The MD Anderson Prostate Cancer Patient-derived Xenograft Series (MDA PCa PDX) Captures the Molecular Landscape of Prostate Cancer and Facilitates Marker-driven Therapy Development.

PURPOSE: Advances in prostate cancer lag behind other tumor types partly due to the paucity of models reflecting key milestones in prostate cancer progression. Therefore, we develop clinically relevant prostate cancer models. EXPERIMENTAL DESIGN: Since 1996, we have generated clinically annotated patient-derived xenografts (PDXs; the MDA PCa PDX series) linked to specific phenotypes reflecting all aspects of clinical prostate cancer. RESULTS: We studied two cell line-derived xenografts and the first 80 PDXs derived from 47 human prostate cancer donors. Of these, 47 PDXs derived from 22 donors are working models and can be expanded either as cell lines (MDA PCa 2a and 2b) or PDXs. The histopathologic, genomic, and molecular characteristics (androgen receptor, ERG, and PTEN loss) maintain fidelity with the human tumor and correlate with published findings. PDX growth response to mouse castration and targeted therapy illustrate their clinical utility. Comparative genomic hybridization and sequencing show significant differences in oncogenic pathways in pairs of PDXs derived from different areas of the same tumor. We also identified a recurrent focal deletion in an area that includes the speckle-type POZ protein-like (SPOPL) gene in PDXs derived from seven human donors of 28 studied (25%). SPOPL is a SPOP paralog, and SPOP mutations define a molecular subclass of prostate cancer. SPOPL deletions are found in 7% of The Cancer Genome Atlas prostate cancers, which suggests that our cohort is a reliable platform for targeted drug development. CONCLUSIONS: The MDA PCa PDX series is a dynamic resource that captures the molecular landscape of prostate cancers progressing under novel treatments and enables optimization of prostate cancer-specific, marker-driven therapy.

Role and regulation of coordinately expressed de novo purine biosynthetic enzymes PPAT and PAICS in lung cancer.

Cancer cells exhibit altered metabolism including aerobic glycolysis that channels several glycolytic intermediates into de novo purine biosynthetic pathway. We discovered increased expression of phosphoribosyl amidotransferase (PPAT) and phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) enzymes of de novo purine biosynthetic pathway in lung adenocarcinomas. Transcript analyses from next-generation RNA sequencing and gene expression profiling studies suggested that PPAT and PAICS can serve as prognostic biomarkers for aggressive lung adenocarcinoma. Immunohistochemical analysis of PAICS performed on tissue microarrays showed increased expression with disease progression and was significantly associated with poor prognosis. Through gene knockdown and over-expression studies we demonstrate that altering PPAT and PAICS expression modulates pyruvate kinase activity, cell proliferation and invasion. Furthermore we identified genomic amplification and aneuploidy of the divergently transcribed PPAT-PAICS genomic region in a subset of lung cancers. We also present evidence for regulation of both PPAT and PAICS and pyruvate kinase activity by L-glutamine, a co-substrate for PPAT. A glutamine antagonist, 6-Diazo-5-oxo-L-norleucine (DON) blocked glutamine mediated induction of PPAT and PAICS as well as reduced pyruvate kinase activity. In summary, this study reveals the regulatory mechanisms by which purine biosynthetic pathway enzymes PPAT and PAICS, and pyruvate kinase activity is increased and exposes an existing metabolic vulnerability in lung cancer cells that can be explored for pharmacological intervention.

Extensive survey of STAT6 expression in a large series of mesenchymal tumors.

OBJECTIVES: Expression of strong nuclear STAT6 is thought to be a specific marker for solitary fibrous tumors (SFTs). Little is known about subtle expression patterns in other mesenchymal lesions. METHODS: We performed immunohistochemical studies against the C-terminus of STAT6 in tissue microarrays and whole sections, comprising 2366 mesenchymal lesions. RESULTS: Strong nuclear STAT6 was expressed in 285 of 2,021 tumors, including 206 of 240 SFTs, 49 of 408 well-differentiated/dedifferentiated liposarcomas, eight of 65 unclassified sarcomas, and 14 of 184 desmoid tumors, among others. Expression in SFTs was predominately limited to the nucleus. Other positive tumors typically expressed both nuclear and cytoplasmic STAT6. Complete absence of STAT6 was most common in pleomorphic liposarcoma and alveolar soft part sarcoma (60% and 72% cases negative, respectively). CONCLUSIONS: Strong nuclear STAT6 is largely specific for SFTs. Physiologic low-level cytoplasmic/nuclear expression is common in mesenchymal neoplasia and is of uncertain significance.

Molecular profiling of ETS and non-ETS aberrations in prostate cancer patients from northern India.

BACKGROUND: Molecular stratification of prostate cancer (PCa) based on genetic aberrations including ETS or RAF gene-rearrangements, PTEN deletion, and SPINK1 over-expression show clear prognostic and diagnostic utility. Gene rearrangements involving ETS transcription factors are frequent pathogenetic somatic events observed in PCa. Incidence of ETS rearrangements in Caucasian PCa patients has been reported, however, occurrence in Indian population is largely unknown. The aim of this study was to determine the prevalence of the ETS and RAF kinase gene rearrangements, SPINK1 over-expression, and PTEN deletion in this cohort. METHODS: In this multi-center study, formalin-fixed paraffin embedded (FFPE) PCa specimens (n = 121) were procured from four major medical institutions in India. The tissues were sectioned and molecular profiling was done using immunohistochemistry (IHC), RNA in situ hybridization (RNA-ISH) and/or fluorescence in situ hybridization (FISH). RESULTS: ERG over-expression was detected in 48.9% (46/94) PCa specimens by IHC, which was confirmed in a subset of cases by FISH. Among other ETS family members, while ETV1 transcript was detected in one case by RNA-ISH, no alteration in ETV4 was observed. SPINK1 over-expression was observed in 12.5% (12/96) and PTEN deletion in 21.52% (17/79) of the total PCa cases. Interestingly, PTEN deletion was found in 30% of the ERG-positive cases (P = 0.017) but in only one case with SPINK1 over-expression (P = 0.67). BRAF and RAF1 gene rearrangements were detected in ∼1% and ∼4.5% of the PCa cases, respectively. CONCLUSIONS: This is the first report on comprehensive molecular profiling of the major spectrum of the causal aberrations in Indian men with PCa. Our findings suggest that ETS gene rearrangement and SPINK1 over-expression patterns in North Indian population largely resembled those observed in Caucasian population but differed from Japanese and Chinese PCa patients. The molecular profiling data presented in this study could help in clinical decision-making for the pursuit of surgery, diagnosis, and in selection of therapeutic intervention.

The miR-124-prolyl hydroxylase P4HA1-MMP1 axis plays a critical role in prostate cancer progression.

Collagen prolyl hydroxylases (C-P4HAs) are a family of enzymes involved in collagen biogenesis. One of the isoforms of P4HA, Prolyl 4-hydroxylase, alpha polypeptide I (P4HA1), catalyzes the formation of 4-hydroxyproline that is essential for the proper three-dimensional folding of newly synthesized procollagen chains. Here, we show the overexpression of P4HA1 in aggressive prostate cancer. Immunohistochemical analysis using tissue microarray demonstrated that P4HA1 expression was correlated with prostate cancer progression. Using in vitro studies, we showed that P4HA1 plays a critical role in prostate cancer cell growth and tumor progression. Expression profiling studies using P4HA1 modulated prostate cells suggested regulation of Matrix metalloproteases 1. The invasive properties of P4HA1 overexpressing cells were reversed by blocking MMP1. Our studies indicate P4HA1 copy number gain in a subset of metastatic prostate tumors and its expression is also regulated by microRNA-124. MiR-124 in turn is negatively regulated by transcriptional repressors EZH2 and CtBP1, both of which are overexpressed in aggressive prostate cancer. Chick chorioallantoic membrane (CAM) assay and mice xenograft investigations show that P4HA1 is required for tumor growth and metastasis in vivo. Our observations suggest that P4HA1 plays a critical role in prostate cancer progression and could serve as a viable therapeutic target.

Evaluation of tissue PCA3 expression in prostate cancer by RNA in situ hybridization--a correlative study with urine PCA3 and TMPRSS2-ERG.

PCA3 is a prostate-specific non-coding RNA, with utility as a urine-based early detection biomarker. Here, we report the evaluation of tissue PCA3 expression by RNA in situ hybridization in a cohort of 41 mapped prostatectomy specimens. We compared tissue PCA3 expression with tissue level ERG expression and matched pre-prostatectomy urine PCA3 and TMPRSS2-ERG levels. Across 136 slides containing 138 foci of prostate cancer, PCA3 was expressed in 55% of cancer foci and 71% of high-grade prostatic intraepithelial neoplasia foci. Overall, the specificity of tissue PCA3 was >90% for prostate cancer and high-grade prostatic intraepithelial neoplasia combined. Tissue PCA3 cancer expression was not significantly associated with urine PCA3 expression. PCA3 and ERG positivity in cancer foci was positively associated (P<0.01). We report the first comprehensive assessment of PCA3 expression in prostatectomy specimens, and find limited correlation between tissue PCA3 and matched urine in prostate cancer.

Diversity of the angiogenic phenotype in non-small cell lung cancer.

Angiogenesis is crucial for tumor biology. There are many mechanisms by which tumors induce angiogenesis. We hypothesize that each individual tumor develops a unique mechanism to induce angiogenesis, and that activation of a particular angiogenic pathway suppresses the evolution of alternative pathways. We characterized 168 human non-small cell lung cancer (NSCLC) specimens for levels of angiogenic factors (angiogenic CXC chemokines, basic fibroblast growth factor, and vascular endothelial growth factor). We also induced lung tumor formation in A/J mice by injecting the tobacco carcinogen NNK. We dissected individual lung tumors and measured expression of angiogenic factors from three distinct families using real-time PCR. Finally, we controlled the angiogenic milieu using in vivo models to determine the resultant phenotype of the angiogenic factors expressed by NSCLC cells. Human tumors displayed marked variation in the expression of angiogenic factors. Individual mouse tumors, even from within the same mouse, displayed variability in their pattern of expression of angiogenic factors. In a sponge model of angiogenesis using murine lung cancer cells, implanting LLC cells with an angiogenic factor suppressed the expression of other angiogenic factors in implanted sponges. This suppressive effect was not seen in vitro. We conclude that lung cancer tumors evolve a unique and dominant angiogenic phenotype. Once an angiogenic pathway is activated, it may allow for tumor growth to proceed in the absence of a selection pressure to activate a second pathway.

The effect of tetrathiomolybdate on cytokine expression, angiogenesis, and tumor growth in squamous cell carcinoma of the head and neck.

OBJECTIVE: To assess the effect of tetrathiomolybdate on cytokine expression, angiogenesis, and tumor growth rate in human squamous cell carcinoma (SCC). DESIGN: Three human SCC cell lines were used in this study for both in vitro and in vivo investigations. Conditioned media from untreated and tetrathiomolybdate-treated cell lines were compared with regard to cytokine levels, endothelial cell chemotaxis, endothelial cell tubule formation, and migration and the ability to induce angiogenesis in a rat aortic ring array. In vivo UM-SCC-38 was seeded onto tissue-engineered scaffolds and surgically implanted into the flanks of immunodeficient mice. Tumor growth rates and the level of angiogenesis were compared after 2 weeks of therapy. SETTING: A tertiary care facility. RESULTS: In this study, we demonstrate that tetrathiomolybdate significantly decreases the secretion of interleukin 6 and basic fibroblast growth factor by head and neck SCC (HNSCC) cell lines in vitro. Furthermore, we demonstrate that tetrathiomolybdate significantly decreases the secretion of interleukin 6 and basic fibroblast growth factor by HNSCC cell lines in vitro. Furthermore, tetrathiomolybdate treatment of HNSCC cell lines results in significantly decreased endothelial cell chemotaxis, tubule formation, and neovascularization in a rat aortic ring assay. This in vitro evidence of decreased angiogenesis by tetrathiomolybdate is confirmed in vivo by using a severe combined immunodeficiency disorder mouse model in which tetrathiomolybdate therapy is shown to prevent human blood vessel formation. Finally, human HNSCC implanted into immunodeficient mice grow to a much larger size in untreated mice compared with those treated with 0.7 mL/kg per day of oral tetrathiomolybdate. CONCLUSIONS: These findings illustrate the ability of tetrathiomolybdate to down-regulate proinflammatory and proangiogenic cytokines in HNSCC. These observations are potentially exciting from a clinical perspective because a global decrease in these cytokines may decrease tumor aggressiveness and reverse the resistance to chemotherapy and radiation therapy seen in this tumor type.

Integrin alpha4beta1 regulates migration across basement membranes by lung fibroblasts: a role for phosphatase and tensin homologue deleted on chromosome 10.

Idiopathic pulmonary fibrosis is a disease that is characterized by fibroblast accumulation and activation in the distal airspaces of the lung. We hypothesized that fibrotic lung fibroblasts migrate/invade across basement membranes by integrin-mediated mechanisms as a means of entering alveoli. We demonstrate that in lung fibroblasts derived from patients with idiopathic pulmonary fibrosis, fibronectin signaling is both necessary and sufficient for basement membrane migration/invasion across basement membranes. This effect is mediated through the alpha5beta1 integrin because blockade of fibronectin-alpha5 integrin ligation attenuated this response. In contrast, ligation of alpha4beta1 integrin inhibits basement membrane invasion by normal lung fibroblasts but not by fibrotic lung fibroblasts. This phenotypic difference is not related to surface expression of the alpha4beta1 integrin, as demonstrated by flow cytometry. In normal lung fibroblasts but not in fibrotic lung fibroblasts, we show that ligation of alpha4beta1 integrin induces a significant increase in phosphatase and tensin homologue deleted on chromosome 10 (PTEN) activity. Fibrotic lung fibroblasts express constitutively less PTEN mRNA and protein as well as phosphatase activity in comparison to normal lung fibroblasts. Together, these data suggest that a loss of alpha4beta1 signaling via PTEN confers a migratory/invasive phenotype to fibrotic lung fibroblasts. Furthermore, this study implicates a loss of PTEN function in the pathophysiology of idiopathic pulmonary fibrosis.