agents, activation of caspase-3 and the induction of double stranded DNA (dsDNA) breaks following treatment were all assessed by flow cytometry using the mitochondrial membrane potential dye DilC1 (5) and propidium iodide (PI) or appropriate antibodies. Results:The IC50 for fludarabine was significantly higher in the 3 cell lines and 13 patient samples with ATM/TP53 lesions than in 4 cell lines or 10 patient samples defined as ATM/TP53 wild-type. Simultaneous exposure to a combination of fludarabine and SNX7081 at a ratio based on the IC50 of the compounds as single agent significantly reduced the IC50 for fludarabine (P<0.01); in 11 patient samples the IC50 for fludarabine was reduced to within a clinically achievable range (<5μM). Synergy between fludarabine and SNX7081 was evident as an effect on the distribution of the cell lines in the cell cycle and as a marked effect on the proportion of apoptotic cells (DilC (1)5 negative/PI negative) in cultures of both the cell lines and patient samples. Furthermore, we show that the combination of the compounds has a greater than additive effect on the activation of caspase-3 and on the formation of dsDNA breaks, as evidenced by the phosphorylation of g-H2Ax. Conclusions:Our studies suggest that inhibition of Hsp90 may overcome fludarabine resistance conferred by mutations of ATM or TP53. The mechanism of the synergy between these compounds appears to be via augmentation of fludarabine-induced dsDNA breaks and is concomitant with an increase in caspase-3 signaling. The data suggest that this combination may represent a promising regimen in the treatment of fludarabine-refractory CLL. Disclosures:Mulligan: Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer Schering, now Genzyme: Honoraria.

Epigenetics and MicroRNAs Combine to Modulate the MDM2/p53 Axis in Myeloma

Avoiding the inhibitory effects of p53 on cell growth is important for tumor progression. In this issue of Cancer Cell, Picchiorri etal. describe epigenetic silencing of MDM2-targeting microRNAs in multiple myeloma (MM), which generally appears to have intact p53 function. This provides the rationale for microRNA-targeted therapy for MM.

Cyclin B1/Cdk1 Phosphorylation of Mitochondrial p53 Induces Anti-Apoptotic Response

The pro-apoptotic function of p53 has been well defined in preventing genomic instability and cell transformation. However, the intriguing fact that p53 contributes to a pro-survival advantage of tumor cells under DNA damage conditions raises a critical question in radiation therapy for the 50% human cancers with intact p53 function. Herein, we reveal an anti-apoptotic role of mitochondrial p53 regulated by the cell cycle complex cyclin B1/Cdk1 in irradiated human colon cancer HCT116 cells with p53+/+ status. Steady-state levels of p53 and cyclin B1/Cdk1 were identified in the mitochondria of many human and mouse cells, and their mitochondrial influx was significantly enhanced by radiation. The mitochondrial kinase activity of cyclin B1/Cdk1 was found to specifically phosphorylate p53 at Ser-315 residue, leading to enhanced mitochondrial ATP production and reduced mitochondrial apoptosis. The improved mitochondrial function can be blocked by transfection of mutant p53 Ser-315-Ala, or by siRNA knockdown of cyclin B1 and Cdk1 genes. Enforced translocation of cyclin B1 and Cdk1 into mitochondria with a mitochondrial-targeting-peptide increased levels of Ser-315 phosphorylation on mitochondrial p53, improved ATP production and decreased apoptosis by sequestering p53 from binding to Bcl-2 and Bcl-xL. Furthermore, reconstitution of wild-type p53 in p53-deficient HCT116 p53−/− cells resulted in an increased mitochondrial ATP production and suppression of apoptosis. Such phenomena were absent in the p53-deficient HCT116 p53−/− cells reconstituted with the mutant p53. These results demonstrate a unique anti-apoptotic function of mitochondrial p53 regulated by cyclin B1/Cdk1-mediated Ser-315 phosphorylation in p53-wild-type tumor cells, which may provide insights for improving the efficacy of anti-cancer therapy, especially for tumors that retain p53.

Nanog, Gli, and p53: a new network of stemness in development and cancer

Nanog and Hedgehog (HH) are both essential regulators of stemness by promoting self-renewal in embryonic stem cells, during early embryonal development (Mitsui et al, 2003) and in cancer cells (Jeter et al, 2009). Two groups have now shown that HH signalling regulates Nanog expression in neural stem cells in the adult brain and in brain tumours. In this issue of The EMBO Journal, Po et al (2010) and Zbinden et al (2010) demonstrate that downstream effectors in the HH pathway, Gli1 and Gli2 directly bind to the Nanog promoter and thus activate Nanog expression (Figure 1A). Po et al (2010) use the cerebellum as experimental paradigm (Figure 1C) and show that Nanog is highly expressed in stem cells derived from the postnatal cerebellum and in medulloblastoma, a malignant brain tumour arising from the cerebellum in children. This work is complemented by a study by Zbinden et al (2010), who use glial tumours and tumour spheres to demonstrate that the Gli-Nanog axis promotes stemness and growth in gliomas (Figure 1B).[GRAPHICS].