CD4 T cells require ICOS-mediated PI3K signaling to increase T-Bet expression in the setting of anti-CTLA-4 therapy.

The transcription factor T-bet controls the Th1 genetic program in T cells for effective antitumor responses. Anti-CTLA-4 immunotherapy elicits dramatic antitumor responses in mice and in human patients; however, factors that regulate T-bet expression during an antitumor response mediated by anti-CTLA-4 remain to be elucidated. We were the first to report that treatment with anti-CTLA-4 led to an increase in the frequency of T cells expressing inducible costimulator (ICOS). In both treated patients and mice, our data revealed that CD4(+)ICOS(hi) T cells can act as effector T cells, which produce the Th1 cytokine IFN-γ. We also showed in a small retrospective analysis that an increased frequency of CD4(+)ICOS(hi) T cells correlated with better clinical outcome and the absence of ICOS or its ligand (ICOSL) in mouse models led to impaired tumor rejection. Here, we show that CD4(+)ICOS(hi) T cells from anti-CTLA-4-treated patients had an increase in signaling via the phospoinositide-3-kinase (PI3K) pathway and an increase in expression of T-bet. An ICOS-specific siRNA transfected into human T cells led to diminished PI3K signaling and T-bet expression. Therefore, we hypothesized that ICOS, and specifically ICOS-mediated PI3K signaling, was required for T-bet expression. We conducted studies in ICOS-deficient and ICOS-YF mice, which have a single amino acid change that abrogates PI3K signaling by ICOS. We found that ICOS-mediated PI3K signaling is required for T-bet expression during an antitumor response elicited by anti-CTLA-4 therapy. Our data provide new insight into the regulation of T-bet expression and suggest that ICOS can be targeted to improve Th1 antitumor responses.

An efficient procedure for protein extraction from formalin-fixed, paraffin-embedded tissues for reverse phase protein arrays.

INTRODUCTION: Protein extraction from formalin-fixed paraffin-embedded (FFPE) tissues is challenging due to extensive molecular crosslinking that occurs upon formalin fixation. Reverse-phase protein array (RPPA) is a high-throughput technology, which can detect changes in protein levels and protein functionality in numerous tissue and cell sources. It has been used to evaluate protein expression mainly in frozen preparations or FFPE-based studies of limited scope. Reproducibility and reliability of the technique in FFPE samples has not yet been demonstrated extensively. We developed and optimized an efficient and reproducible procedure for extraction of proteins from FFPE cells and xenografts, and then applied the method to FFPE patient tissues and evaluated its performance on RPPA. RESULTS: Fresh frozen and FFPE preparations from cell lines, xenografts and breast cancer and renal tissues were included in the study. Serial FFPE cell or xenograft sections were deparaffinized and extracted by six different protein extraction protocols. The yield and level of protein degradation were evaluated by SDS-PAGE and Western Blots. The most efficient protocol was used to prepare protein lysates from breast cancer and renal tissues, which were subsequently subjected to RPPA. Reproducibility was evaluated and Spearman correlation was calculated between matching fresh frozen and FFPE samples.The most effective approach from six protein extraction protocols tested enabled efficient extraction of immunoreactive protein from cell line, breast cancer and renal tissue sample sets. 85% of the total of 169 markers tested on RPPA demonstrated significant correlation between FFPE and frozen preparations (p < 0.05) in at least one cell or tissue type, with only 23 markers common in all three sample sets. In addition, FFPE preparations yielded biologically meaningful observations related to pathway signaling status in cell lines, and classification of renal tissues. CONCLUSIONS: With optimized protein extraction methods, FFPE tissues can be a valuable source in generating reproducible and biologically relevant proteomic profiles using RPPA, with specific marker performance varying according to tissue type.

Effects of abiraterone acetate on androgen signaling in castrate-resistant prostate cancer in bone.

PURPOSE: Persistent androgen signaling is implicated in castrate-resistant prostate cancer (CRPC) progression. This study aimed to evaluate androgen signaling in bone marrow-infiltrating cancer and testosterone in blood and bone marrow and to correlate with clinical observations. PATIENTS AND METHODS: This was an open-label, observational study of 57 patients with bone-metastatic CRPC who underwent transiliac bone marrow biopsy between October 2007 and March 2010. Patients received oral abiraterone acetate (1 g) once daily and prednisone (5 mg) twice daily. Androgen receptor (AR) and CYP17 expression were assessed by immunohistochemistry, testosterone concentration by mass spectrometry, AR copy number by polymerase chain reaction, and TMPRSS2-ERG status by fluorescent in situ hybridization in available tissues. RESULTS: Median overall survival was 555 days (95% CI, 440 to 965+ days). Maximal prostate-specific antigen decline ≥ 50% occurred in 28 (50%) of 56 patients. Homogeneous, intense nuclear expression of AR, combined with ≥ 10% CYP17 tumor expression, was correlated with longer time to treatment discontinuation (> 4 months) in 25 patients with tumor-infiltrated bone marrow samples. Pretreatment CYP17 tumor expression ≥ 10% was correlated with increased bone marrow aspirate testosterone. Blood and bone marrow aspirate testosterone concentrations declined to less than picograms-per-milliliter levels and remained suppressed at progression. CONCLUSION: The observed pretreatment androgen-signaling signature is consistent with persistent androgen signaling in CRPC bone metastases. This is the first evidence that abiraterone acetate achieves sustained suppression of testosterone in both blood and bone marrow aspirate to less than picograms-per-milliliter levels. Potential admixture of blood with bone marrow aspirate limits our ability to determine the origin of measured testosterone.

Dual inhibition of tumor energy pathway by 2-deoxyglucose and metformin is effective against a broad spectrum of preclinical cancer models.

Tumor cell proliferation requires both growth signals and sufficient cellular bioenergetics. The AMP-activated protein kinase (AMPK) pathway seems dominant over the oncogenic signaling pathway suppressing cell proliferation. This study investigated the preclinical efficacy of targeting the tumor bioenergetic pathway using a glycolysis inhibitor 2-deoxyglucose (2DG) and AMPK agonists, AICAR and metformin. We evaluated the in vitro antitumor activity of 2DG, metformin or AICAR alone, and 2DG in combination either with metformin or AICAR. We examined in vivo efficacy using xenograft mouse models. 2DG alone was not sufficient to promote tumor cell death, reflecting the limited efficacy showed in clinical trials. A combined use of 2DG and AICAR also failed to induce cell death. However, 2DG and metformin led to significant cell death associated with decrease in cellular ATP, prolonged activation of AMPK, and sustained autophagy. Gene expression analysis and functional assays revealed that the selective AMPK agonist AICAR augments mitochondrial energy transduction (OXPHOS) whereas metformin compromises OXPHOS. Importantly, forced energy restoration with methyl pyruvate reversed the cell death induced by 2DG and metformin, suggesting a critical role of energetic deprivation in the underlying mechanism of cell death. The combination of 2DG and metformin inhibited tumor growth in mouse xenograft models. Deprivation of tumor bioenergetics by dual inhibition of energy pathways might be an effective novel therapeutic approach for a broad spectrum of human tumors.

Selenium and vitamin E: cell type- and intervention-specific tissue effects in prostate cancer.

BACKGROUND: Secondary analyses of two randomized, controlled phase III trials demonstrated that selenium and vitamin E could reduce prostate cancer incidence. To characterize pharmacodynamic and gene expression effects associated with use of selenium and vitamin E, we undertook a randomized, placebo-controlled phase IIA study of prostate cancer patients before prostatectomy and created a preoperative model for prostatectomy tissue interrogation. METHODS: Thirty-nine men with prostate cancer were randomly assigned to treatment with 200 microg of selenium, 400 IU of vitamin E, both, or placebo. Laser capture microdissection of prostatectomy biopsy specimens was used to isolate normal, stromal, and tumor cells. Gene expression in each cell type was studied with microarray analysis and validated with a real-time polymerase chain reaction (PCR) and immunohistochemistry. An analysis of variance model was fit to identify genes differentially expressed between treatments and cell types. A beta-uniform mixture model was used to analyze differential expression of genes and to assess the false discovery rate. All statistical tests were two-sided. RESULTS: The highest numbers of differentially expressed genes by treatment were 1329 (63%) of 2109 genes in normal epithelial cells after selenium treatment, 1354 (66%) of 2051 genes in stromal cells after vitamin E treatment, and 329 (56%) of 587 genes in tumor cells after combination treatment (false discovery rate = 2%). Validation of 21 representative genes across all treatments and all cell types yielded Spearman correlation coefficients between the microarray analysis and the PCR validation ranging from 0.64 (95% confidence interval [CI] = 0.31 to 0.79) for the vitamin E group to 0.87 (95% CI = 0.53 to 0.99) for the selenium group. The increase in the mean percentage of p53-positive tumor cells in the selenium-treated group (26.3%), compared with that in the placebo-treated group (5%), showed borderline statistical significance (difference = 21.3%; 95% CI = 0.7 to 41.8; P = .051). CONCLUSIONS: We have demonstrated the feasibility and efficiency of the preoperative model and its power as a hypothesis-generating engine. We have also identified cell type- and zone-specific tissue effects of interventions with selenium and vitamin E that may have clinical implications.

Cytoplasmic sequestration of p27 via AKT phosphorylation in renal cell carcinoma.

PURPOSE: p27 localization and expression has prognostic and predictive value in cancer. Little is known regarding expression patterns of p27 in renal cell carcinoma (RCC) or how p27 participates in disease progression or response to therapy. EXPERIMENTAL DESIGN: RCC-derived cell lines, primary tumors, and normal renal epithelial cells were analyzed for p27 expression, phosphorylation (T157 of the NLS), and subcellular localization. RCC-derived cell lines were treated with phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitors and effects on p27 localization were assessed. The potential contribution of cytoplasmic p27 to resistance to apoptosis was also evaluated. RESULTS: p27 was elevated in tumors compared with matched controls, and cytoplasmic mislocalization of p27 was associated with increasing tumor grade. Cytoplasmic localization of p27 correlated with phosphorylation at T157, an AKT phosphorylation site in the p27 NLS. In RCC cell lines, activated PI3K/AKT signaling was accompanied by mislocalization of p27. AKT activation and phosphorylation of p27 was associated with resistance to apoptosis, and small interfering RNA knockdown of p27 or relocalization to the nucleus increased apoptosis in RCC cells. Treatment with the PI3K inhibitors LY294002 or wortmannin resulted in nuclear relocalization of p27, whereas mTOR inhibition by rapamycin did not. CONCLUSIONS: In RCC, p27 is phosphorylated at T157 of the NLS, with increasing tumor grade associated with cytoplasmic p27. PI3K inhibition (which reduces AKT activity) reduces T157 phosphorylation and induces nuclear relocalization of p27, whereas mTOR inhibition does not. Clinical testing of these findings may provide a rational approach for use of mTOR and PI3K/AKT pathway inhibitors in patients with RCC.

SPRY2 is an inhibitor of the ras/extracellular signal-regulated kinase pathway in melanocytes and melanoma cells with wild-type BRAF but not with the V599E mutant.

BRAF mutations result in constitutively active BRAF kinase activity and increased extracellular signal-regulated kinase (ERK) signaling and cell proliferation. Initial studies have shown that BRAF mutations occur at a high frequency in melanocytic nevi and metastatic lesions, but recent data have revealed much lower incidence of these mutations in early-stage melanoma, implying that other factors may contribute to melanoma pathogenesis in a wild-type (WT) BRAF context. To identify such contributing factors, we used microarray gene expression profiling to screen for differences in gene expression between a panel of melanocytic and melanoma cell lines with WT BRAF and a group of melanoma cell lines with the V599E BRAF mutation. We found that SPRY2, an inhibitor homologous to SPRY4, which was previously shown to suppress Ras/ERK signaling via direct binding to Raf-1, had reduced expression in WT BRAF cells. Using small interfering RNA-mediated SPRY2 knockdown, we showed that SPRY2 acts as an inhibitor of ERK signaling in melanocytes and WT BRAF melanoma cells, but not in cell lines with the V599E mutation. We also show that SPRY2 and SPRY4 directly bind WT BRAF but not the V599E and other exon 15 BRAF mutants. These data suggest that SPRY2, an inhibitor of ERK signaling, may be bypassed in melanoma cells either by down-regulation of its expression in WT BRAF cells, or by the presence of the BRAF mutation.

Modeling and simulation of pathways in menopause.

The analytical representation and simulation of complex molecular pathways can contribute to understanding and evaluating physiological as well as pathological processes. We are interested in modeling the processes of menopause to stratify women in terms of the genotypic and environmental components and their implications for development of individualized risk of postmenopausal disorders, e.g., breast and ovarian cancer, cardiovascular disease, and osteoporosis. We have initiated this study using the UltraSAN package to analyze the pathway associated with estrogen production. This model incorporates detailed information about the hormone factors affecting estrogen production, and the simulations carried out are based on published experimental data corresponding to hormone levels during the course of the normal female reproductive cycle. The agreement between the experimental data and the simulation is typically less than 2 ng/ml or 2 pg/ml respectively for progesterone and estradiol output. This approach further permits inclusion of information about an SNP observed in the gene coding for the enzyme aromatase as a model to study the impact of reduced enzymatic activity on hormone levels.