Showing papers by "Carlos Cordon-Cardo published in 2007"

Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas

Abstract: Although cancer arises from a combination of mutations in oncogenes and tumour suppressor genes, the extent to which tumour suppressor gene loss is required for maintaining established tumours is poorly understood. p53 is an important tumour suppressor that acts to restrict proliferation in response to DNA damage or deregulation of mitogenic oncogenes, by leading to the induction of various cell cycle checkpoints, apoptosis or cellular senescence. Consequently, p53 mutations increase cell proliferation and survival, and in some settings promote genomic instability and resistance to certain chemotherapies. To determine the consequences of reactivating the p53 pathway in tumours, we used RNA interference (RNAi) to conditionally regulate endogenous p53 expression in a mosaic mouse model of liver carcinoma. We show that even brief reactivation of endogenous p53 in p53-deficient tumours can produce complete tumour regressions. The primary response to p53 was not apoptosis, but instead involved the induction of a cellular senescence program that was associated with differentiation and the upregulation of inflammatory cytokines. This program, although producing only cell cycle arrest in vitro, also triggered an innate immune response that targeted the tumour cells in vivo, thereby contributing to tumour clearance. Our study indicates that p53 loss can be required for the maintenance of aggressive carcinomas, and illustrates how the cellular senescence program can act together with the innate immune system to potently limit tumour growth.

Mutational loss of PTEN induces resistance to NOTCH1 inhibition in T-cell leukemia

Abstract: Gain-of-function mutations in NOTCH1 are common in T-cell lymphoblastic leukemias and lymphomas (T-ALL), making this receptor a promising target for drugs such as γ-secretase inhibitors, which block a proteolytic cleavage required for NOTCH1 activation. However, the enthusiasm for these therapies has been tempered by tumor resistance and the paucity of information on the oncogenic programs regulated by oncogenic NOTCH1. Here we show that NOTCH1 regulates the expression of PTEN (encoding phosphatase and tensin homolog) and the activity of the phosphoinositol-3 kinase (PI3K)-AKT signaling pathway in normal and leukemic T cells. Notch signaling and the PI3K-AKT pathway synergize in vivo in a Drosophila melanogaster model of Notch-induced tumorigenesis, and mutational loss of PTEN is associated with human T-ALL resistance to pharmacological inhibition of NOTCH1. Overall, these findings identify transcriptional control of PTEN and regulation of the PI3K-AKT pathway as key elements of the leukemogenic program activated by NOTCH1 and provide the basis for the design of new therapeutic strategies for T-ALL.

The AKT-mTOR pathway plays a critical role in the development of leiomyosarcomas.

Abstract: We analyzed the PI3K-AKT signaling cascade in a cohort of sarcomas and found a marked induction of insulin receptor substrate-2 (IRS2) and phosphorylated AKT and a concomitant upregulation of downstream effectors in most leiomyosarcomas. To determine the role of aberrant PI3K-AKT signaling in leiomyosarcoma pathogenesis, we genetically inactivated Pten in the smooth muscle cell lineage by cross-breeding Pten(loxP/loxP) mice with Tagln-cre mice. Mice carrying homozygous deletion of Pten alleles developed widespread smooth muscle cell hyperplasia and abdominal leiomyosarcomas, with a very rapid onset and elevated incidence (approximately 80%) compared to other animal models. Constitutive mTOR activation was restricted to the leiomyosarcomas, revealing the requirement for additional molecular events besides Pten loss. The rapamycin derivative everolimus substantially decelerated tumor growth on Tagln-cre/Pten(loxP/loxP) mice and prolonged their lifespan. Our data show a new and critical role for the AKT-mTOR pathway in smooth muscle transformation and leiomyosarcoma genesis, and support treatment of selected sarcomas by the targeting of this pathway with new compounds or combinations of these with conventional chemotherapy agents.

Declining p53 function in the aging process: A possible mechanism for the increased tumor incidence in older populations

Abstract: Cancer is a disease of aging. The accumulation of mutations in individual cells over a lifetime is thought to be the reason. In this work, we explored an additional hypothesis: could p53 function decline with age, which would contribute to an enhanced mutation frequency and tumorigenesis in the aging process? The efficiency of the p53 response to γ-irradiation was found to decline significantly in various tissues of aging mice from several inbred strains, including lower p53 transcriptional activity and p53-dependent apoptosis. This decline resulted from a decreased stabilization of the p53 protein after stress. The function of the Ataxia-telangiectasia mutated (ATM) kinase declined significantly with age, which may then be responsible for the decline of the p53 response to radiation. Declining p53 responses to other stresses were also observed in the cultured splenocytes from aging mice. Interestingly, the time of onset of this decreased p53 response correlated with the life span of mice; mice that live longer delay their onset of decreased p53 activity with time. These results suggest an enhanced fixation of mutations in older individuals because of the declining fidelity of p53-mediated apoptosis or senescence in response to stress, and they suggest a plausible explanation for the correlation between tumorigenesis and the aging process.

Derivation of sarcomas from mesenchymal stem cells via inactivation of the Wnt pathway

Abstract: Malignant fibrous histiocytoma (MFH), now termed high-grade undifferentiated pleomorphic sarcoma, is a commonly diagnosed mesenchymal tumor, yet both the underlying molecular mechanisms of tumorigenesis and cell of origin remain unidentified. We present evidence demonstrating that human mesenchymal stem cells (hMSCs) are the progenitors of MFH. DKK1, a Wnt inhibitor and mediator of hMSC proliferation, is overexpressed in MFH. Using recombinant proteins, antibody depletion, and siRNA knockdown strategies of specific Wnt elements, we show that DKK1 inhibits hMSC commitment to differentiation via Wnt2/β-catenin canonical signaling and that Wnt5a/JNK noncanonical signaling regulates a viability checkpoint independent of Dkk1. Finally, we illustrate that hMSCs can be transformed via inhibition of Wnt signaling to form MFH-like tumors in nude mice, and conversely, MFH cells in which Wnt signaling is appropriately reestablished can differentiate along mature connective tissue lineages. Our results provide mechanistic insights regarding the cell of origin of MFH, establish what we believe is a novel tumor suppressor role for Wnt signaling, and identify a potential therapeutic differentiation strategy for sarcomas.

Tissue-specific and reversible RNA interference in transgenic mice

Abstract: Genetically engineered mice provide powerful tools for understanding mammalian gene function. These models traditionally rely on gene overexpression from transgenes or targeted, irreversible gene mutation. By adapting the tetracycline (tet)-responsive system previously used for gene overexpression, we have developed a simple transgenic system to reversibly control endogenous gene expression using RNA interference (RNAi) in mice. Transgenic mice harboring a tet-responsive RNA polymerase II promoter driving a microRNA-based short hairpin RNA targeting the tumor suppressor Trp53 reversibly express short hairpin RNA when crossed with existing mouse strains expressing general or tissue-specific 'tet-on' or 'tet-off' transactivators. Reversible Trp53 knockdown can be achieved in several tissues, and restoring Trp53 expression in lymphomas whose development is promoted by Trp53 knockdown leads to tumor regression. By leaving the target gene unaltered, this approach permits tissue-specific, reversible regulation of endogenous gene expression in vivo, with potential broad application in basic biology and drug target validation.

Role of the chromobox protein CBX7 in lymphomagenesis

Abstract: Chromobox 7 (CBX7) is a chromobox family protein and a component of the Polycomb repressive complex 1 (PRC1) that extends the lifespan of cultured epithelial cells and can act independently of BMI-1 to repress the INK4a/ARF tumor suppressor locus. To determine whether CBX7 might be oncogenic, we examined its expression pattern in a range of normal human tissues and tumor samples. CBX7 was expressed at high levels in germinal center lymphocytes and germinal center-derived follicular lymphomas, where elevated expression correlated with high c-Myc expression and a more advanced tumor grade. By targeting Cbx7 expression to the lymphoid compartment in mice, we showed that Cbx7 can initiate T cell lymphomagenesis and cooperate with c-Myc to produce highly aggressive B cell lymphomas. Furthermore, Cbx7 repressed transcription from the Ink4a/Arf locus and acted epistatically to the Arf-p53 pathway during tumorigenesis. These data identify CBX7 as a chromobox protein causally linked to cancer development and may help explain the low frequency of INK4a/ARF mutations observed in human follicular lymphoma.

Hyperactivation of Ha-ras oncogene, but not Ink4a/Arf deficiency, triggers bladder tumorigenesis

Abstract: Although ras is a potent mitogenic oncogene, its tumorigenicity depends on cellular context and cooperative events. Here we show that low-level expression of a constitutively active Ha-ras in mouse urothelium induces simple urothelial hyperplasia that is resistant to progression to full-fledged bladder tumors even in the absence of Ink4a/Arf. In stark contrast, doubling of the gene dosage of the activated Ha-ras triggered early-onset, rapidly growing, and 100% penetrant tumors throughout the urinary tract. Tumor initiation required superseding a rate-limiting step between simple and nodular hyperplasia, the latter of which is marked by the emergence of mesenchymal components and the coactivation of AKT and STAT pathways as well as PTEN inactivation. These results indicate that overactivation of Ha-ras is both necessary and sufficient to induce bladder tumors along a low-grade, noninvasive papillary pathway, and they shed light on the recent findings that ras activation, via point mutation, overexpression, or intensified signaling from FGF receptor 3, occurs in 70%-90% of these tumors in humans. Our results highlight the critical importance of the dosage/strength of Ha-ras activation in dictating its tumorigenicity--a mechanism of oncogene activation not fully appreciated to date. Finally, our results have clinical implications, as inhibiting ras and/or its downstream effectors, such as AKT and STAT3/5, could provide alternative means to treat low-grade, superficial papillary bladder tumors, the most common tumor in the urinary system.

Improved prediction of prostate cancer recurrence through systems pathology

Abstract: We have developed an integrated, multidisciplinary methodology, termed systems pathology, to generate highly accurate predictive tools for complex diseases, using prostate cancer for the prototype. To predict the recurrence of prostate cancer following radical prostatectomy, defined by rising serum prostate-specific antigen (PSA), we used machine learning to develop a model based on clinicopathologic variables, histologic tumor characteristics, and cell type-specific quantification of biomarkers. The initial study was based on a cohort of 323 patients and identified that high levels of the androgen receptor, as detected by immunohistochemistry, were associated with a reduced time to PSA recurrence. The model predicted recurrence with high accuracy, as indicated by a concordance index in the validation set of 0.82, sensitivity of 96%, and specificity of 72%. We extended this approach, employing quantitative multiplex immunofluorescence, on an expanded cohort of 682 patients. The model again predicted PSA recurrence with high accuracy, concordance index being 0.77, sensitivity of 77% and specificity of 72%. The androgen receptor was selected, along with 5 clinicopathologic features (seminal vesicle invasion, biopsy Gleason score, extracapsular extension, preoperative PSA, and dominant prostatectomy Gleason grade) as well as 2 histologic features (texture of epithelial nuclei and cytoplasm in tumor only regions). This robust platform has broad applications in patient diagnosis, treatment management, and prognostication.

Gene amplification is a relatively frequent event leading to ZBTB7A (Pokemon) overexpression in non-small cell lung cancer.

Abstract: ZBTB7A (Pokemon) is a member of the POK family of transcriptional repressors. Its main function is the suppression of the p14ARF tumour suppressor gene. Although ZBTB7A expression has been found to be increased in various types of lymphoma, there are no reports dealing with its expression in solid tumours. Given that p14(ARF) inhibits MDM2, the main negative regulator of p53, we hypothesized that overexpression of ZBTB7A could lead indirectly to p53 inactivation. To this end, we examined the status of ZBTB7A and its relationship with tumour kinetics (proliferation and apoptosis) and nodal members of the p53 network in a panel of 83 non-small cell lung carcinomas (NSCLCs). We observed, in the majority of the samples, prominent expression of ZBTB7A in the cancerous areas compared to negligible presence in the adjacent normal tissue elements. Gene amplification (two- to five-fold) was found in 27.7% of the cases, denoting its significance as a mechanism driving ZBTB7A overproduction in NSCLCs. In the remaining non-amplified group of carcinomas, analysis of the mRNA and protein expression patterns suggested that deregulation at the transcriptional and post-translational level accounts for ZBTB7A overexpression. Proliferation was associated with ZBTB7A expression (p = 0.033) but not apoptosis. The association with proliferation was reflected in the positive correlation between ZBTB7A expression and tumour size (p = 0.018). The overexpression of ZBTB7A in both p53 mutant and p53 wild-type cases, implies either a synergistic effect or that ZBTB7A exerts its oncogenic properties independently of the p14(ARF)-MDM2-p53 axis. The concomitant expression of ZBTB7A with p14(ARF) (p = 0.039), instead of the anticipated inverse relation, supports the latter notion. In conclusion, regardless of the pathway followed, the distinct expression of ZBTB7A in cancerous areas and the association with proliferation and tumour size pinpoints a role for this novel cell cycle regulator in the pathogenesis of lung cancer.

A polymorphism in HDM2 (SNP309) associates with early onset in superficial tumors, TP53 mutations, and poor outcome in invasive bladder cancer.

Abstract: Purpose: The HDM2 gene represents one of the central nodes in the p53 pathway. A recent study reported the association of a single nucleotide polymorphism (SNP309) in the HDM2 promoter region with accelerated tumor formation in both hereditary and sporadic cancers. In this study, we aim to evaluate the SNP309 in bladder cancer and to link it to TP53 status. Experimental Design: SNP309 genotyping and TP53 mutation status were done on 141 bladder tumors and 8 bladder cancer cell lines using a RFLP strategy and TP53 genotyping arrays, respectively. Transcript profiling of a subset of cases ( n = 41) was done using oligonucleotide arrays to identify genes differentially expressed regarding their SNP309 status. Results: Of 141 bladder tumors analyzed, 36.9% displayed the SNP309 wild-type (WT; T/T) genotype, whereas 11.3% were homozygous (G/G) and 51.8% were heterozygous (T/G) cases. Patients with superficial disease and the G/G genotype had an earlier age on onset than those with the T/G or T/T genotypes ( P = 0.029). Tumors with SNP309 WT genotype significantly displayed TP53 mutations when compared with tumors harboring G/G or T/G genotypes ( P P TP53 mutation status provided enhanced prognostic value ( P TP53 targets among those differentially expressed between tumors displaying G/G or T/G SNP309 versus WT cases. Conclusions: SNP309 is a frequent event in bladder cancer, related to earlier onset of superficial disease and TP53 mutation status. SNP309 genotypes were found to be associated with clinical outcome.

Relation between human papillomavirus positivity and p16 expression in head and neck carcinomas--a tissue microarray study.

Abstract: Overexpression of p16 has been demonstrated to be strongly related to the presence of HPV16,18. Squamous cell carcinoma of the head and neck has been shown to be associated with human papillomavirus (HPV) infection. The main aim of this study was to investigate the relationship between HPV presence and p16 expression in a representative collection of 60 head and neck carcinomas by tissue microarrays. The presence of HPV (HPV6,11-low risk, HPV16,18-high risk) was detected by applying in situ hybridisation. P-16 protein expression was detected imuunohistochemically. HPV6,11 positivity was observed in 10 out of 60 carcinomas. HPV16,18 presence was found in 30 out of 60 tumours. P-16 expression was detected in 35 out of 60 tumours. A statistically significant relationship was demonstrated between HPV16,18 presence and increased expression of p16. Also the HPV6,11 presence was significantly correlated with p16 immunoreactivity. Additionally, this study demonstrates that it is possible to analyse p16 expression and HPV presence by tissue microarrays.

Somatic cell type specific gene transfer reveals a tumor‐promoting function for p21Waf1/Cip1

Abstract: How proteins participate in tumorigenesis can be obscured by their multifunctional nature. For example, depending on the cellular context, the cdk inhibitors can affect cell proliferation, cell motility, apoptosis, receptor tyrosine kinase signaling, and transcription. Thus, to determine how a protein contributes to tumorigenesis, we need to evaluate which functions are required in the developing tumor. Here we demonstrate that the RCAS/TvA system, originally developed to introduce oncogenes into somatic cells of mice, can be adapted to allow us to define the contribution that different functional domains make to tumor development. Studying the development of growth-factor-induced oligodendroglioma, we identified a critical role for the Cy elements in p21, and we showed that cyclin D1T286A, which accumulates in the nucleus of p21-deficient cells and binds to cdk4, could bypass the requirement for p21 during tumor development. These genetic results suggest that p21 acts through the cyclin D1–cdk4 complex to support tumor growth, and establish the utility of using a somatic cell modeling system for defining the contribution proteins make to tumor development.

Technology insight: will systems pathology replace the pathologist?

Abstract: By using systems pathology, it might be possible to provide a predictive, personalized therapeutic recommendation for patients with prostate cancer. Systems pathology integrates quantitative data and information from many sources to generate a reliable prediction of the expected natural course of the disease and response to different therapeutic options. In other words, through the integration of relatively large data sets and the use of knowledge engineering, systems pathology aims at predicting the future behavior of tumors and their interaction with the host. In this Review, we introduce the methods used in systems pathology and summarize a recent study providing the first evidence of a concept for this strategy. The results show that systems pathology can provide a personalized prediction of the risk of recurrence after prostatectomy for cancer.

Prognostic significance of p27Kip1 expression in bladder cancer.

Abstract: The importance of markers in urological cancer is well recognised and many attempts are being made to find one which will be of prognostic significance. Authors from New York found that low expression of p27Kip1 in patients with bladder cancer was a significant predictor of pelvic recurrence, progression to metastasis and death. Authors from Switzerland examined patients with a primary solitary distal ureteric TCC; they found that distal ureteric resection in such patients is feasible, and that the long-term oncological outcome appears to be comparable to more radical treatment of this condition. OBJECTIVE To define the prognostic significance of p27Kip1 expression in bladder cancer for overall, disease-specific, metastasis-free and pelvic recurrence-free survival, and to identify clinical and pathological correlates of p27Kip1 immunophenotypes. PATIENTS AND METHODS Tumour samples from 128 evaluable patients with bladder cancer were assessed by immunohistochemistry for p27Kip1 and E2F-1 expression. Immunoreactivity of p27Kip1 was correlated with clinicopathological variables, E2F-1 immunoreactivity, and outcome. Multivariate analysis was used to assess predictors of outcome. The median follow-up was 30.9 months overall and 105.7 months in the 32 patients alive at the last follow-up. RESULTS The fraction of tumour cells with p27Kip1 nuclear immunoreactivity was <5% in 15, 5–25% in 30, 25–50% in 19, 50–75% in 51, and ≥ 75% in 13 patients. High-grade tumours and those with lower E2F-1 nuclear reactivity had a lower mean percentage p27Kip1 reactivity (P = 0.047 and 0.011, respectively). On multivariate analysis, the percentage p27Kip1 reactivity was a significant independent predictor of pelvic recurrence (P = 0.017), progression to metastases (P = 0.046), death from disease (P = 0.008), and death from any cause (P = 0.017), with a low expression portending a worse prognosis. Suspicion of vascular invasion was a significant independent predictor of progression to metastases (P = 0.002), death from disease, and death from any cause (both P < 0.001). Lymph node involvement was a significant independent predictor of progression to metastases (P = 0.006). CONCLUSIONS Low expression of p27Kip1 was a significant independent predictor of pelvic recurrence, progression to metastasis, death from disease and death from any cause, in patients with bladder cancer.

Highly efficient gene delivery for bladder cancers by intravesically administered replication-competent retroviral vectors.

Abstract: Purpose: In an attempt to improve viral delivery of potentially therapeutic genes via an intravesical route, we have recently developed murine leukemia virus-based replication-competent retrovirus (RCR) vectors. Experimental Design: We evaluated the transduction efficiency of intravesically administered RCR vectors to bladder tumor using orthotopic animal models to determine their potential as delivery vectors for bladder cancer. Results: The RCR vector containing green fluorescent protein (GFP) marker gene achieved efficient in vitro transmission of the GFP transgene. Murine bladder tumor-2 mouse bladder tumors exposed to intravesically administered RCR vectors exhibited 0%, 9.2 ± 2.9%, and 30.0 ± 6.2% of GFP expression at 9, 18, and 27 days after exposure in the orthotopic model, respectively. Orthotopic KU-19-19 human bladder tumors exposed to intravesically administered RCR vectors exhibited 3%, 85 ± 1.0%, and 100% of GFP expression at 7, 21, and 35 days after exposure, respectively. GFP staining was observed only in the tumor cells in the bladder. No detectable PCR products of GFP gene could be observed in distant organs. Treatment with RCR vectors containing yeast cytosine deaminase ( CD ) gene plus 5-fluorocytosine (5-FC) dramatically inhibited the growth of preestablished murine bladder tumor-2 tumors. A single course of 5-FC treatment resulted in a 50% animal survival in mice exposed to RCR-CD compared with a 0% survival in all controls over a 70-day follow-up period. Conclusions: Intravesically administered RCR vectors can efficiently deliver genes to orthotopic bladder tumor without viral spread in distant organs. RCR-CD/5-FC suicide gene therapy promises to be a novel and potentially therapeutic modality for bladder cancer.

Delivery of replication-competent retrovirus expressing Escherichia coli purine nucleoside phosphorylase increases the metabolism of the prodrug, fludarabine phosphate and suppresses the growth of bladder tumor xenografts.

Abstract: We have developed unique replication-competent retroviral (RCR) vectors based on murine leukemia virus that provide improved efficiency of viral delivery, allow for long-term transgene expression and demonstrate an intrinsic selectivity for transduction of rapidly dividing tumor cells. The purpose of this study was to evaluate the in vivo transduction efficiency and the therapeutic efficacy of the RCR vector mediated delivery of Escherichia coli purine nucleoside phosphorylase (PNP) in combination with fludarabine phosphate for bladder cancer. We constructed vectors containing green fluorescent protein (GFP) gene (ACE)-GFP) or PNP gene (ACE-PNP). KU-19-19 bladder tumors exhibited 28.3+/-16.1, 46.6+/-5.8 and 93.7+/-7.8% of GFP expression on 14, 18 and 26 days after intratumoral injection of ACE-GFP, respectively. GFP expression could not be observed in normal tissues surrounding the injected tumors. No detectable polymerase chain reaction products of GFP gene could be observed in any distant organs. Intratumoral injection of ACE-PNP, followed by systemically administered fludarabine phosphate, significantly inhibited the growth of pre-established KU-19-19 tumors. Our results indicate that RCR vectors are a potentially efficient gene delivery method and that the RCR vector mediated PNP gene transfer and fludarabine phosphate treatment might be a novel and potentially therapeutic modality for bladder cancer.

Genomic and Proteomic Profiles Reveal the Association of Gelsolin to TP53 Status and Bladder Cancer Progression

Abstract: Bladder cancer transformation and immortalization require the inactivation of key regulatory genes, including TP53. Genotyping of a large cohort of bladder cancer patients (n = 256) using the TP53 GeneChip showed mutations in 103 cases (40.2%), the majority of them mapping to the DNA-binding core domain. TP53 mutation status was significantly associated with tumor stage (P = 0.0001) and overall survival for patients with advanced disease (P = 0.01). Transcript profiling using oligonucleotide arrays was performed on a subset of these cases (n = 46). Supervised analyses identified genes differentially expressed between invasive bladder tumors with wild-type (n = 24) and mutated TP53 (n = 22). Pathway analyses of top-ranked genes supported the central role of TP53 in the functional network of such gene patterns. A proteomic strategy using reverse phase arrays with protein extracts of bladder cancer cell lines validated the association of identified differentially expressed genes, such as gelsolin, to TP53 status. Immunohistochemistry on tissue microarrays (n = 294) revealed that gelsolin was associated with tumor stage and overall survival, correlating positively with TP53 status in a subset of these patients. This study further reveals that TP53 mutations are frequent events in bladder cancer progression and identified gelsolin related to TP53 status, tumor staging, and clinical outcome by independent high-throughput strategies.

Androgen receptor (AR) level in the prostatectomy specimen predicts time to disease progression post androgen suppression therapy

Abstract: 5065 Background: The androgen receptor (AR) plays an important role in the initiation and growth of prostate cancer and its subsequent response to hormone therapy. Previous analyses, including our ...