The 5-Hydroxymethylcytosine Landscape of Prostate Cancer.

Analysis of DNA methylation is a valuable tool to understand disease progression and is increasingly being used to create diagnostic and prognostic clinical biomarkers. While conversion of cytosine to 5-methylcytosine (5mC) commonly results in transcriptional repression, further conversion to 5-hydroxymethylcytosine (5hmC) is associated with transcriptional activation. Here we perform the first study integrating whole-genome 5hmC with DNA, 5mC, and transcriptome sequencing in clinical samples of benign, localized, and advanced prostate cancer. 5hmC is shown to mark activation of cancer drivers and downstream targets. Furthermore, 5hmC sequencing revealed profoundly altered cell states throughout the disease course, characterized by increased proliferation, oncogenic signaling, dedifferentiation, and lineage plasticity to neuroendocrine and gastrointestinal lineages. Finally, 5hmC sequencing of cell-free DNA from patients with metastatic disease proved useful as a prognostic biomarker able to identify an aggressive subtype of prostate cancer using the genes TOP2A and EZH2, previously only detectable by transcriptomic analysis of solid tumor biopsies. Overall, these findings reveal that 5hmC marks epigenomic activation in prostate cancer and identify hallmarks of prostate cancer progression with potential as biomarkers of aggressive disease. SIGNIFICANCE: In prostate cancer, 5-hydroxymethylcytosine delineates oncogene activation and stage-specific cell states and can be analyzed in liquid biopsies to detect cancer phenotypes. See related article by Wu and Attard, p. 3880.

Predicting cancer drug TARGETS - TreAtment Response Generalized Elastic-neT Signatures.

We are now in an era of molecular medicine, where specific DNA alterations can be used to identify patients who will respond to specific drugs. However, there are only a handful of clinically used predictive biomarkers in oncology. Herein, we describe an approach utilizing in vitro DNA and RNA sequencing and drug response data to create TreAtment Response Generalized Elastic-neT Signatures (TARGETS). We trained TARGETS drug response models using Elastic-Net regression in the publicly available Genomics of Drug Sensitivity in Cancer (GDSC) database. Models were then validated on additional in-vitro data from the Cancer Cell Line Encyclopedia (CCLE), and on clinical samples from The Cancer Genome Atlas (TCGA) and Stand Up to Cancer/Prostate Cancer Foundation West Coast Prostate Cancer Dream Team (WCDT). First, we demonstrated that all TARGETS models successfully predicted treatment response in the separate in-vitro CCLE treatment response dataset. Next, we evaluated all FDA-approved biomarker-based cancer drug indications in TCGA and demonstrated that TARGETS predictions were concordant with established clinical indications. Finally, we performed independent clinical validation in the WCDT and found that the TARGETS AR signaling inhibitors (ARSI) signature successfully predicted clinical treatment response in metastatic castration-resistant prostate cancer with a statistically significant interaction between the TARGETS score and PSA response (p = 0.0252). TARGETS represents a pan-cancer, platform-independent approach to predict response to oncologic therapies and could be used as a tool to better select patients for existing therapies as well as identify new indications for testing in prospective clinical trials.

An integrated functional and clinical genomics approach reveals genes driving aggressive metastatic prostate cancer.

Genomic sequencing of thousands of tumors has revealed many genes associated with specific types of cancer. Similarly, large scale CRISPR functional genomics efforts have mapped genes required for cancer cell proliferation or survival in hundreds of cell lines. Despite this, for specific disease subtypes, such as metastatic prostate cancer, there are likely a number of undiscovered tumor specific driver genes that may represent potential drug targets. To identify such genetic dependencies, we performed genome-scale CRISPRi screens in metastatic prostate cancer models. We then created a pipeline in which we integrated pan-cancer functional genomics data with our metastatic prostate cancer functional and clinical genomics data to identify genes that can drive aggressive prostate cancer phenotypes. Our integrative analysis of these data reveals known prostate cancer specific driver genes, such as AR and HOXB13, as well as a number of top hits that are poorly characterized. In this study we highlight the strength of an integrated clinical and functional genomics pipeline and focus on two top hit genes, KIF4A and WDR62. We demonstrate that both KIF4A and WDR62 drive aggressive prostate cancer phenotypes in vitro and in vivo in multiple models, irrespective of AR-status, and are also associated with poor patient outcome.

Autoantibody Landscape in Patients with Advanced Prostate Cancer.

PURPOSE: Autoantibody responses in cancer are of great interest, as they may be concordant with T-cell responses to cancer antigens or predictive of response to cancer immunotherapies. Thus, we sought to characterize the antibody landscape of metastatic castration-resistant prostate cancer (mCRPC). EXPERIMENTAL DESIGN: Serum antibody epitope repertoire analysis (SERA) was performed on patient serum to identify tumor-specific neoepitopes. Somatic mutation-specific neoepitopes were investigated by associating serum epitope enrichment scores with whole-genome sequencing results from paired solid tumor metastasis biopsies and germline blood samples. A protein-based immunome-wide association study (PIWAS) was performed to identify significantly enriched epitopes, and candidate serum antibodies enriched in select patients were validated by ELISA profiling. A distinct cohort of patients with melanoma was evaluated to validate the top cancer-specific epitopes. RESULTS: SERA was performed on 1,229 serum samples obtained from 72 men with mCRPC and 1,157 healthy control patients. Twenty-nine of 6,636 somatic mutations (0.44%) were associated with an antibody response specific to the mutated peptide. PIWAS analyses identified motifs in 11 proteins, including NY-ESO-1 and HERVK-113, as immunogenic in mCRPC, and ELISA confirmed serum antibody enrichment in candidate patients. Confirmatory PIWAS, Identifying Motifs Using Next-generation sequencing Experiments (IMUNE), and ELISA analyses performed on serum samples from 106 patients with melanoma similarly revealed enriched cancer-specific antibody responses to NY-ESO-1. CONCLUSIONS: We present the first large-scale profiling of autoantibodies in advanced prostate cancer, utilizing a new antibody profiling approach to reveal novel cancer-specific antigens and epitopes. Our study recovers antigens of known importance and identifies novel tumor-specific epitopes of translational interest.

The DNA methylation landscape of advanced prostate cancer.

Although DNA methylation is a key regulator of gene expression, the comprehensive methylation landscape of metastatic cancer has never been defined. Through whole-genome bisulfite sequencing paired with deep whole-genome and transcriptome sequencing of 100 castration-resistant prostate metastases, we discovered alterations affecting driver genes that were detectable only with integrated whole-genome approaches. Notably, we observed that 22% of tumors exhibited a novel epigenomic subtype associated with hypermethylation and somatic mutations in TET2, DNMT3B, IDH1 and BRAF. We also identified intergenic regions where methylation is associated with RNA expression of the oncogenic driver genes AR, MYC and ERG. Finally, we showed that differential methylation during progression preferentially occurs at somatic mutational hotspots and putative regulatory regions. This study is a large integrated study of whole-genome, whole-methylome and whole-transcriptome sequencing in metastatic cancer that provides a comprehensive overview of the important regulatory role of methylation in metastatic castration-resistant prostate cancer.

Copy Number Loss of 17q22 Is Associated with Enzalutamide Resistance and Poor Prognosis in Metastatic Castration-Resistant Prostate Cancer.

PURPOSE: The purpose of this study was to measure genomic changes that emerge with enzalutamide treatment using analyses of whole-genome sequencing and RNA sequencing. EXPERIMENTAL DESIGN: One hundred and one tumors from men with metastatic castration-resistant prostate cancer (mCRPC) who had not been treated with enzalutamide (n = 64) or who had enzalutamide-resistant mCRPC (n = 37) underwent whole genome sequencing. Ninety-nine of these tumors also underwent RNA sequencing. We analyzed the genomes and transcriptomes of these mCRPC tumors. RESULTS: Copy number loss was more common than gain in enzalutamide-resistant tumors. Specially, we identified 124 protein-coding genes that were more commonly lost in enzalutamide-resistant samples. These 124 genes included eight putative tumor suppressors located at nine distinct genomic regions. We demonstrated that focal deletion of the 17q22 locus that includes RNF43 and SRSF1 was not present in any patient with enzalutamide-naïve mCRPC but was present in 16% (6/37) of patients with enzalutamide-resistant mCRPC. 17q22 loss was associated with lower RNF43 and SRSF1 expression and poor overall survival from time of biopsy [median overall survival of 19.3 months in 17q22 intact vs. 8.9 months in 17q22 loss, HR, 3.44 95% confidence interval (CI), 1.338-8.867, log-rank P = 0.006]. Finally, 17q22 loss was linked with activation of several targetable factors, including CDK1/2, Akt, and PLK1, demonstrating the potential therapeutic relevance of 17q22 loss in mCRPC. CONCLUSIONS: Copy number loss is common in enzalutamide-resistant tumors. Focal deletion of chromosome 17q22 defines a previously unappreciated molecular subset of enzalutamide-resistant mCRPC associated with poor clinical outcome.

Down-regulation of ADRB2 expression is associated with small cell neuroendocrine prostate cancer and adverse clinical outcomes in castration-resistant prostate cancer.

OBJECTIVES: The net oncogenic effect of ß2-adrenergic receptor ADRB2, whose downstream elements induce neuroendocrine differentiation and whose expression is regulated by EZH2, is unclear. ADRB2 expression and associated clinical outcomes in metastatic castration-resistant prostate cancer (mCRPC) are unknown. METHODS AND MATERIALS: This was a retrospective analysis of a multi-center, prospectively enrolled cohort of mCRPC patients. Metastatic biopsies were obtained at progression, and specimens underwent laser capture microdissection and RNA-seq. ADRB2 expression was stratified by histology and clustering based on unsupervised hierarchical transcriptome analysis and correlated with EZH2 expression; an external dataset was used for validation. The association between ADRB2 expression and overall survival (OS) was assessed by log-rank test and a multivariable Cox proportional hazard model. RESULTS: One hundred and twenty-seven patients with progressive mCRPC had sufficient metastatic tumor for RNA-seq. ADRB2 expression was lowest in the small cell-enriched transcriptional cluster (P < 0.01) and correlated inversely with EZH2 expression (r = -0.28, P < 0.01). These findings were validated in an external cohort enriched for neuroendocrine differentiation. Patients with tumors harboring low ADRB2 expression (lowest quartile) had a shorter median OS than those with higher (9.5 vs. 20.5 months, P = 0.02). In multivariable analysis, low ADRB2 expression was associated with a trend toward shorter OS (HR for death = 1.54, 95%CI 0.98-2.44). Conversely, higher expression of upstream transcriptional regulator EZH2 was associated with shortened OS (HR for death = 3.01, 95%CI 1.12-8.09). CONCLUSIONS: Low ADRB2 expression is associated with neuroendocrine differentiation and is associated with shortened survival. EZH2 is a potential therapeutic target for preventing neuroendocrine transdifferentiation and improving outcomes in mCRPC. Further studies of agents targeting ß-adrenergic signaling are warranted.

Genomic and clinical characterization of stromal infiltration markers in prostate cancer.

BACKGROUND: The progression of prostate cancer is a complex, multistep process that involves molecular alterations in cells of the tumor and the microenvironment, with associated interactions between the stroma and epithelium. Genomic expression analyses of stromal infiltration markers were performed to determine the significance thereof in prostate cancer. METHODS: Genome-wide expression profiles of formalin-fixed, paraffin-embedded radical prostatectomy samples were evaluated from a prospective registry cohort (n = 5239) and 3 retrospective institutional cohorts (n = 1135). Two independent stromal gene expression signatures implied stromal infiltration. Cox proportional hazards regression defined the association between stromal infiltration expression and metastasis-free survival (MFS). RESULTS: Stromal expression scores were correlated with stromal signature genes and with other key stromal markers (CAV1, VIM, and TAGLN), basal activity, and CD3 and CD4 immune biomarkers (r > 0.5 for all). The top decile of stromal expression was associated with high genomic risk scores (Decipher ≥ 0.6) , high Cancer of the Prostate Risk Assessment-Postsurgical scores, Gleason 9 to 10 disease, and a higher risk for metastasis (hazard ratio, 2.35; 95% CI, 1.37-4.02; P = .001). A higher stromal infiltration score was also associated with decreased expression of DNA repair genes and higher radiation sensitivity genomic scores. Postoperative radiation therapy (RT) was associated with an MFS benefit for patients with high stromal scores, but not for patients with low stromal scores (Pinteraction  = .02). CONCLUSIONS: Expression of stromal infiltration markers is correlated with prostate cancer aggressiveness/progression and may be predictive of a response to RT. Stromal infiltration markers should be studied and considered for incorporation into clinical prognostication and decision making.

Uncovering axes of variation among single-cell cancer specimens.

While several tools have been developed to map axes of variation among individual cells, no analogous approaches exist for identifying axes of variation among multicellular biospecimens profiled at single-cell resolution. For this purpose, we developed 'phenotypic earth mover's distance' (PhEMD). PhEMD is a general method for embedding a 'manifold of manifolds', in which each datapoint in the higher-level manifold (of biospecimens) represents a collection of points that span a lower-level manifold (of cells). We apply PhEMD to a newly generated drug-screen dataset and demonstrate that PhEMD uncovers axes of cell subpopulational variation among a large set of perturbation conditions. Moreover, we show that PhEMD can be used to infer the phenotypes of biospecimens not directly profiled. Applied to clinical datasets, PhEMD generates a map of the patient-state space that highlights sources of patient-to-patient variation. PhEMD is scalable, compatible with leading batch-effect correction techniques and generalizable to multiple experimental designs.

Germline polymorphisms associated with impaired survival outcomes and somatic tumor alterations in advanced prostate cancer.

INTRODUCTION: Germline variants in androgen metabolism genes may influence clinical response to androgen deprivation therapy (ADT) in advanced prostate cancer. We sought to investigate the prognostic significance of germline variants in androgen metabolism genes with respect to overall survival (OS) after ADT, and to associate germline variants with tumor genomic features. METHODS: Germline and somatic whole-genome sequencing (WGS) data were evaluated in a cohort of 101 men with metastatic castration-resistant prostate cancer (mCRPC). Survival analyses were performed to identify polymorphisms associated with impaired OS after primary ADT. Germline variants found to be prognostic of OS were associated with tumor somatic DNA-sequence alterations based on WGS performed on paired metastasis biopsies from the same 101 patients. Gene set enrichment analysis was performed based on tumor RNA-sequencing data to identify genomic pathways differentially expressed in patients with germline variants. RESULTS: A comprehensive literature review identified 17 candidate polymorphisms in nine androgen metabolism genes that have been previously shown to have an association with response to ADT in prostate cancer. Of these, the variant rs1856888 allele located 13 kb upstream of HSD3B1 was found to be significantly associated with impaired OS (P = 0.029). Variant rs1856888 was commonly co-inherited with the well-characterized HSD3B1(1245A>C) polymorphism, and there was a trend toward shorter median OS in patients with HSD3B1(1245A>C) compared with homozygous wild-type patients (P = 0.052). While HSD3B1 germline variants were not associated with common somatic tumor DNA alterations, they were associated with increased tumor expression of cell proliferation and cell cycle genes. CONCLUSIONS: This study presents a comprehensive assessment of germline variants in androgen metabolism genes and highlights HSD3B1 polymorphisms as prognostic of OS after ADT and associated with an aggressive gene expression tumor profile in mCRPC.

Transcriptomic and Clinical Characterization of Neuropeptide Y Expression in Localized and Metastatic Prostate Cancer: Identification of Novel Prostate Cancer Subtype with Clinical Implications.

BACKGROUND: Tumor microenvironment and its interaction with neuroendocrine modulators contribute to prostate carcinogenesis and progression. OBJECTIVE: We sought to define the transcriptomic and clinical implications of neuropeptide Y (NPY) expression in prostate cancer progression. DESIGN, SETTING, AND PARTICIPANTS: Genome-wide expression profiling of three localized prostate cancer (total n=18818) and five metastatic castrate-resistant prostate cancer (mCRPC; total n=495) cohorts was used to characterize NPY expression. All men underwent radical prostatectomy (RP) for localized prostate cancer. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Patients were grouped into those with low NPY and high NPY based on NPY expression. Associations between these groups and histological, genomic, and clinical outcomes including progression-free survival (PFS) and metastases-free survival (MFS) were examined. Combining ERG-fusion status with NPY expression, four groups were defined (lowNPY/ERG+, lowNPY/ERG-, highNPY/ERG+, and highNPY/ERG-). Cox proportional hazards modeled the time to distant metastasis after RP. Genomic risk scores for metastasis were calculated for prospective samples, based on a 22-gene signature. RESULTS AND LIMITATIONS: Across cancers, NPY showed the highest expression in prostate cancer in The Cancer Genome Atlas (TCGA) PAN-Cancer cohort (n=9483, p<0.0001). In 17967 prospective samples, low NPY expression was associated with aggressive grade group 5 disease and a higher genomic risk (p<0.0001). In the retrospective (n=355) and TCGA (n=497) cohorts, low NPY was associated with shorter MFS and PFS, respectively (p=0.001 for both). In mCRPC cohorts, low NPY was associated with neuroendocrine development (p<0.01). NPY was highly correlated to ERG; thus, we defined four groups based on NPY expression and ERG fusion. The lowNPY/ERG+ subtype was associated with the highest genomic risk for metastasis (p<0.0001) and the highest rate of metastasis compared with all other subtypes (hazard ratio [HR]: 2.2 [1.22-4.03], p=0.008), while the highNPY/ERG- subtype was associated with the lowest genomic risk for metastasis (p<0.0001) and the lowest rate of metastasis (HR: 0.53 [0.35-0.81], p=0.003). CONCLUSIONS: Low NPY expression is associated with adverse genomic features and clinical correlates and outcomes. The lowNPY/ERG+ subtype was associated with the highest risk of developing metastasis. Prognostic subgrouping and tailored treatments by NPY expression and ERG fusion status warrant further study. PATIENT SUMMARY: The low neuropeptide Y prostate cancer subtype appears to be aggressive with a high risk of developing metastasis.

Genomic Drivers of Poor Prognosis and Enzalutamide Resistance in Metastatic Castration-resistant Prostate Cancer.

BACKGROUND: Metastatic castration-resistant prostate cancer (mCRPC) is the lethal form of the disease. Several recent studies have identified genomic alterations in mCRPC, but the clinical implications of these genomic alterations have not been fully elucidated. OBJECTIVE: To use whole-genome sequencing (WGS) to assess the association between key driver gene alterations and overall survival (OS), and to use whole-transcriptome RNA sequencing to identify genomic drivers of enzalutamide resistance. DESIGN, SETTING, AND PARTICIPANTS: We performed survival analyses and gene set enrichment analysis (GSEA) on WGS and RNA sequencing results for a cohort of 101 mCRPC patients. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: OS was the clinical endpoint for all univariate and multivariable survival analyses. Candidate drivers of enzalutamide resistance were identified in an unbiased manner, and mutations of the top candidate were further assessed for enrichment among enzalutamide-resistant patients using Fisher's exact test. RESULTS AND LIMITATIONS: Harboring two DNA alterations in RB1 was independently predictive of poor OS (median 14.1 vs 42.0mo; p=0.007) for men with mCRPC. GSEA identified the Wnt/ß-catenin pathway as the top differentially modulated pathway among enzalutamide-resistant patients. Furthermore, ß-catenin mutations were exclusive to enzalutamide-resistant patients (p=0.01) and independently predictive of poor OS (median 13.6 vs 41.7mo; p=0.025). CONCLUSIONS: The presence of two RB1 DNA alterations identified in our WGS analysis was independently associated with poor OS among men with mCRPC. The Wnt/ß-catenin pathway plays an important role in enzalutamide resistance, with differential pathway expression and enrichment of ß-catenin mutations in enzalutamide-resistant patients. Moreover, ß-catenin mutations were predictive of poor OS in our cohort. PATIENT SUMMARY: We observed a correlation between genomic findings for biopsy samples from metastases from men with metastatic castration-resistant prostate cancer (mCRPC) and clinical outcomes. This work sheds new light on clinically relevant genomic alterations in mCRPC and provides a roadmap for the development of new personalized treatment regimens in mCRPC.

Novel RB1-Loss Transcriptomic Signature Is Associated with Poor Clinical Outcomes across Cancer Types.

PURPOSE: Rb-pathway disruption is of great clinical interest, as it has been shown to predict outcomes in multiple cancers. We sought to develop a transcriptomic signature for detecting biallelic RB1 loss (RBS) that could be used to assess the clinical implications of RB1 loss on a pan-cancer scale. EXPERIMENTAL DESIGN: We utilized data from the Cancer Cell Line Encyclopedia (N = 995) to develop the first pan-cancer transcriptomic signature for predicting biallelic RB1 loss (RBS). Model accuracy was validated using The Cancer Genome Atlas (TCGA) Pan-Cancer dataset (N = 11,007). RBS was then used to assess the clinical relevance of biallelic RB1 loss in TCGA Pan-Cancer and in an additional metastatic castration-resistant prostate cancer (mCRPC) cohort. RESULTS: RBS outperformed the leading existing signature for detecting RB1 biallelic loss across all cancer types in TCGA Pan-Cancer (AUC, 0.89 vs. 0.66). High RBS (RB1 biallelic loss) was associated with promoter hypermethylation (P = 0.008) and gene body hypomethylation (P = 0.002), suggesting RBS could detect epigenetic gene silencing. TCGA Pan-Cancer clinical analyses revealed that high RBS was associated with short progression-free (P < 0.00001), overall (P = 0.0004), and disease-specific (P < 0.00001) survival. On multivariable analyses, high RBS was predictive of shorter progression-free survival in TCGA Pan-Cancer (P = 0.03) and of shorter overall survival in mCRPC (P = 0.004) independently of the number of DNA alterations in RB1. CONCLUSIONS: Our study provides the first validated tool to assess RB1 biallelic loss across cancer types based on gene expression. RBS can be useful for analyzing datasets with or without DNA-sequencing results to investigate the emerging prognostic and treatment implications of Rb-pathway disruption.See related commentary by Choudhury and Beltran, p. 4199.

Clinical and Genomic Implications of Luminal and Basal Subtypes Across Carcinomas.

PURPOSE: Carcinomas originate from epithelial tissues, which have apical (luminal) and basal orientations. The degree of luminal versus basal differentiation in cancer has been shown to be biologically important in some carcinomas and impacts treatment response. EXPERIMENTAL DESIGN: Although prior studies have focused on individual cancer types, we used a modified clinical-grade classifier (PAM50) to subtype 8,764 tumors across 22 different carcinomas into luminal A, luminal B, and basal-like tumors. RESULTS: We found that all epithelial tumors demonstrated similar gene expression-based luminal/basal subtypes. As expected, basal-like tumors were associated with increased expression of the basal markers KRT5/6 and KRT14, and luminal-like tumors were associated with increased expression of the luminal markers KRT20. Luminal A tumors consistently had improved outcomes compared with basal across many tumor types, with luminal B tumors falling between the two. Basal tumors had the highest rates of TP53 and RB1 mutations and copy number loss. Luminal breast, cervical, ovarian, and endometrial tumors had increased ESR1 expression, and luminal prostate, breast, cervical, and bladder tumors had increased androgen receptor (AR) expression. Furthermore, luminal B tumors had the highest rates of AR and ESR1 mutations and had increased sensitivity in vitro to bicalutamide and tamoxifen. Luminal B tumors were more sensitive to gemcitabine, and basal tumors were more sensitive to docetaxel. CONCLUSIONS: This first pan-carcinoma luminal/basal subtyping across epithelial tumors reveals global similarities across carcinomas in the transcriptome, genome, clinical outcomes, and drug sensitivity, emphasizing the biological and translational importance of these luminal versus basal subtypes.

CDKN2A Copy Number Loss Is an Independent Prognostic Factor in HPV-Negative Head and Neck Squamous Cell Carcinoma.

BACKGROUND: HPV infection is associated with high p16 expression and good prognosis in head and neck squamous cell carcinomas (HNSCCs). Analysis of CDKN2A, the gene encoding p16, may further elucidate the association between p16 expression and prognosis. We sought to determine whether CDKN2A copy number loss was associated with poor survival in HPV-negative HNSCCs. METHODS: The Cancer Genome Atlas HNSCC clinical and genomic data were obtained and integrated. Patients <80 years old with a primary tumor in the oral cavity, oropharynx, hypopharynx, or larynx were included. Stratifying by copy number loss status, CDKN2A mRNA and p16 protein expression levels were examined and overall survival (OS) and disease-free survival (DFS) were evaluated. RESULTS: 401 patients with HPV-negative HNSCC were identified. 146 patients demonstrated CDKN2A copy number loss. The CDKN2A copy number loss group expressed significantly lower levels of CDKN2A mRNA and p16 protein than did the non-copy number loss group. Median OS for patients with and without CDKN2A copy number loss was 16.5 and 46.6 months, respectively (p = 0.007). Median DFS for both groups was 11.6 and 19.2 months, respectively (p = 0.03). In both univariate and multivariable analyses, stage IV designation, receipt of chemotherapy and CDKN2A copy number loss were predictive of OS. CONCLUSION: CDKN2A copy number loss predicted poor survival independently of other patient and treatment factors and may be a clinically useful prognostic factor.

Leptin deficiency and beta-cell dysfunction underlie type 2 diabetes in compound Akt knockout mice.

Phenotypic analyses of mice null for the individual Akt isoforms suggested that they are functionally distinct and that only Akt2 plays a role in diabetes. We show here that Akt isoforms play compensatory and complementary roles in glucose homeostasis and diabetes. Insulin resistance in Akt2(-/-) mice was inhibited by haplodeficiency of Pten, suggesting that other Akt isoforms can compensate for Akt2 function. Haplodeficiency of Akt1 in Akt2(-/-) mice, however, converts prediabetes to overt type 2 diabetes, which is also reversed by haplodeficiency of Pten. Akt3 does not appear to contribute significantly to diabetes. Overt type 2 diabetes in Akt1(+/-) Akt2(-/-) mice is manifested by hyperglycemia due to beta-cell dysfunction combined with impaired glucose homeostasis due to markedly decreased leptin levels. Restoring leptin levels was sufficient to restore normal blood glucose and insulin levels in Akt1(+/-) Akt2(-/-) and Akt2(-/-) mice, suggesting that leptin-deficiency is the predominant cause of diabetes in these mice. These results uncover a new mechanism linking Akt to diabetes, provide a therapeutic strategy, and show that diabetes induced as a consequence of cancer therapy, via Akt inhibition, could be reversed by leptin therapy.

Akt1 in osteoblasts and osteoclasts controls bone remodeling.

Bone mass and turnover are maintained by the coordinated balance between bone formation by osteoblasts and bone resorption by osteoclasts, under regulation of many systemic and local factors. Phosphoinositide-dependent serine-threonine protein kinase Akt is one of the key players in the signaling of potent bone anabolic factors. This study initially showed that the disruption of Akt1, a major Akt in osteoblasts and osteoclasts, in mice led to low-turnover osteopenia through dysfunctions of both cells. Ex vivo cell culture analyses revealed that the osteoblast dysfunction was traced to the increased susceptibility to the mitochondria-dependent apoptosis and the decreased transcriptional activity of runt-related transcription factor 2 (Runx2), a master regulator of osteoblast differentiation. Notably, our findings revealed a novel role of Akt1/forkhead box class O (FoxO) 3a/Bim axis in the apoptosis of osteoblasts: Akt1 phosphorylates the transcription factor FoxO3a to prevent its nuclear localization, leading to impaired transactivation of its target gene Bim which was also shown to be a potent proapoptotic molecule in osteoblasts. The osteoclast dysfunction was attributed to the cell autonomous defects of differentiation and survival in osteoclasts and the decreased expression of receptor activator of nuclear factor-kappaB ligand (RANKL), a major determinant of osteoclastogenesis, in osteoblasts. Akt1 was established as a crucial regulator of osteoblasts and osteoclasts by promoting their differentiation and survival to maintain bone mass and turnover. The molecular network found in this study will provide a basis for rational therapeutic targets for bone disorders.

Akt1 governs breast cancer progression in vivo.

The serine threonine kinase Akt1 has been implicated in the control of cellular metabolism, survival and growth. Here, disruption of the ubiquitously expressed member of the Akt family of genes, Akt1, in the mouse demonstrates a requirement for Akt1 in ErbB2-induced mammary tumorigenesis. Akt1 deficiency delayed tumor growth and reduced lung metastases, correlating with a reduction in phosphorylation of the Akt1 target, tuberous sclerosis 2 (TSC2) at Ser-939. Akt1-deficient mammary epithelial tumor cells (MEC) were reduced in size and proliferative capacity, with reduced cyclin D1 and p27(KIP1) abundance. Akt1 deficiency abrogated the oncogene-induced changes in polarization of MEC in three-dimensional culture and reverted oncogene-induced relocalization of the phosphorylated ezrin-radixin-moesin proteins. Akt1 increased MEC migration across an endothelial cell barrier, enhancing the persistence of migratory directionality. An unbiased proteomic analysis demonstrated Akt1 mediated MEC migration through paracrine signaling via induction of expression and secretion of CXCL16 and MIP1gamma. Akt1 governs MEC polarity, migratory directionality and breast cancer onset induced by ErbB2 in vivo.

Akt deficiency impairs normal cell proliferation and suppresses oncogenesis in a p53-independent and mTORC1-dependent manner.

Akt contributes to tumorigenesis by inhibiting apoptosis. Here we establish that Akt is required for normal cell proliferation and susceptibility to oncogenesis independently of its antiapoptotic activity. Partial ablation of Akt activity by deleting Akt1 inhibits cell proliferation and oncogenesis. These effects are compounded by deleting both Akt1 and Akt2. In vivo, Akt1 null mice are resistant to MMTV-v-H-Ras-induced tumors and to skin carcinogenesis. Thus, partial ablation of Akt activity is sufficient to suppress tumorigenesis in vitro and in vivo. The effect of Akt deficiency on cell proliferation and oncogenesis is p53 independent but mTORC1 dependent. Surprisingly, upon mTORC1 hyperactivation, the reduction in Akt activity does not impair cell proliferation and susceptibility to oncogenic transformation; thus, Akt may mediate these processes exclusively via mTORC1.