Mumtaz V. Rojiani

Identification of molecular markers specific for pancreatic neuroendocrine tumors by genetic profiling of core biopsies

AbstractBackground: There is a paucity of known molecular markers that distinguish pancreatic neuroendocrine tumors from other pancreatic tumor types. We hypothesized that novel markers for pancreatic neuroendocrine tumors could be identified with molecular fingerprinting of pooled RNA samples from core biopsies. Methods: Total RNA was harvested from nine core biopsies of normal pancreas, pancreatitis, pancreatic adenocarcinoma, pancreatic adenocarcinoma metastases, and pancreatic neuroendocrine tumors. RNA from each group of samples was pooled and hybridized to an oligonucleotide-based microarray. Four genes (ANG2, NPDC1, ELOVL4, and CALCR) were selected for further investigation by reverse transcriptase polymerase chain reaction from the top 20 highest expressed genes, on the basis of potential as novel markers. Results: Neuroendocrine tumors were most unique from normal pancreas. Pancreatitis, pancreatic adenocarcinoma, and metastases are more closely related to each other and to normal pancreas. ANG2 was overexpressed in 89% of neuroendocrine tumors, compared with 22% of normal pancreas, making it the best potential molecular marker or therapeutic target of the four genes selected for analysis. Conclusion: We have identified a specific set of molecular markers for pancreatic neuroendocrine tumors distinct from pancreatitis and pancreatic adenocarcinoma. These novel markers may prove useful as molecular markers or therapeutic targets unique to pancreatic neuroendocrine tumors.

Gender-specific differential expression of exosomal miRNA in synovial fluid of patients with osteoarthritis

The pathogenesis of osteoarthritis (OA) is poorly understood, and therapeutic approaches are limited to preventing progression of the disease. Recent studies have shown that exosomes play a vital role in cell-to-cell communication, and pathogenesis of many age-related diseases. Molecular profiling of synovial fluid derived exosomal miRNAs may increase our understanding of OA progression and may lead to the discovery of novel biomarkers and therapeutic targets. In this article we report the first characterization of exosomes miRNAs from human synovial fluid. The synovial fluid exosomes share similar characteristics (size, surface marker, miRNA content) with previously described exosomes in other body fluids. MiRNA microarray analysis showed OA specific exosomal miRNA of male and female OA. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified gender-specific target genes/signaling pathways. These pathway analyses showed that female OA specific miRNAs are estrogen responsive and target TLR (toll-like receptor) signaling pathways. Furthermore, articular chondrocytes treated with OA derived extracellular vesicles had decreased expression of anabolic genes and elevated expression of catabolic and inflammatory genes. In conclusion, synovial fluid exosomal miRNA content is altered in patients with OA and these changes are gender specific.

Extracellular Matrix Microenvironment in Glioma Progression

Malignant gliomas are primary brain tumors, which are highly invasive but not known to metastasize outside the central nervous system (CNS). The median survival time of patients with glioma is only 6 months to 2 years depending on various patient, tumor and treatment parameters (Louis et al. 2007). The highly aggressive character of gliomas with glioblastoma multiforme (GBM) being the most aggressive subtype are characterized by their diffuse infiltration into the normal brain parenchyma and interaction with the extracellular matrix (ECM) components in the brain. Standard brain tumor therapies, which include surgery followed by chemotherapy and radiation are not effective in eradicating single glioma cells that migrated into the normal brain establishing new tumor foci. Glioma cells are locally invasive and when migrating through the ECM within several millimeters or centimeters from the main lesion they initiate recurrent tumors often distant to the primary lesion (Bolteus et al. 2001). The infiltrative path of glioma into the normal brain parenchyma involves the basement membrane of blood vessels and myelinated nerve fibers of white matter tracts (Rao 2003, Lefranc et al. 2005). The pattern of glioma cell invasion is related to the unique composition of the cerebral ECM microenvironment, which is remodeled during invasion by activated matrix metalloproteinases (MMPs) (reviewed by Rojiani et al. 2011). In addition, new ECM molecules are secreted and receptor adhesion molecules are expressed by glioma promoting the glioma cell-ECM interaction and signaling. Some of the secreted ECM molecules such as tenascin-C are known to be associated with cell motility and angiogenesis which are both essential for tumor development. Another important microenvironment component affecting glioma development was found to be mechanical force determined by ECM rigidity. More rigid ECM promotes glioma migration and proliferation and lower rigidity of ECM (similar to that of normal brain) would have an opposite effect (Ulrich et al. 2009). The recent sequencing data presented by the Cancer Genome Atlas Research Network (2008) revealed genomic abnormalities in GBM that relate to several signaling pathways such as Epidermal Growth Factor Receptor (EGFR) /Ras /PI3K known to be associated with ECM-related signaling (Ulrich et al. 2009). In addition, the recent integrated genomic analysis identified clinically relevant subtypes of GBM with its characteristic abnormalities

TIMP-1 overexpression in lung carcinoma enhances tumor kinetics and angiogenesis in brain metastasis

Abstract Tissue inhibitors of matrix metalloproteinase (TIMP) orchestrate many biologic activities, including inhibition of matrix metalloproteinase activity, activation of pro–matrix metalloproteinases, and regulation of cell proliferation, angiogenesis, and apoptosis induction. Tissue inhibitors of matrix metalloproteinase can play a protective role during tumor invasion and metastasis, but elevated TIMP messenger RNA levels have also been associated with aggressive cancers and poor clinical outcome. We examined the potential roles of TIMP-1 in H2009 lung adenocarcinoma cells and in cells transfected with a human TIMP-1–overexpressing vector (HB-6 and HB-1). Tumors resulting from the implantation of parental cell lines and transfected HB-1 cells into the brains of nude mice had a typical carcinoma profile, but human TIMP-1–overexpressing tumors showed enhanced tumor kinetics and focally more infiltrative features; vessel density assessed with anti-CD31 immunohistochemistry was also greater within HB-1 tumor implants. Similar effects on HB-6 and HB-1 cells versus parental cell lines and empty vector clones were observed in endothelial cell assays. Anchorage-independent growth and invasion through Matrigel were also increased in TIMP-1–overexpressing cells. Together, these results indicate tumor-promoting functions of TIMP-1 through alterations in angiogenesis, increased tumorigenicity, and invasive behavior. Although matrix metalloproteinase inhibition has been the traditionally identified function of TIMP-1, matrix metalloproteinase–independent interactions may contribute to the growth of metastatic carcinomas in the brain.

TIMP-1 Inhibits Apoptosis in Lung Adenocarcinoma Cells via Interaction with Bcl-2

Tissue inhibitors of metalloproteinases (TIMPs) are multifaceted molecules that exhibit properties beyond their classical proteinase inhibitory function. Although TIMP-1 is a known inhibitor of apoptosis in mammalian cells, the mechanisms by which it exerts its effects are not well-established. Our earlier studies using H2009 lung adenocarcinoma cells, implanted in the CNS, showed that TIMP-1 overexpressing H2009 cells (HB-1), resulted in more aggressive tumor kinetics and increased vasculature. The present study was undertaken to elucidate the role of TIMP-1 in the context of apoptosis, using the same lung cancer cell lines. Overexpressing TIMP-1 in a lung adenocarcinoma cell line H2009 resulted in an approximately 3-fold increased expression of Bcl-2, with a marked reduction in apoptosis upon staurosporine treatment. This was an MMP-independent function as a clone expressing TIMP-1 mutant T2G, lacking MMP inhibition activity, inhibited apoptosis as strongly as TIMP1 overexpressing clones, as determined by inhibition of PARP cleavage. Immunoprecipitation of Bcl-2 from cell lysates also co-immunoprecipitated TIMP-1, indicative of an interaction between these two proteins. This interaction was specific for TIMP-1 as TIMP-2 was not present in the Bcl-2 pull-down. Additionally, we show a co-dependency of TIMP-1 and Bcl-2 RNA and protein levels, such that abrogating Bcl-2 causes a downregulation of TIMP-1 but not TIMP-2. Finally, we demonstrate that TIMP-1 dependent inhibition of apoptosis occurs through p90RSK, with phosphorylation of the pro-apoptotic protein BAD at serine 112, ultimately reducing Bax levels and increasing mitochondrial permeability. Together, these studies define TIMP-1 as an important cancer biomarker and demonstrate the potential TIMP-1 as a crucial therapeutic target.

Cell proliferation index determination by immunohistochemical detection of hCDC47 protein.

A member of the human minichromosome maintenance complex protein family, hCDC47 (alias MCM7) has been identified as a component of the regulatory mechanism in cell proliferation. The expression of this protein, as determined by immunohistochemistry, was investigated to determine its application as a proliferation marker. A mouse monoclonal antibody (Clone 47DC141, NeoMarkers, Fremont CA) raised against recombinant hCDC47 protein was tested against a wide range of tissues. Immunoreaction patterns were determined in normal and neoplastic, human tissues, including skin, tonsils and lymph nodes, primary, and metastatic brain tumors. The protein was detected in the nuclei of both, normal and neoplastic proliferating cells. Similarly, we also examined the distribution of hCDC47 in normal rat and mouse tissues, and rodent and human tumors grown in nude mice.The pattern of immunolocalization was identical to that seen in human tissue, with positive nuclear immunoreaction readily identified in proliferating cells. Western immunoblot was carried out on extracts from PANC cells (human pancreatic adenocarcinoma cell line) to confirm the specificity of the protein. To correlate Ki67 protein immunoexpression with hCDC47 antibody reactivity, semiquantitative comparisons were carried out on parallel tissue sections. There was excellent correlation in the distribution pattern of the 2 markers, although hCDC47 was more sensitive.Thus this marker may have important clinical and research applications because of its activity in formalin-fixed, paraffin-embedded, proliferating, normal, and neoplastic tissue. More significantly, its application to animal tissue makes it a reliable and easy to use, proliferation marker for experimental studies.

TIMP-1 downregulation modulates miR-125a-5p expression and triggers the apoptotic pathway

Matrix metalloproteinases and their natural inhibitors (TIMPs) are important elements in a wide range of oncology settings. Elevated levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) have often been associated with increased tumorigenesis. This has been demonstrated in a number of clinical and experimental models which include breast, gastric, colorectal and non-small cell lung carcinoma (NSCLC). Our earlier studies have identified increased angiogenic activity and aggressive tumor kinetics in TIMP-1 overexpressing H2009 lung adenocarcinoma cells. TIMP-1 overexpression has also been implicated in antiapoptotic responses, inducing a significant upregulation of Bcl-2. These TIMP-1 functions have been shown to be MMP-independent and provide insight into its pleiotropic activities. The current study examines microRNA (miRNA) interactions with this molecule. We have sought to define the relationship between TIMP-1 and miRNA by knocking down TIMP-1 in high TIMP-1 expressing lung adenocarcinoma cell lines. TIMP-1 knockdown resulted in increased expression of miR-125a-5p with a concomitant increase in apoptosis and attenuation of the tumorigenic features of these cells. We have identified TIMP-1 as a bona fide target of miR-125a-5p, and their interaction resulted in an increase in p53 expression. We further corroborated our in vitro data with patient samples, which exhibited an inverse correlation between TIMP-1 and miR-125a-5p expression. Our study lends support to the notion that elevated TIMP-1 levels, which are frequently associated with poor prognosis, cause aberrant modulation of miRNAs.

Suprasellar Ganglioglioma: Expanding the Differential Diagnosis

This case study describes a young man with symptoms suggestive of the presence of a space-occupying lesion within the cranial cavity. Imaging studies confirmed a lesion in the suprasellar region and surgical intervention to remove the tumor yielded an unexpected diagnosis. Neuroimaging characteristics and histopathology including immunohistochemistry are described. Gangliogliomas are uncommon CNS neoplasms and are most commonly found in the temporal and frontal lobes of young, male adults. They are rarely seen in the suprasellar region and only a handful of cases have been reported to date. The differential diagnoses associated with these suprasellar region lesions can be dependent on the age of the patient and neuroimaging characteristics. The present report highlights the importance of histopathological examination and the need to consider a wide range of diagnostic entities in the differential diagnosis of lesions in this topographic distribution, including rarely encountered tumors such as gangliogliomas.

Morphologic and Elemental Analysis of Primary Melanosis of the Dentate Nucleus: Review and Correlation With Neuromelanin

Primary melanosis of the dentate nucleus is a rarely described entity with neither known cause nor definitive clinicopathologic correlation. We revisit this previously reported phenomenon by presenting one such case with a review of the pathology as well as additional investigations including elemental analysis by energy-dispersive X-ray, immunohistochemistry and electron microscopy. The lesion presented macroscopically as a sharply defined, black pigmentation that was restricted to the dentate nucleus of the cerebellum. Other deep nuclei were uninvolved. Similarly, other areas of the cerebellum, brainstem, and supratentorial regions were macroscopically free of pigment. Microscopically, however, the pigment was noted to be present, albeit in microscopic deposits, within layers of the cerebellar cortex. Additionally, immunohistochemistry and electron microscopy defined an intracellular component within astrocytes. X-ray analysis of the pigment showed it to consist almost entirely of sulfur, an element known to be prominent in neuromelanin. This report also describes an association of the pigment with astrocytes by ultrastructural examination. We discuss the results of our findings in the context of etiopathogenetic considerations, seeking to gain a better understanding of this abnormal pigmentation and its relationship to neuromelanin.

Abstract 3368: TIMP-1 and angiogenesis: A role for CDH5 and eNOS

Tissue inhibitors of metalloproteinases (TIMP) have emerged as diverse molecules with novel roles in apoptosis, angiogenesis and metastasis. TIMP-1, a well-documented inhibitor of apoptosis, has been shown to inhibit or promote angiogenesis. In recent years, a strong association has also been demonstrated between high levels of TIMP-1 and poor prognosis in many cancers. It has been recognized as a multifunctional protein affecting cell growth and angiogenesis. Conflicting studies have shown it to function either as a negative or a positive regulator of angiogenesis. In earlier studies, we have documented that lung adenocarcinoma cell line H2009, when transfected to overexpress TIMP-1 and injected into nude mice resulted in larger, more aggressive tumors with increased microvessel density, which was supported by in vitro angiogenesis assays whereby enhanced capillary network formation was seen. We have also shown that TIMP-1 expression levels can be correlated with KRAS independency in non-small-cell lung carcinoma (NSCLC) cell lines harboring KRAS mutations. The present study was undertaken to address the role of TIMP-1 in angiogenesis in the context of KRAS in NSCLC cell lines. KRAS dependent cell line H2009 and its TIMP-1 overexpressing clone HB1 and a KRAS independent cell line A549 and its TIMP-1 knockdown (KD) clone SH3 were examined by angiogenesis specific PCR array. Comparison of the angiogenesis associated profiles of these cell lines identified a marked increase in vascular endothelial cadherin CDH5 in TIMP-1 over-expressing clone HB1. KRAS independent A549 cells expressed high level of CDH5, however its TIMP-1 KD clone exhibited a marked decrease in CDH5 level. A similar profile was identified for endothelial nitric oxide synthase (eNOS/NOS3). CDH5 is an indispensable element of normal vascular development and maintenance, and any perturbations of normal levels will impact vessel sprouting and growth. eNOS is primarily active through production of nitric oxide, maintaining vascular tone as well as expressing anti-proliferative and antithrombotic properties. We confirmed our earlier observation that serum free conditioned media (SFCM) from TIMP-1 overexpressing HBI cells caused increased and more complex network formation. SFCM from A549 TIMP-1 KD SH3 clone showed a marked inhibition of endothelial network formation compared to SFCM from A549 cells. To confirm that TIMP-1 was responsible for the changes observed we purified secretory TIMP-1 by combined fast protein liquid chromatography and gel filtration from another high TIMP-1 producing NSCLC cell line (H460) and also observed more capillary network formation in human umbilical vein endothelial cells. Our studies suggest that TIMP-1 modulates angiogenesis by impacting CDH5 and eNOS positively. These results define an important function of TIMP-1 in angiogenesis and provide additional therapeutic targets for managing NSCLC. Citation Format: Sampa Ghoshal-Gupta, Ian A. Coe, Ammar Kutiyanawalla, Byung R. Lee, Amyn M. Rojiani, Mumtaz V. Rojiani. TIMP-1 and angiogenesis: A role for CDH5 and eNOS. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3368.