Mireia Castillo-Martin

Targeting AKT/mTOR and ERK MAPK signaling inhibits hormone-refractory prostate cancer in a preclinical mouse model

The AKT/mammalian target of rapamycin (AKT/mTOR) and ERK MAPK signaling pathways have been shown to cooperate in prostate cancer progression and the transition to androgen-independent disease. We have now tested the effects of combinatorial inhibition of these pathways on prostate tumorigenicity by performing preclinical studies using a genetically engineered mouse model of prostate cancer. We report here that combination therapy using rapamycin, an inhibitor of mTOR, and PD0325901, an inhibitor of MAPK kinase 1 (MEK; the kinase directly upstream of ERK), inhibited cell growth in cultured prostate cancer cell lines and tumor growth particularly for androgen-independent prostate tumors in the mouse model. We further showed that such inhibition leads to inhibition of proliferation and upregulated expression of the apoptotic regulator Bcl-2-interacting mediator of cell death (Bim). Furthermore, analyses of human prostate cancer tissue microarrays demonstrated that AKT/mTOR and ERK MAPK signaling pathways are often coordinately deregulated during prostate cancer progression in humans. We therefore propose that combination therapy targeting AKT/mTOR and ERK MAPK signaling pathways may be an effective treatment for patients with advanced prostate cancer, in particular those with hormone-refractory disease.

Inactivation of p53 and Pten promotes invasive bladder cancer

Although bladder cancer represents a serious health problem worldwide, relevant mouse models for investigating disease progression or therapeutic targets have been lacking. We show that combined deletion of p53 and Pten in bladder epithelium leads to invasive cancer in a novel mouse model. Inactivation of p53 and PTEN promotes tumorigenesis in human bladder cells and is correlated with poor survival in human tumors. Furthermore, the synergistic effects of p53 and Pten deletion are mediated by deregulation of mammalian target of rapamycin (mTOR) signaling, consistent with the ability of rapamycin to block bladder tumorigenesis in preclinical studies. Our integrated analyses of mouse and human bladder cancer provide a rationale for investigating mTOR inhibition for treatment of patients with invasive disease.

A NOTCH1-driven MYC enhancer promotes T cell development, transformation and acute lymphoblastic leukemia.

Efforts to identify and annotate cancer driver genetic lesions have been focused primarily on the analysis of protein-coding genes; however, most genetic abnormalities found in human cancer are located in intergenic regions. Here we identify a new long range–acting MYC enhancer controlled by NOTCH1 that is targeted by recurrent chromosomal duplications in human T cell acute lymphoblastic leukemia (T-ALL). This highly conserved regulatory element, hereby named N-Me for NOTCH MYC enhancer, is located within a broad super-enhancer region +1.47 Mb from the MYC transcription initiating site, interacts with the MYC proximal promoter and induces orientation-independent MYC expression in reporter assays. Moreover, analysis of N-Me knockout mice demonstrates a selective and essential role of this regulatory element during thymocyte development and in NOTCH1-induced T-ALL. Together these results identify N-Me as a long-range oncogenic enhancer implicated directly in the pathogenesis of human leukemia and highlight the importance of the NOTCH1-MYC regulatory axis in T cell transformation and as a therapeutic target in T-ALL.Efforts to identify and annotate cancer driver genetic lesions have been almost exclusively focused on the analysis of protein coding genes. Here we identify a new long-range acting MYC enhancer controlled by NOTCH1, targeted by recurrent chromosomal duplications in human T-cell acute lymphoblastic leukemia (T-ALL). This highly conserved regulatory element, hereby named N-Me for NOTCH MYC enhancer, is located within a broad super-enhancer region +1.47 Mb from the MYC transcription initiating site, interacts with the MYC proximal promoter and induces orientation-independent MYC expression in reporter assays. Moreover, analysis of N-Me knockout mice demonstrates a selective and essential role of this regulatory element during thymocyte development and in NOTCH1-induced T-ALL. Altogether, these results identify N-Me as a long range oncogenic enhancer directly implicated in the pathogenesis of human leukemia and highlight the fundamental importance of the NOTCH1-MYC regulatory axis in T-cell transformation and as therapeutic target in T-ALL.

Cross-Species Regulatory Network Analysis Identifies a Synergistic Interaction between FOXM1 and CENPF that Drives Prostate Cancer Malignancy

To identify regulatory drivers of prostate cancer malignancy, we have assembled genome-wide regulatory networks (interactomes) for human and mouse prostate cancer from expression profiles of human tumors and of genetically engineered mouse models, respectively. Cross-species computational analysis of these interactomes has identified FOXM1 and CENPF as synergistic master regulators of prostate cancer malignancy. Experimental validation shows that FOXM1 and CENPF function synergistically to promote tumor growth by coordinated regulation of target gene expression and activation of key signaling pathways associated with prostate cancer malignancy. Furthermore, co-expression of FOXM1 and CENPF is a robust prognostic indicator of poor survival and metastasis. Thus, genome-wide cross-species interrogation of regulatory networks represents a valuable strategy to identify causal mechanisms of human cancer.

Distinct expression profiles of p63 variants during urothelial development and bladder cancer progression.

The TP63 gene, a member of the TP53 tumor suppressor gene family, can be expressed as at least six isoforms due to alternative promoter use and alternative splicing. The lack of p63 isoform-specific antibodies has limited the analysis of the biological significance of p63. We report a novel set of well-defined antibodies to examine p63 isoforms in mouse and human urothelium during embryogenesis and tumor progression, respectively. We provide evidence that basal and intermediate urothelial cells express p63 isoforms, with the TAp63 variant the first to be detected during development, whereas umbrella cells are characterized by a p63-negative phenotype. Notably, we report that p63-null mice develop a bladder with an abnormal urothelium, constituted by a single layer of cells that express uroplakin II and low molecular weight cytokeratins, consistent with an umbrella cell phenotype. Finally, analysis of 202 human bladder carcinomas revealed a new categorization of invasive tumors into basal-like (positive for ΔNp63 and high molecular weight cytokeratins and negative for low molecular weight cytokeratins) versus luminal-like (negative for ΔNp63 and high molecular weight cytokeratins and positive for low molecular weight cytokeratins) phenotypes, with ΔNp63 expression associated with an aggressive clinical course and poor prognosis. This study highlights the relevance of p63 isoforms in both urothelial development and bladder carcinoma progression, with ΔNp63 acting as an oncogene in certain invasive bladder tumors.

Massive parallel sequencing uncovers actionable FGFR2–PPHLN1 fusion and ARAF mutations in intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma (iCCA) is a fatal bile duct cancer with dismal prognosis and limited therapeutic options. By performing RNA- and exome-sequencing analyses, we report a novel fusion event, FGFR2-PPHLN1 (16%), and damaging mutations in the ARAF oncogene (11%). Here we demonstrate that the chromosomal translocation t(10;12)(q26;q12) leading to FGFR2-PPHLN1 fusion possesses transforming and oncogenic activity, which is successfully inhibited by a selective FGFR2 inhibitor in vitro. Among the ARAF mutations, N217I and G322S lead to activation of the pathway and N217I shows oncogenic potential in vitro. Screening of a cohort of 107 iCCA patients reveals that FGFR2 fusions represent the most recurrent targetable alteration (45%, 17/107), while they are rarely present in other primary liver tumours (0/100 of hepatocellular carcinoma (HCC); 1/21 of mixed iCCA-HCC). Taken together, around 70% of iCCA patients harbour at least one actionable molecular alteration (FGFR2 fusions, IDH1/2, ARAF, KRAS, BRAF and FGF19) that is amenable for therapeutic targeting.

A Molecular Signature Predictive of Indolent Prostate Cancer

A three-gene panel derived from mechanistic models of cell senescence predicts outcome of low Gleason score prostate tumors. To Treat or Not to Treat...* ...That is often the question for prostate cancer patients and their caretakers. Now, Irshad et al. describe a gene signature that may guide treatment choices when prognosis is unclear. Along with other clinical and molecular parameters, pathologists use the Gleason grading system to stage prostate cancers and predict patient prognosis. A Gleason score is assigned to a cancer on the basis of its microscopic features and is directly related to tumor aggressiveness and poor prognosis. Most newly diagnosed prostate cancers with low Gleason scores require no treatment intervention and are monitored with active surveillance (indolent tumors). However, the pinpointing of tumors that are aggressive and lethal despite having low Gleason scores is a clinical challenge. In these cases, new tools are needed to answer the title question. Irshad and colleagues show that low Gleason score prostate tumors can be separated into distinct indolent and aggressive subgroups on the basis of their expression of aging and senescence genes. Using patient tissue samples and gene expression data along with computational biology techniques, including a decision tree learning model, the authors identified three genes—FGFR1, PMP22, and CDKN1A—that predicted the clinical outcome of low Gleason score prostate tumors. The prognostic power of the three-gene signature was validated in independent patient cohorts, and expression of the FGFR1, PMP22, and CDKN1A proteins in biopsy samples identified Gleason 6 patients who had failed surveillance over a 10-year period. Just as Hamlet laments in his famous soliloquy, oncologists and patients need more information about the unknown before making a decision. The new signature might aid in the choice between “bear[ing] those ills [they] have” with active surveillance or actively treating—and hopefully thwarting—aggressive tumors. *Paraphrased from the “To be, or not to be” soliloquy in Hamlet by William Shakespeare. Many newly diagnosed prostate cancers present as low Gleason score tumors that require no treatment intervention. Distinguishing the many indolent tumors from the minority of lethal ones remains a major clinical challenge. We now show that low Gleason score prostate tumors can be distinguished as indolent and aggressive subgroups on the basis of their expression of genes associated with aging and senescence. Using gene set enrichment analysis, we identified a 19-gene signature enriched in indolent prostate tumors. We then further classified this signature with a decision tree learning model to identify three genes—FGFR1, PMP22, and CDKN1A—that together accurately predicted outcome of low Gleason score tumors. Validation of this three-gene panel on independent cohorts confirmed its independent prognostic value as well as its ability to improve prognosis with currently used clinical nomograms. Furthermore, protein expression of this three-gene panel in biopsy samples distinguished Gleason 6 patients who failed surveillance over a 10-year period. We propose that this signature may be incorporated into prognostic assays for monitoring patients on active surveillance to facilitate appropriate courses of treatment.

The metabolic co-regulator PGC1α suppresses prostate cancer metastasis

Cellular transformation and cancer progression is accompanied by changes in the metabolic landscape. Master co-regulators of metabolism orchestrate the modulation of multiple metabolic pathways through transcriptional programs, and hence constitute a probabilistically parsimonious mechanism for general metabolic rewiring. Here we show that the transcriptional co-activator peroxisome proliferator-activated receptor gamma co-activator 1α (PGC1α) suppresses prostate cancer progression and metastasis. A metabolic co-regulator data mining analysis unveiled that PGC1α is downregulated in prostate cancer and associated with disease progression. Using genetically engineered mouse models and xenografts, we demonstrated that PGC1α opposes prostate cancer progression and metastasis. Mechanistically, the use of integrative metabolomics and transcriptomics revealed that PGC1α activates an oestrogen-related receptor alpha (ERRα)-dependent transcriptional program to elicit a catabolic state and metastasis suppression. Importantly, a signature based on the PGC1α–ERRα pathway exhibited prognostic potential in prostate cancer, thus uncovering the relevance of monitoring and manipulating this pathway for prostate cancer stratification and treatment.

Preclinical Analysis of the γ-Secretase Inhibitor PF-03084014 in Combination with Glucocorticoids in T-cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemias (T-ALL) and lymphomas are aggressive hematologic cancers frequently associated with activating mutations in NOTCH1. Early studies identified NOTCH1 as an attractive therapeutic target for the treatment of T-ALL through the use of γ-secretase inhibitors (GSI). Here, we characterized the interaction between PF-03084014, a clinically relevant GSI, and dexamethasone in preclinical models of glucocorticoid-resistant T-ALL. Combination treatment of the GSI PF-03084014 with glucocorticoids induced a synergistic antileukemic effect in human T-ALL cell lines and primary human T-ALL patient samples. Mechanistically PF-03084014 plus glucocorticoid treatment induced increased transcriptional upregulation of the glucocorticoid receptor and glucocorticoid target genes. Treatment with PF-03084014 and glucocorticoids in combination was highly efficacious in vivo, with enhanced reduction of tumor burden in a xenograft model of T-ALL. Finally, glucocorticoid treatment effectively reversed PF-03084014–induced gastrointestinal toxicity via inhibition of goblet cell metaplasia. These results warrant the analysis of PF-03084014 and glucocorticoids in combination for the treatment of glucocorticoid-resistant T-ALL. Mol Cancer Ther; 11(7); 1565–75. ©2012 AACR.

PTEN counteracts FBXL2 to promote IP3R3- and Ca2+-mediated apoptosis limiting tumour growth

In response to environmental cues that promote IP3 (inositol 1,4,5-trisphosphate) generation, IP3 receptors (IP3Rs) located on the endoplasmic reticulum allow the ‘quasisynaptical’ feeding of calcium to the mitochondria to promote oxidative phosphorylation. However, persistent Ca2+ release results in mitochondrial Ca2+ overload and consequent apoptosis. Among the three mammalian IP3Rs, IP3R3 appears to be the major player in Ca2+-dependent apoptosis. Here we show that the F-box protein FBXL2 (the receptor subunit of one of 69 human SCF (SKP1, CUL1, F-box protein) ubiquitin ligase complexes) binds IP3R3 and targets it for ubiquitin-, p97- and proteasome-mediated degradation to limit Ca2+ influx into mitochondria. FBXL2-knockdown cells and FBXL2-insensitive IP3R3 mutant knock-in clones display increased cytosolic Ca2+ release from the endoplasmic reticulum and sensitization to Ca2+-dependent apoptotic stimuli. The phosphatase and tensin homologue (PTEN) gene is frequently mutated or lost in human tumours and syndromes that predispose individuals to cancer. We found that PTEN competes with FBXL2 for IP3R3 binding, and the FBXL2-dependent degradation of IP3R3 is accelerated in Pten−/− mouse embryonic fibroblasts and PTEN-null cancer cells. Reconstitution of PTEN-null cells with either wild-type PTEN or a catalytically dead mutant stabilizes IP3R3 and induces persistent Ca2+ mobilization and apoptosis. IP3R3 and PTEN protein levels directly correlate in human prostate cancer. Both in cell culture and xenograft models, a non-degradable IP3R3 mutant sensitizes tumour cells with low or no PTEN expression to photodynamic therapy, which is based on the ability of photosensitizer drugs to cause Ca2+-dependent cytotoxicity after irradiation with visible light. Similarly, disruption of FBXL2 localization with GGTi-2418, a geranylgeranyl transferase inhibitor, sensitizes xenotransplanted tumours to photodynamic therapy. In summary, we identify a novel molecular mechanism that limits mitochondrial Ca2+ overload to prevent cell death. Notably, we provide proof-of-principle that inhibiting IP3R3 degradation in PTEN-deregulated cancers represents a valid therapeutic strategy.