Nandini Dey

A Vascular Targeted Pan Phosphoinositide 3-Kinase Inhibitor Prodrug, SF1126, with Antitumor and Antiangiogenic Activity

PTEN and the pan phosphoinositide 3-kinase (PI3K) inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1 benzopyran-4-one (LY294002) exert significant control over tumor-induced angiogenesis and tumor growth in vivo. The LY294002 compound is not a viable drug candidate due to poor pharmacologic variables of insolubility and short half-life. Herein, we describe the development and antitumor activity of a novel RGDS-conjugated LY294002 prodrug, termed SF1126, which is designed to exhibit increased solubility and bind to specific integrins within the tumor compartment, resulting in enhanced delivery of the active compound to the tumor vasculature and tumor. SF1126 is water soluble, has favorable pharmacokinetics, and is well tolerated in murine systems. The capacity of SF1126 to inhibit U87MG and PC3 tumor growth was enhanced by the RGDS integrin (alpha v beta 3/alpha 5 beta 1) binding component, exhibiting increased activity compared with a false RADS-targeted prodrug, SF1326. Antitumor activity of SF1126 was associated with the pharmacokinetic accumulation of SF1126 in tumor tissue and the pharmacodynamic knockdown of phosphorylated AKT in vivo. Furthermore, SF1126 seems to exhibit both antitumor and antiangiogenic activity. The results support SF1126 as a viable pan PI3K inhibitor for phase I clinical trials in cancer and provide support for a new paradigm, the application of pan PI3K inhibitory prodrugs for the treatment of cancer.

The Protein Phosphatase Activity of PTEN Regulates Src Family Kinases and Controls Glioma Migration

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is mutated or lost in 60% to 70% of advanced gliomas and is associated with malignant phenotypic changes such as migration, which contribute to the morbidity and mortality of this disease. Most of the tumor suppressor function of PTEN has been attributed to its ability to dephosphorylate the second messenger, phosphatidylinositol 3,4,5-triphosphate, resulting in the biological control of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. Despite recent work suggesting that the protein phosphatase activity of PTEN controls glioma cell migration, the mechanisms by which this occurs are unclear. Herein, we show using glioma cell lines (U87MG and U373MG) stably transfected with wild-type PTEN or catalytically altered mutants of PTEN that PTEN controls integrin-directed migration in a lipid phosphatase, PI3K/AKT-independent manner. Confirming this observation, we show that the stable overexpression of COOH-terminal Src kinase, the physiologic negative regulator of SRC family kinases (SFK), or treatment with the SFK inhibitor PP1 abrogates glioma migration. The results provide direct evidence that the downstream effect of the protein phosphatase activity of PTEN is to suppress SFK and FYN, and to regulate RAC-GTPase activity after alpha(v) integrin stimulation. Furthermore, studying vitronectin-directed migration using (a) Fyn small interfering RNA and (b) astrocytes from Fyn heterozygous (+/-) mice, Pten heterozygous (+/-) mice, Pten and Fyn double heterozygous (+/-) mice, or Fyn knockout (-/-) mice confirmed a role of FYN in alpha(v) integrin-mediated haptotaxis in glial cells. Our combined results provide direct biochemical and genetic evidence that PTENs protein phosphatase activity controls FYN kinase function in glioma cells and regulates migration in a PI3K/AKT-independent manner.

Wnt signaling in triple negative breast cancer is associated with metastasis

BackgroundTriple Negative subset of (TN) Breast Cancers (BC), a close associate of the basal-like subtype (with limited discordance) is an aggressive form of the disease which convey unpredictable, and poor prognosis due to limited treatment options and lack of proven effective targeted therapies.MethodsWe conducted an expression study of 240 formalin-fixed, paraffin-embedded (FFPE) primary biopsies from two cohorts, including 130 TN tumors, to identify molecular mechanisms of TN disease.ResultsThe annotation of differentially expressed genes in TN tumors contained an overrepresentation of canonical Wnt signaling components in our cohort and others. These observations were supported by upregulation of experimentally induced oncogenic Wnt/β-catenin genes in TN tumors, recapitulated using targets induced by Wnt3A. A functional blockade of Wnt/β-catenin pathway by either a pharmacological Wnt-antagonist, WntC59, sulidac sulfide, or β-catenin (functional read out of Wnt/β-catenin pathway) SiRNA mediated genetic manipulation demonstrated that a functional perturbation of the pathway is causal to the metastasis- associated phenotypes including fibronectin-directed migration, F-actin organization, and invasion in TNBC cells. A classifier, trained on microarray data from β-catenin transfected mammary cells, identified a disproportionate number of TNBC breast tumors as compared to other breast cancer subtypes in a meta-analysis of 11 studies and 1,878 breast cancer patients, including the two cohorts published here. Patients identified by the Wnt/β-catenin classifier had a greater risk of lung and brain, but not bone metastases.ConclusionThese data implicate transcriptional Wnt signaling as a hallmark of TNBC disease associated with specific metastatic pathways.

CSK Controls Retinoic Acid Receptor (RAR) Signaling: a RAR-c-SRC Signaling Axis Is Required for Neuritogenic Differentiation

ABSTRACT Herein, we report the first evidence that c-SRC is required for retinoic acid (RA) receptor (RAR) signaling, an observation that suggests a new paradigm for this family of nuclear hormone receptors. We observed that CSK negatively regulates RAR functions required for neuritogenic differentiation. CSK overexpression inhibited RA-mediated neurite outgrowth, a result which correlated with the inhibition of the SFK c-SRC. Consistent with an extranuclear effect of CSK on RAR signaling and neurite outgrowth, CSK overexpression blocked the downstream activation of RAC1. The conversion of GDP-RAC1 to GTP-RAC1 parallels the activation of c-SRC as early as 15 min following all-trans-retinoic acid treatment in LA-N-5 cells. The cytoplasmic colocalization of c-SRC and RARγ was confirmed by immunofluorescence staining and confocal microscopy. A direct and ligand-dependent binding of RAR with SRC was observed by surface plasmon resonance, and coimmunoprecipitation studies confirmed the in vivo binding of RARγ to c-SRC. Deletion of a proline-rich domain within RARγ abrogated this interaction in vivo. CSK blocked the RAR-RA-dependent activation of SRC and neurite outgrowth in LA-N-5 cells. The results suggest that transcriptional signaling events mediated by RA-RAR are necessary but not sufficient to mediate complex differentiation in neuronal cells. We have elucidated a nongenomic extranuclear signal mediated by the RAR-SRC interaction that is negatively regulated by CSK and is required for RA-induced neuronal differentiation.

Differential activation of Wnt-β-catenin pathway in triple negative breast cancer increases MMP7 in a PTEN dependent manner.

Mutations of genes in tumor cells of Triple Negative subset of Breast Cancer (TNBC) deregulate pathways of signal transduction. The loss of tumor suppressor gene PTEN is the most common first event associated with basal-like subtype (Martins, De, Almendro, Gonen, and Park, 2012). Here we report for the first time that the functional upregulation of secreted-MMP7, a transcriptional target of Wnt-β-catenin signature pathway in TNBC is associated to the loss of PTEN. We identified differential expression of mRNAs in several key-components genes, and transcriptional target genes of the Wnt-β-catenin pathway (WP), including beta-catenin, FZD7, DVL1, MMP7, c-MYC, BIRC5, CD44, PPARD, c-MET, and NOTCH1 in FFPE tumors samples from TNBC patients of two independent cohorts. A similar differential upregulation of mRNA/protein for beta-catenin, the functional readout of WP, and for MMP7, a transcriptional target gene of beta-catenin was observed in TNBC cell line models. Genetic or pharmacological attenuation of beta-catenin by SiRNA or WP modulators (XAV939 and sulindac sulfide) and pharmacological mimicking of PTEN following LY294002 treatment downregulated MMP7 levels as well as enzymatic function of the secreted MMP7 in MMP7 positive PTEN-null TNBC cells. Patient data revealed that MMP7 mRNA was high in only a subpopulation of TNBC, and this subpopulation was characterized by a concurrent low expression of PTEN mRNA. In cell lines, a high expression of casein-zymograph-positive MMP7 was distinguished by an absence of functional PTEN. A similar inverse relationship between MMP7 and PTEN mRNA levels was observed in the PAM50 data set (a correlation coefficient of -0.54). The PAM50 subtype and outcome data revealed that the high MMP7 group had low pCR (25%) and High Rd (74%) in clinical stage T3 pathologic response in contrast to the high pCR (40%) and low residual disease (RD) (60%) of the low MMP7 group.

PI3K-AKT-mTOR inhibitors in breast cancers: From tumor cell signaling to clinical trials.

&NA; Breast cancer (BC) is the most common women cancer and second most common cause of cancer death in women. A woman living in the United States has 12.3% lifetime risk of being diagnosed with BC. From the genomics point of view, the most common three subtypes of BC encountered in clinics are HR +, HER2 +, and TNBC or basal‐like BC. Estrogen receptor (ER) status or HER2 amplification or chemotherapy is not sufficient to understand the underlying mechanisms of disease progression and resistance (de novo or acquire). Although hormonal therapy and HER2‐directed therapies have produced a considerable positive outcome in HR + and HER2 + BC respectively, there are no established targeted agents for TNBC and basal‐like BC. While PARP inhibitors have shown promising activity in BRCA‐related cancers, its value in the treatment of TNBC remains to be demonstrated. The PI3K‐AKT‐mTOR signaling pathway plays a crucial role in the initiation and progress in tumorigenesis including breast tumorigenesis and regulates critical cellular functions including survival, proliferation, and metabolism. This article aims to understand the role of PI3K‐mTORC1/C2 alterations in determining the clinical outcome in the specific breast cancer subtypes. The understanding of the tumor cell signaling will help us in the decision‐making the process for obtaining the treatment modalities towards further advancement of the precision medicine. In this review, we will restrict our discussion to a basic understanding of the biology of subtype‐specific BC and several targeted agents under development for the treatment of BC.

Promise of rapalogues versus mTOR kinase inhibitors in subset specific breast cancer: Old targets new hope

The PI3K-AKT-mTOR network has been the major focus of attention for cancer researchers (both in the clinic and the laboratory) in the last decade. An incomplete knowledge of the molecular biology of this complex network has seen an expansion of first generation allosteric mTOR inhibitors, rapalogues, but also biomarker studies designed to identify the best responders of these agents. Currently, research in this pathway has focused on the dual nature of mTOR that is integrated by mTOR-RAPTOR complex (mTORC1) and mTOR-RICTOR complex (mTORC2). These two complexes are regulated by different upstream proteins and also regulated by multiple different compensatory feedback loops. The related advantage of feedback regulation of signaling systems is that it allows diversification in signal response. This deeper understanding has facilitated the development of a novel second generation of inhibitors that are able to affect both mTORC1 and mTORC2, and their downstream effectors, through inhibition of their catalytic activity (ATP competitive inhibitors, attacking the kinase domain of this protein) than binding to the FKBP12 regulatory proteins as for rapalogues. This article reviews the newest insight in the signaling network of the mTOR pathway, preclinical/clinical status of mTOR inhibitors (including second generation of kinase inhibitors) and then focuses on the development of a new wave of research related to combination therapies in subset specific breast tumors.

Wnt-beta-catenin pathway signals metastasis-associated tumor cell phenotypes in triple negative breast cancers.

Tumor cells acquire metastasis-associated (MA) phenotypes following genetic alterations in them which cause deregulation of different signaling pathways. Earlier, we reported that an upregulation of the Wnt-beta-catenin pathway (WP) is one of the genetic salient features of triple-negative breast cancer (TNBC), and WP signaling is associated with metastasis in TNBC. Using cBioPortal, here we found that collective % of alteration(s) in WP genes, CTNNB1, APC and DVL1 among breast-invasive-carcinomas was 21% as compared to 56% in PAM50 Basal. To understand the functional relevance of WP in the biology of heterogeneous/metastasizing TNBC cells, we undertook this comprehensive study using 15 cell lines in which we examined the role of WP in the context of integrin-dependent MA-phenotypes. Directional movement of tumor cells was observed by confocal immunofluorescence microscopy and quantitative confocal-video-microscopy while matrigel-invasion was studied by MMP7-specific casein-zymography. WntC59, XAV939, sulindac sulfide and beta-catenin siRNA (1) inhibited fibronectin-directed migration, (2) decreased podia-parameters and motility-descriptors, (3) altered filamentous-actin, (4) decreased matrigel-invasion and (5) inhibited cell proliferation as well as 3D clonogenic growth. Sulindac sulfide and beta-catenin siRNA decreased beta-catenin/active-beta-catenin and MMP7. LWnt3ACM-stimulated proliferation, clonogenicity, fibronection-directed migration and matrigel-invasion were perturbed by WP-modulators, sulindac sulfide and GDC-0941. We studied a direct involvement of WP in metastasis by stimulating brain-metastasis-specific MDA-MB231BR cells to demonstrate that LWnt3ACM-stimulated proliferation, clonogenicity and migration were blocked following sulindac sulfide, GDC-0941 and beta-catenin knockdown. We present the first evidence showing a direct functional relationship between WP activation and integrin-dependent MA-phenotypes. By proving the functional relationship between WP activation and MA-phenotypes, our data mechanistically explains (1) why different components of WP are upregulated in TNBC, (2) how WP activation is associated with metastasis and (3) how integrin-dependent MA-phenotypes can be regulated by mitigating the WP.

Mutation matters in precision medicine: A future to believe in

As a genetic disease [1] cancer dysregulates key oncogenic pathways that influence cell growth, proliferation, survival, angiogenesis, and metastasis. Among the major determinants that enable cancer cells to acquire malignant traits are genomic diversity and instability. In the post human genome project era, cancer-specific genomic maps are redesigning tumor taxonomy. The treatment modalities, as well as the overall management of cancer as a disease in todays clinic, have started depending heavily on the molecular pathology of the individual tumor(s) in addition to the fundamental classification of cancers by histopathology. The enrichment tumor taxonomy by genomic morphology has also opened up the possibilities for genomics-driven drug development. The success of a cancer drug today is fundamentally based on the success in identifying target genes that control tumorigenic pathways. One primary goal of precision cancer medicine is to make clinical decisions based on genomic/proteomic data, which can identify a target or targets for therapy, and subsequent inevitable development of therapeutic resistance to the drug. The ability to exploit tumor genetic information for its full clinical potential has only recently become evident. Over the last decade, the convergence of discovery, technology, and therapeutic development has created an unparalleled opportunity to test the hypothesis that systematic knowledge of genomic and proteomic information from individual tumor(s) may significantly improve clinical outcomes for many patients with unmanageable tumor burden. This review presents the signaling logic behind the ground rules for the rational approach to the genomics-driven precision medicine.

Enriched transcription factor signatures in triple negative breast cancer indicates possible targeted therapies with existing drugs

Purpose Triple negative (TN) breast cancers which lack expression of the estrogen (ER), progesterone (PR), and human epidermal growth factor 2 (HER2) receptors convey a poor prognosis due in part to a lack of targeted therapies. Methods To identify viable targets for the treatment of TN disease, we have conducted a gene set enrichment analysis (GSEA) on seven different breast cancer whole genome gene expression cohorts comparing TN vs. ER + HER2 − to identify consistently enriched genes that share a common promoter motif. The seven cohorts were profiled on three different genome expression platforms (Affymetrix, Illumina and RNAseq) consisting in total of 2088 samples with IHC metadata. Results GSEA identified enriched gene expression patterns in TN samples that share common promoter motifs associated with SOX9, E2F1, HIF1A, HMGA1, MYC BACH2, CEBPB, and GCNF/NR6A1. Unexpectedly, NR6A1 an orphan nuclear receptor normally expressed in germ cells of gonads is highly expressed in TN and ER + HER2 − samples making it an ideal drug target. Conclusion With the increasing number of large sample size breast cancer cohorts, an exploratory analysis of genes that are consistently enriched in TN sharing common promoter motifs allows for the identification of possible therapeutic targets with extensive validation in patient derived data sets.