PI3K Akt mTOR Signaling Research Articles

Circulating miR-1246 Targeting UBE2C, TNNI3, TRAIP, UCHL1 Genes and Key Pathways as a Potential Biomarker for Lung Adenocarcinoma: Integrated Biological Network Analysis.

Analysis of circulating miRNAs (cmiRNAs) before surgical operation (BSO) and after the surgical operation (ASO) has been informative for lung adenocarcinoma (LUAD) diagnosis, progression, and outcomes of treatment. Thus, we performed a biological network analysis to identify the potential target genes (PTGs) of the overexpressed cmiRNA signatures from LUAD samples that had undergone surgical therapy. Differential expression (DE) analysis of microarray datasets, including cmiRNAs (GSE137140) and cmRNAs (GSE69732), was conducted using the Limma package. cmiR-1246 was predicted as a significantly upregulated cmiRNA of LUAD samples BSO and ASO. Then, 9802 miR-1246 target genes (TGs) were predicted using 12 TG prediction platforms (MiRWalk, miRDB, and TargetScan). Briefly, 425 highly expressed overlapping miRNA-1246 TGs were observed between the prediction platform and the cmiRNA dataset. ClueGO predicted cell projection morphogenesis, chemosensory behavior, and glycosaminoglycan binding, and the PI3K–Akt signaling pathways were enriched metabolic interactions regulating miRNA-1245 overlapping TGs in LUAD. Using 425 overlapping miR-1246 TGs, a protein–protein interaction network was constructed. Then, 12 PTGs of three different Walktrap modules were identified; among them, ubiquitin-conjugating enzyme E2C (UBE2C), troponin T1(TNNT1), T-cell receptor alpha locus interacting protein (TRAIP), and ubiquitin c-terminal hydrolase L1(UCHL1) were positively correlated with miR-1246, and the high expression of these genes was associated with better overall survival of LUAD. We conclude that PTGs of cmiRNA-1246 and key pathways, namely, ubiquitin-mediated proteolysis, glycosaminoglycan binding, the DNA metabolic process, and the PI3K–Akt–mTOR signaling pathway, the neurotrophin and cardiomyopathy signaling pathway, and the MAPK signaling pathway provide new insights on a noninvasive prognostic biomarker for LUAD.

Integrated analysis of mRNA and miRNA expression profiles reveals muscle growth differences between fast- and slow-growing king ratsnakes (Elaphe carinata).

In some countries, snakes are important protein sources in human diets, and their economic value depends predominantly on their muscle production, including in the king ratsnake (Elaphe carinata). Muscle growth in the king ratsnake clearly differs among individuals. To date, few potential molecular mechanisms underlying these differences in muscle growth and development have been reported. Here, we integrated mRNA and miRNA expression profiles to screen for genes, pathways, and predicted miRNA-mRNA networks associated with muscle growth and development in fast-growing and slow-growing King ratsnakes. Six hundred eight differentially expressed genes (DEGs) were identified, 48 of which were associated with muscle growth. The 37 genes upregulated in fast-growing individuals (FGIs) may be related to the promotion of muscle growth, whereas the 11 upregulated genes in slow-growing individuals (SGIs) may be related to the inhibition of muscle growth. Seven DEGs were enriched in the PI3K-AKT-MTOR signaling pathway, which appears to promote muscle growth in FGIs. Eleven DEGs were enriched in the ubiquitin-proteasome pathway, which appears to inhibit muscle growth in SGIs. It may interpret why muscle growth differences. Furthermore, 698 miRNA were identified, including 125 novel miRNAs. 63 differentially expressed miRNA (DEMs) were screened, and 950 negative miRNA-mRNA interactions with the 63 DEMs and 608 DEGs were predicted. The miRNA-targeted genes were enriched in pathways related to muscle growth, protein synthesis, and protein degradation. Therefore, in addition to the identified DEGs, miRNAs may play important roles in the differential regulation of muscle growth in FGIs and SGIs of the king ratsnake.

Single-cell profiling of pediatric T-cell acute lymphoblastic leukemia: Impact of PTEN exon 7 mutation on PI3K/Akt and JAK-STAT signaling pathways.

The PI3K/Akt/mTOR (PI3K) signaling pathway has a crucial role in T-cell acute lymphoblastic leukemias (T-ALLs). Although loss-of-function of phosphatase and tensin homolog (PTEN) is a common event in pediatric T-ALLs, the exact role of this tumor suppressor in T-ALL development has yet to be defined. Here, we report an optimized cytometric method for accurate proteomic profiling of T-ALL leukemic blasts at single-cell level. We determined the expression of PI3K and JAK-STAT signaling components in both primary and immortalized T-ALL cells as well as in normal T cells. We observed that PTEN exon 7 mutated T-ALL cells retain a distinct PI3K activation; in particular, these cells show higher pAkt levels and a lower pS6 expression. Interestingly, we demonstrated for the first time that PTEN exon 7 mutated T-ALL are nonresponsive to IL7 in vitro as assessed by lack of pSTAT5 activation, although they do express IL7R. Phosphoflow analysis represents a fast, reliable, and accurate method to study the signaling profile of T-ALL. PTEN exon 7 mutated T-ALL cells are nonresponsive to IL7 in vitro suggesting that they may activate other mechanisms to support their viability and proliferation such as a higher constitutive PI3K/Akt signaling. Further investigations are necessary to elucidate the significance of this peculiar signaling behavior. Our observations should be taken into account in future studies aiming at molecular targeting of PI3K and/or JAK/STAT pathways for pharmacological intervention in T-ALL.

Cerebral organoid and mouse models reveal a RAB39b-PI3K-mTOR pathway-dependent dysregulation of cortical development leading to macrocephaly/autism phenotypes.

Dysregulation of early neurodevelopment is implicated in macrocephaly/autism disorders. However, the mechanism underlying this dysregulation, particularly in human cells, remains poorly understood. Mutations in the small GTPase gene RAB39b are associated with X-linked macrocephaly, autism spectrum disorder (ASD), and intellectual disability. The in vivo roles of RAB39b in the brain remain unknown. We generated Rab39b knockout (KO) mice and found that they exhibited cortical neurogenesis impairment, macrocephaly, and hallmark ASD behaviors, which resembled patient phenotypes. We also produced mutant human cerebral organoids that were substantially enlarged due to the overproliferation and impaired differentiation of neural progenitor cells (NPCs), which resemble neurodevelopmental deficits in KO mice. Mechanistic studies reveal that RAB39b interacts with PI3K components and its deletion promotes PI3K-AKT-mTOR signaling in NPCs of mouse cortex and cerebral organoids. The mTOR activity is robustly enhanced in mutant outer radial glia cells (oRGs), a subtype of NPCs barely detectable in rodents but abundant in human brains. Inhibition of AKT signaling rescued enlarged organoid sizes and NPC overproliferation caused by RAB39b mutations. Therefore, RAB39b mutation promotes PI3K-AKT-mTOR activity and alters cortical neurogenesis, leading to macrocephaly and autistic-like behaviors. Our studies provide new insights into neurodevelopmental dysregulation and common pathways associated with ASD across species.

Gene microarray expression profile analysis of differentially expressed genes of potential alternative pathways in non–small cell lung cancer: In search of biomarkers

In this study, we aimed to identify genes that could be involved in alternative pathways linked to the pathogenesis of non–small cell lung cancer (NSCLC), which could provide novel molecular biomarkers. Owing to NSCLC subtypes, three individual NSCLC data sets were downloaded and analyzed. Gene–subsetting subsequent meta–analysis was performed to screen differentially expressed genes (DEGs). The functional enrichment and pathway analyses were then executed using comprehensive bioinformatics analysis. Accordingly, the three hub genes of alternative pathways and their strong correlation with NSCLC pathway were initially identified as potential candidates (common DEGs) for the progression of NSCLC. The clinicopathological functions of common DEGs in NSCLC were validated by analyzing transcription factors targeting common DEGs, and their effectiveness was validated by analysis of protein/gene interactions and biological processes annotations. Finally, elucidated genes, such as protein phosphatase 3 catalytic subunit alpha (PPP3CA) of calcium signaling pathway, ubiquitin conjugating enzyme E2–Q1 (UBE2Q1) of β–catenin–EGFR–PI3K–Akt–mTOR signaling pathway and TATA–Box binding protein like–1 (TBPL1) of RNA polymerase–II transcription initiation and promoter clearance and PI3K–Akt signaling pathway were recognized as alternative networks mostly associated with the progression of NSCLC by co–occurred with oncogenes of the extracellular–signal–regulated kinase (ERK) pathway; and these genes can confer resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in NSCLC therapy through various mechanisms. Together, these results indicate that mutation–activated alternative pathway genes and related checkpoints can confer progression to NSCLC and drug resistance; therefore, which could provide as promising biomarkers for early detection and treatment of NSCLC.

Simulated Microgravity Influences VEGF, MAPK, and PAM Signaling in Prostate Cancer Cells.

Prostate cancer is one of the leading causes of cancer mortality in men worldwide. An unusual but unique environment for studying tumor cell processes is provided by microgravity, either in space or simulated by ground-based devices like a random positioning machine (RPM). In this study, prostate adenocarcinoma-derived PC-3 cells were cultivated on an RPM for time periods of 3 and 5 days. We investigated the genes associated with the cytoskeleton, focal adhesions, extracellular matrix, growth, survival, angiogenesis, and metastasis. The gene expression of signaling factors of the vascular endothelial growth factor (VEGF), mitogen-activated protein kinase (MAPK), and PI3K/AKT/mTOR (PAM) pathways was investigated using qPCR. We performed immunofluorescence to study the cytoskeleton, histological staining to examine the morphology, and a time-resolved immunofluorometric assay to analyze the cell culture supernatants. When PC-3 cells were exposed to simulated microgravity (s-µg), some cells remained growing as adherent cells (AD), while most cells detached from the cell culture flask bottom and formed multicellular spheroids (MCS). After 3-day RPM exposure, PC-3 cells revealed significant downregulation of the VEGF, SRC1, AKT, MTOR, and COL1A1 gene expression in MCS, whereas FLT1, RAF1, MEK1, ERK1, FAK1, RICTOR, ACTB, TUBB, and TLN1 mRNAs were not significantly changed. ERK2 and TLN1 were elevated in AD, and FLK1, LAMA3, COL4A5, FN1, VCL, CDH1, and NGAL mRNAs were significantly upregulated in AD and MCS after 3 days. After a 5-day culture in s-µg, the PC-3 cells showed significant downregulations of VEGF mRNA in AD and MCS, and FN1, CDH1, and LAMA3 in AD and SCR1 in MCS. In addition, we measured significant upregulations in FLT1, AKT, ERK1, ERK2, LCN2, COL1A1, TUBB, and VCL mRNAs in AD and MCS, and increases in FLK1, FN1, and COL4A5 in MCS as well as LAMB2, CDH1, RAF1, MEK1, SRC1, and MTOR mRNAs in AD. FAK1 and RICTOR were not altered by s-µg. In parallel, the secretion rate of VEGFA and NGAL proteins decreased. Cytoskeletal alterations (F-actin) were visible, as well as a deposition of collagen in the MCS. In conclusion, RPM-exposure of PC-3 cells induced changes in their morphology, cytoskeleton, and extracellular matrix protein synthesis, as well as in their focal adhesion complex and growth behavior. The significant upregulation of genes belonging to the PAM pathway indicated their involvement in the cellular changes occurring in microgravity.

Autophagy-related genes prognosis signature as potential predictive markers for immunotherapy in hepatocellular carcinoma.

Autophagy-related genes (ATGs) depress tumorigenesis. However, in tumor tissue, it promotes tumor progression. Here, we demonstrated that 63 ATGs were differentially expressed in normal tissues and tumor tissues of hepatocellular carcinoma (HCC), and seven prognostic-related genes were chosen to establish prognostic risk signatures. It is not just an independent prognostic factor for HCC, but also closely related to the degree of malignancy of HCC. Further, the hallmarks of PI3K–AKT–mTOR signaling was significantly enriched in the high-risk group. Moreover, AKT–pS473 and mTOR–pS2448 expression was down-regulated and correlated with patient prognosis in high-risk group. Finally, we demonstrate that the prognosis signature of ATGs is closely related to immune cell infiltration and PD-L1 expression. In conclusion, ATGs are a crucial factor in the malignant progression of HCC and will be a new prognostic marker for diagnosis and treatment. ATGs prognostic signatures are potentially useful for predicting PD-L1 therapeutic effects.

Genomic profiling of the residual disease of advanced high‐grade serous ovarian cancer after neoadjuvant chemotherapy

The goal of our study was to demonstrate the spectrum of genomic alterations present in the residual disease of patients with advanced high-grade serous ovarian cancer (HGSOC) after neoadjuvant chemotherapy (NAC), including matched pretreatment biopsies. During the study period between 2006 and 2017, we collected pre-NAC and post-NAC tumor tissue samples from patients with advanced HGSOC. We performed combined next-generation sequencing and immunohistochemistry to identify actionable targets and pathway activation in post-NAC residual tumors. We also examined whether post-NAC profiling of residual HGSOC identified targetable molecular lesions in the chemotherapy-resistant component of tumors. Among 102 post-NAC samples, 41 (40%) of patients had mutations in homologous recombination repair (HRR) genes (HRR deficiency). Patients with HRR mutations had higher tumor mutation burdens (p < 0.001) and higher alterations in the PI3K-AKT-mTOR pathway (p = 0.004) than patients without these HRR mutations. Nevertheless, we found no significant differences in progression-free survival (p = 0.662) and overall survival (OS; p = 0.828) between the two groups. Most patients (91%) had alterations in at least one of the targetable pathways, and those patients with cell cycle (p = 0.004) and PI3K-AKT-mTOR signaling (p = 0.005) pathway alterations had poorer OS (Bonferroni-corrected threshold = 0.0083, 0.05/6). We showed the genomic landscape of tumor cells remaining in advanced HGSOC after NAC. Once validated, these data can help inform biomarker-driven adjuvant studies in targeting residual tumors to improve the outcomes of patients with advanced HGSOC after NAC.

1698P - Reversion of resistance to mTOR inhibitors with the addition of exemestane in patients with malignant PEComa

1698P - Reversion of resistance to mTOR inhibitors with the addition of exemestane in patients with malignant PEComa

Genetic drivers of oncogenic pathways in molecular subgroups of peripheral T-cell lymphoma

AbstractPeripheral T-cell lymphoma (PTCL) is a group of complex clinicopathological entities, often associated with an aggressive clinical course. Angioimmunoblastic T-cell lymphoma (AITL) and PTCL-not otherwise specified (PTCL-NOS) are the 2 most frequent categories, accounting for >50% of PTCLs. Gene expression profiling (GEP) defined molecular signatures for AITL and delineated biological and prognostic subgroups within PTCL-NOS (PTCL-GATA3 and PTCL-TBX21). Genomic copy number (CN) analysis and targeted sequencing of these molecular subgroups revealed unique CN abnormalities (CNAs) and oncogenic pathways, indicating distinct oncogenic evolution. PTCL-GATA3 exhibited greater genomic complexity that was characterized by frequent loss or mutation of tumor suppressor genes targeting the CDKN2A/B-TP53 axis and PTEN-PI3K pathways. Co-occurring gains/amplifications of STAT3 and MYC occurred in PTCL-GATA3. Several CNAs, in particular loss of CDKN2A, exhibited prognostic significance in PTCL-NOS as a single entity and in the PTCL-GATA3 subgroup. The PTCL-TBX21 subgroup had fewer CNAs, primarily targeting cytotoxic effector genes, and was enriched in mutations of genes regulating DNA methylation. CNAs affecting metabolic processes regulating RNA/protein degradation and T-cell receptor signaling were common in both subgroups. AITL showed lower genomic complexity compared with other PTCL entities, with frequent co-occurring gains of chromosome 5 (chr5) and chr21 that were significantly associated with IDH2R172 mutation. CN losses were enriched in genes regulating PI3K–AKT–mTOR signaling in cases without IDH2 mutation. Overall, we demonstrated that novel GEP-defined PTCL subgroups likely evolve by distinct genetic pathways and provided biological rationale for therapies that may be investigated in future clinical trials.

Construction of Breast Cancer-Related LncSLGP Models for Systematically Deciphering Their Functions and Improving Prognosis

With the rapid development of systematic biological approaches, researchers pay more attention to uncover the complex interplaying mechanisms of different molecular models constructed by multiple coding or non-coding RNAs in cancers, such as synthetic lethal gene pairs (SLGPs) or competing endogenous RNAs (ceRNAs). To further investigate more complex framework, we attempted to construct a LncRNA Synthetic Lethal Gene Pair (LncSLGP) model with the combination of one lncRNA and two synthetic lethal genes in health and disease conditions. We established LncSLGPs in breast cancer and expected the LncSLGPs may play more crucial roles during the development of cancers than individual RNA molecules. Through the integration of analysis of RNA-Seq, copy number variant (CNV), somatic mutation, and DNA methylation data of 411 BC samples and 83 normal samples in the TCGA database, 119 LncSLGP models containing 97 lncRNAs, 71 genes (87 SLGPs) were generated. We found the majority of lncRNAs and genes in these LncSLGP models were disease related. Function enrichment analysis showed that genes in LncSLGPs were enriched in 'IL2 STAT5 SIGNALING', 'WNT BETA-CATENIN SIGNALING', 'MYC TARGETS', and 'PI3K AKT MTOR SIGNALING' pathways. Survival analysis results of three candidate LncSLGPs, including 'LINC01133/CDC6-KRAS', 'RP11-626G11.1/KRAS-MAP3K7' and 'RP11-483H20.4/TOP1-KRAS' (log-rank pvalues: 0.002, 0.009 and 0.007, respectively) showed that they were significantly related to poor prognosis of BC patients. We developed a systematic approach to construct and identify LncSLGP models, which could be served as predictive factors for improving prognosis and guiding the clinical applications for BC patients. Funding Statement: This work was supported in part by the National Natural Science Foundation of China [Grant Nos. 31871336, 61573122, 31701159]. Wu lien-teh youth science fund project of Harbin medical university [Grant Nos. WLD-QN1407]. The Health Department Science Foundation of Heilongjiang Province (Grant Nos. 2013128), the Education Department Science Foundation of Heilongjiang Province (Grant Nos. 12541415), the Postdoctoral project of Heilongjiang Province (Grant Nos. LBH-Z14130). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: Not needed.

CUL1 promotes breast cancer metastasis through regulating EZH2-induced the autocrine expression of the cytokines CXCL8 and IL11

CUL1 is an essential component of SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex. Our previous study has showed that CUL1 is positively associated with poor overall and disease-specific survival of breast cancer patients. Here, we further explored its roles in breast cancer metastasis. Our data showed that CUL1 significantly promoted breast cancer cell migration, invasion, tube formation in vitro, as well as angiogenesis and metastasis in vivo. In mechanism, the human gene expression profiling was used to determine global transcriptional changes in MDA-MB-231 cells, and we identified autocrine expression of the cytokines CXCL8 and IL11 as the target genes of CUL1 in breast cancer cell migration, invasion, metastasis, and angiogenesis. CUL1 regulated EZH2 expression to promote the production of cytokines, and finally significantly aggravating the breast cancer cell metastasis and angiogenesis through the PI3K–AKT–mTOR signaling pathway. Combined with the previous report about CUL1, we proposed that CUL1 may serve as a promising therapeutic target for breast cancer metastasis.

Foxo-dependent Par-4 Upregulation Prevents Long-term Survival of Residual Cells Following PI3K-Akt Inhibition.

Tumor recurrence is a leading cause of death and is thought to arise from a population of residual cells that survive treatment. These residual cancer cells can persist, locally or at distant sites, for years or decades. Therefore, understanding the pathways that regulate residual cancer cell survival may suggest opportunities for targeting these cells to prevent recurrence. Previously, it was observed that the proapoptotic protein (PAWR/Par-4) negatively regulates residual cell survival and recurrence in mice and humans. However, the mechanistic underpinnings on how Par-4 expression is regulated are unclear. Here, it is demonstrated that Par-4 is transcriptionally upregulated following treatment with multiple drugs targeting the PI3K-Akt-mTOR signaling pathway, and identify the Forkhead family of transcription factors as mediators of this upregulation. Mechanistically, Foxo3a directly binds to the Par-4 promoter and activates its transcription following inhibition of the PI3K-Akt pathway. This Foxo-dependent Par-4 upregulation limits the long-term survival of residual cells following treatment with therapeutics that target the PI3K-Akt pathway. Taken together, these results indicate that residual breast cancer tumor cell survival and recurrence requires circumventing Foxo-driven Par-4 upregulation and suggest that approaches to enforce Par-4 expression may prevent residual cell survival and recurrence. Mol Cancer Res; 16(4); 599-609. ©2018 AACR.

Abstract P5-03-03: Cytotoxic t-lymphocyte density increased within the tumor immune microenvironment of patients with breast cancer following treatment with Akt inhibitor MK-2206

Abstract Background: The PAM (PI3K/Akt/mTOR) signaling pathway has been implicated in the oncogenesis of multiple solid malignancies, including breast cancer (BC). Tumor infiltrating lymphocytes (TILs) found within the tumor immune microenvironment (TME) are both prognostic of overall survival as well as predictive of response to neoadjuvant chemotherapy in BC. Our aim is to characterize the TME in a series of patients with operable stage I-III BC treated with MK-2206, an allosteric Akt inhibitor as part of a presurgical, window of opportunity, trial. In our presurgical trial (clinicaltrials.gov #: NCT01319539), patients received 2 doses of MK-2206 with first dose at day -9 and second at day -2 from surgery. Methods: Quantitative multiplex immunofluorescence (qmIF) was performed using immune biomarkers (DAPI, CD3, CD8, CD4, FOXP3, CD68, Pancytokeratin) on full section (4uM) tissue slides from core biopsy specimens and postsurgical specimens of 10 patients - 5 patient treated with MK-2206, and 5 prospectively collected untreated controls. Images were taken using Vectra, a novel pathology workstation and analyzed using inForm software to perform cell classification and phenotyping. Student T- test was used to compare biomarker changes before and after MK-2206. Results: Preliminary analysis demonstrates that patients treated with MK-2206 exhibited a significantly increased median cytotoxic T-cells (CD3CD8+) density, as compared to the matched control cohort (87% vs.0.2%, p &amp;lt; 0.05). We did not identify a change in macrophage (CD68) or T helper/T reg (CD4/CD4FOXP3+) density following MK-2206 treatment in this small cohort. Proximity comparison, using nearest neighbor analysis was used to assess for potential impact of therapy on interactions between CD3CD8+ cells and BC cells. The median distance from CD3+CD8+ cells to nearest neighboring tumor cell was determined in both the pre and post tissues specimens for both groups. In patients treated with MK-2206, a 12.5% reduction in median distance was observed between CD3+CD8+ cells and tumor cells following treatment, suggestive that the increased effector T cells are not relegated to the periphery. This observation was not seen in the pre and post samples of the control cohort. Conclusions: In our study, we found that presurgical Akt inhibition lead to a significant increase in the cytotoxic T-cell population which was in similar proximity, if not closer, to tumor cells as compared to matched controls. Limitations of this exploratory study include a small patient cohort size and use of a single pathology evaluation technique. We are currently expanding our characterization of the TME with a more comprehensive myeloid panel as well as performing additional tissue analysis to validate our findings. At present, there are currently FDA approved therapies, as well as agents in clinical development that exert antineoplastic activity through the PAM pathway. Investigations that endeavor to understand the impact of these therapies on the TME may lead to both an increased understanding of the bioactivity of these agents and potentially identify aspects of the immune response which can be exploited by future immunotherapy therapeutics. Citation Format: Marks DK, Gartrell RD, Asmar ME, Hart TD, Lu Y, Esancy CL, Hibshoosh H, Connolly EP, Kalinsky KM, Saenger YM. Cytotoxic t-lymphocyte density increased within the tumor immune microenvironment of patients with breast cancer following treatment with Akt inhibitor MK-2206 [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-03-03.

Metabolic reprogramming ensures cancer cell survival despite oncogenic signaling blockade.

There is limited knowledge about the metabolic reprogramming induced by cancer therapies and how this contributes to therapeutic resistance. Here we show that although inhibition of PI3K-AKT-mTOR signaling markedly decreased glycolysis and restrained tumor growth, these signaling and metabolic restrictions triggered autophagy, which supplied the metabolites required for the maintenance of mitochondrial respiration and redox homeostasis. Specifically, we found that survival of cancer cells was critically dependent on phospholipase A2 (PLA2) to mobilize lysophospholipids and free fatty acids to sustain fatty acid oxidation and oxidative phosphorylation. Consistent with this, we observed significantly increased lipid droplets, with subsequent mobilization to mitochondria. These changes were abrogated in cells deficient for the essential autophagy gene ATG5 Accordingly, inhibition of PLA2 significantly decreased lipid droplets, decreased oxidative phosphorylation, and increased apoptosis. Together, these results describe how treatment-induced autophagy provides nutrients for cancer cell survival and identifies novel cotreatment strategies to override this survival advantage.

Enrichment of PI3K-AKT-mTOR Pathway Activation in Hepatic Metastases from Breast Cancer.

Purpose: Little is known about the molecular signatures associated with specific metastatic sites in breast cancer. Using comprehensive multi-omic molecular profiling, we assessed whether alterations or activation of the PI3K-AKT-mTOR pathway is associated with specific sites of breast cancer metastasis.Experimental Design: Next-generation sequencing-based whole-exome sequencing was coupled with reverse-phase protein microarray (RPPA) functional signaling network analysis to explore the PI3K-AKT-mTOR axis in 32 pretreated breast cancer metastases. RPPA-based signaling data were further validated in an independent cohort of 154 metastatic lesions from breast cancer and 101 unmatched primary breast tumors. The proportion of cases with PI3K-AKT-mTOR genomic alterations or signaling network activation were compared between hepatic and nonhepatic lesions.Results:PIK3CA mutation and activation of AKT (S473) and p70S6K (T389) were detected more frequently among liver metastases than nonhepatic lesions (P < 0.01, P = 0.056, and P = 0.053, respectively). However, PIK3CA mutations alone were insufficient in predicting protein activation (P = 0.32 and P = 0.19 for activated AKT and p70S6K, respectively). RPPA analysis of an independent cohort of 154 tumors confirmed the relationship between pathway activation and hepatic metastasis [AKT (S473), mTOR (S2448), and 4EBP1 (S65); P < 0.01, P = 0.02, and P = 0.01, respectively]. Similar results were also seen between liver metastases and primary breast tumors [AKT (S473) P < 0.01, mTOR (S2448) P < 0.01, 4EBP1 (S65) P = 0.01]. This signature was lost when primary tumors were compared with all metastatic sites combined.Conclusions: Breast cancer patients with liver metastasis may represent a molecularly homogenized cohort with increased incidence of PIK3CA mutations and activation of the PI3K-AKT-mTOR signaling network. Clin Cancer Res; 23(16); 4919-28. ©2017 AACR.

Characterization of the tumor immune microenvironment (TIM) with multiplex immunohistochemistry (mIHC) in patients with breast cancer following treatment with MK-2206.

e12109 Background: The PAM (PI3K/Akt/mTOR) signaling pathway has been implicated in the oncogenesis of multiple solid malignancies, including breast cancer (BC). Our aim is to characterize the TIM in a series of patients with operable stage I-III BC treated with MK-2206, an allosteric Akt inhibitor within a presurgical trial. In NCT01319539 , patients received 2 doses of MK-2206 with first dose at day -9 and second at day -2 from surgery. Methods: mIHC was performed using immune biomarkers (DAPI, CD3, CD8, CD4, FOXP3, CD68, Pancytokeratin) on full section (4uM) tissue slides from core biopsy specimens and postsurgical specimens of 10 patients - 5 treated with MK-2206, 5 paired controls. Images were taken using Vectra, a novel pathology workstation and analyzed using inForm software to perform cell classification and phenotyping. T- tests were used to compare biomarker changes before and after MK-2206. Results: Our preliminary analysis demonstrates that patients treated with MK-2206 exhibited an increase in median cytotoxic T-cells (CD3CD8+) density, as compared to the control population , which was statistically significant (87% vs.0.2%, p &lt; 0.05). We did not identify a change in macrophage (CD68) or T helper/T reg (CD4/CD4FOXP3+) density following MK-2206 treatment in this small cohort. We are currently expanding our myeloid panel as well as performing additional tissue analysis to validate our findings. As data is exploratory no correction was made for multiple comparisons. Conclusions: Tumor infiltrating lymphocytes (TILs) are both prognostic of overall survival as well as predictive of response to neoadjuvant chemotherapy in BC. At present, there are currently both FDA approved therapies, as well as agents in clinical development that exert antineoplastic activity through the PAM pathway. Investigations that endeavour to understand the impact of these therapies on the tumor immune microenvironment may lead to both an increased understanding of the bioactivity of these agents and potentially identify aspects of the immune response which can be exploited in future therapeutics.

PI3K-mTOR Pathway Inhibition Exhibits Efficacy Against High-grade Glioma in Clinically Relevant Mouse Models.

Purpose: The PI3K-AKT-mTOR signaling pathway is frequently activated in glioblastoma and offers several druggable targets. However, clinical efficacy of PI3K/mTOR inhibitors in glioblastoma has not yet been demonstrated. Insufficient drug delivery may limit the efficacy of PI3K/mTOR inhibitors against glioblastoma. The presence of the efflux transporters ABCB1/Abcb1 (P-glycoprotein, MDR1) and ABCG2/Abcg2 (BCRP) at the blood-brain barrier (BBB) restricts the brain penetration of many drugs.Experimental Design: We used in vitro drug transport assays and performed pharmacokinetic/pharmacodynamic studies in wild-type and ABC-transporter knockout mice. The efficacy of PI3K-mTOR inhibition was established using orthotopic allograft and genetically engineered spontaneous glioblastoma mouse models.Results: The mTOR inhibitors rapamycin and AZD8055 are substrates of ABCB1, whereas the dual PI3K/mTOR inhibitor NVP-BEZ235 and the PI3K inhibitor ZSTK474 are not. Moreover, ABCG2 transports NVP-BEZ235 and AZD8055, but not ZSTK474 or rapamycin. Concordantly, Abcb1a/b-/-;Abcg2-/- mice revealed increased brain penetration of rapamycin (13-fold), AZD8055 (7.7-fold), and NVP-BEZ235 (4.5-fold), but not ZSTK474 relative to WT mice. Importantly, ABC transporters limited rapamycin brain penetration to subtherapeutic levels, while the reduction in NVP-BEZ235 brain penetration did not prevent target inhibition. NVP-BEZ235 and ZSTK474 demonstrated antitumor efficacy with improved survival against U87 orthotopic gliomas, although the effect of ZSTK474 was more pronounced. Finally, ZSTK474 prolonged overall survival in Cre-LoxP conditional transgenic Pten;p16Ink4a/p19Arf;K-Rasv12;LucR mice, mainly by delaying tumor onset.Conclusions: PI3K/mTOR inhibitors with weak affinities for ABC transporters can achieve target inhibition in brain (tumors), but have modest single-agent efficacy and combinations with (BBB penetrable) inhibitors of other activated pathways may be required. Clin Cancer Res; 23(5); 1286-98. ©2016 AACR.

SOX4 Is Essential for Prostate Tumorigenesis Initiated by PTEN Ablation.

Understanding remains incomplete of the mechanisms underlying initiation and progression of prostate cancer, the most commonly diagnosed cancer in American men. The transcription factor SOX4 is overexpressed in many human cancers, including prostate cancer, suggesting it may participate in prostate tumorigenesis. In this study, we investigated this possibility by genetically deleting Sox4 in a mouse model of prostate cancer initiated by loss of the tumor suppressor Pten. We found that specific homozygous deletion of Sox4 in the adult prostate epithelium strongly inhibited tumor progression initiated by homozygous loss of Pten. Mechanistically, Sox4 ablation reduced activation of AKT and β-catenin, leading to an attenuated invasive phenotype. Furthermore, SOX4 expression was induced by Pten loss as a result of the activation of PI3K-AKT-mTOR signaling, suggesting a positive feedback loop between SOX4 and PI3K-AKT-mTOR activity. Collectively, our findings establish that SOX4 is a critical component of the PTEN/PI3K/AKT pathway in prostate cancer, with potential implications for combination-targeted therapies against both primary and advanced prostate cancers.

Fisetin, a phytochemical, potentiates sorafenib-induced apoptosis and abrogates tumor growth in athymic nude mice implanted with BRAF-mutated melanoma cells.

Melanoma is the most deadly form of cutaneous malignancy, and its incidence rates are rising worldwide. In melanoma, constitutive activation of the BRAF/MEK/ERK (MAPK) and PI3K/AKT/mTOR (PI3K) signaling pathways plays a pivotal role in cell proliferation, survival and tumorigenesis. A combination of compounds that lead to an optimal blockade of these critical signaling pathways may provide an effective strategy for prevention and treatment of melanoma. The phytochemical fisetin is known to possess anti-proliferative and pro-apoptotic activities. We found that fisetin treatment inhibited PI3K signaling pathway in melanoma cells. Therefore, we investigated the effect of fisetin and sorafenib (an RAF inhibitor) alone and in combination on cell proliferation, apoptosis and tumor growth. Combination treatment (fisetin + sorafenib) more effectively reduced the growth of BRAF-mutated human melanoma cells at lower doses when compared to individual agents. In addition, combination treatment resulted in enhanced (i) apoptosis, (ii) cleavage of caspase-3 and PARP, (iii) expression of Bax and Bak, (iv) inhibition of Bcl2 and Mcl-1, and (v) inhibition of expression of PI3K, phosphorylation of MEK1/2, ERK1/2, AKT and mTOR. In athymic nude mice subcutaneously implanted with melanoma cells (A375 and SK-MEL-28), we found that combination therapy resulted in greater reduction of tumor growth when compared to individual agents. Furthermore, combination therapy was more effective than monotherapy in: (i) inhibition of proliferation and angiogenesis, (ii) induction of apoptosis, and (iii) inhibition of the MAPK and PI3K pathways in xenograft tumors. These data suggest that simultaneous inhibition of both these signaling pathways using combination of fisetin and sorafenib may serve as a therapeutic option for the management of melanoma.