No small matter: microRNAs -- key regulators of cancer stem cells

This is the published version, also available here: http://www.ijcem.com/1002003A.html.Emerging evidence demonstrates that both tumor suppressor and oncogenic miRNAs play an essential role in stem cell self-renewal and differentiation by negatively regulating the expression of certain key genes in stem cells. It seems logical that they may also be critical players in cancer stem cells. Though small in size, miRNAs play a key role in the epigenetic regulation of cancer stem cells. Specifically, the imbalance of oncogenic vs. tumor suppressor miRNAs may lead to dysregulation of cancer stem cells, thus causing excessive self-renewal and survival of cancer stem cells, and resistance to chemo/radiotherapy. We postulate that restoring the balance of miRNAs will correct this dysregulation via the direct and simultaneous modulation of downstream stem cell pathways involved in cancer stem cell self-renewal and/or differentiation. The resultant restoration of key regulatory pathways could improve therapeutic response. Restoring tumor suppressor miRNAs and/or inhibiting oncogenic miRNAs may provide a novel molecular therapy for human cancers, potentially via modulating cancer stem cells. (IJCEM1002003)

Structural basis for L27 domain‐mediated assembly of signaling and cell polarity complexes

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102252/1/emboj7600294.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102252/2/emboj7600294-sup-0001.pd

HAb18G/CD147 Promotes pSTAT3-Mediated Pancreatic Cancer Development via CD44s

Purpose STAT3 plays a critical role in initiation and progression of pancreatic cancer. However, therapeutically targeting STAT3 is failure in clinic. We previously identified HAb18G/CD147 as an effective target for cancer treatment. In this study, we aimed to investigate potential role of HAb18G/CD147 in STAT3-involved pancreatic tumorigenesis in vitro and in vivo. Experimental Design The expression of HAb18G/CD147, pSTAT3 and CD44s were determined in tissue microarrays. The tumorigenic function and molecular signaling mechanism of HAb18G/CD147 was assessed by in vitro cellular and clonogenic growth, reporter assay, immunoblot, immunofluorescence staining, immunoprecipitation, and in vivo tumor formationusing loss or gain-of-function strategies. Results Highly expressed HAb18G/CD147 promoted cellular and clonogenic growth in vitro and tumorigenicity in vivo. CyPA, a ligand of CD147, stimulated STAT3 phosphorylation and its downstream genes cyclin D1/survivin through HAb18G/CD147 dependent mechanisms. HAb18G/CD147 was associated and co-localized with cancer stem cell marker CD44s in lipid rafts. The inhibitors of STAT3 and survivin, as well as CD44s neutralizing antibodies suppressed the HAb18G/CD147-induced cell growth. High HAb18G/CD147 expression in pancreatic cancer was significantly correlated with the poor tumor differentiation, and the high co-expression of HAb18G/CD147-CD44s-STAT3 associated with poor survival of patients with pancreatic cancer. Conclusions We identified HAb18G/CD147 as a novel upstream activator of STAT3 via interacts with CD44s and plays a critical role in the development of pancreatic cancer. The data suggest HAb18G/CD147 could be a promising therapeutic target for highly aggressive pancreatic cancer and a surrogate marker in the STAT3-targeted molecular therapies

Improved prediction of radiation pneumonitis by combining biological and radiobiological parameters using a data-driven Bayesian network analysis

Grade 2 and higher radiation pneumonitis (RP2) is a potentially fatal toxicity that limits efficacy of radiation therapy (RT). We wished to identify a combined biomarker signature of circulating miRNAs and cytokines which, along with radiobiological and clinical parameters, may better predict a targetable RP2 pathway. In a prospective clinical trial of response-adapted RT for patients (n = 39) with locally advanced non-small cell lung cancer, we analyzed patients\u27 plasma, collected pre- and during RT, for microRNAs (miRNAs) and cytokines using array and multiplex enzyme linked immunosorbent assay (ELISA), respectively. Interactions between candidate biomarkers, radiobiological, and clinical parameters were analyzed using data-driven Bayesian network (DD-BN) analysis. We identified alterations in specific miRNAs (miR-532, -99b and -495, let-7c, -451 and -139-3p) correlating with lung toxicity. High levels of soluble tumor necrosis factor alpha receptor 1 (sTNFR1) were detected in a majority of lung cancer patients. However, among RP patients, within 2 weeks of RT initiation, we noted a trend of temporary decline in sTNFR1 (a physiological scavenger of TNFα) and ADAM17 (a shedding protease that cleaves both membrane-bound TNFα and TNFR1) levels. Cytokine signature identified activation of inflammatory pathway. Using DD-BN we combined miRNA and cytokine data along with generalized equivalent uniform dose (gEUD) to identify pathways with better accuracy of predicting RP2 as compared to either miRNA or cytokines alone. This signature suggests that activation of the TNFα-NFκB inflammatory pathway plays a key role in RP which could be specifically ameliorated by etanercept rather than current therapy of non-specific leukotoxic corticosteroids

Biochemical and structural studies of the LIN-2 and 7 (L27) dimerization domain: Linking proteins important to cell polarity.

Asymmetric distribution of proteins and other molecules within cells allows for efficient completion of myriad complex tasks necessary for life. In mammalian epithelia, apical and basolateral proteins and lipids are prevented from mixing by tight junctions, a network of interacting transmembrane and peripheral membrane proteins comprising a physical barrier preventing passage of inorganic solutes and soluble growth factors between cells. Protein-protein interaction domains organize tight junctions and many other multimolecular complexes important for cell signaling and cellular polarity. One protein-protein interaction module of primary importance to these processes is the P&barbelow;ost-synaptic-density-95, D&barbelow;iscs Large, Z&barbelow;onula-occludens-1 (PDZ) domain. In C. elegans, the PDZ proteins LIN-2, LIN-7, and LIN-10 act in concert to localize LET-23 to the basolateral surface of body epithelia. A complex of the mammalian orthologs of these proteins, mLIN-2/CASK, mLIN-7/Veli and mLIN-10/X11alpha, was immunoprecipitated from brain lysate, but the moeities responsible for complex formation were unknown. Here, we chronicle the elucidation of the L&barbelow;IN-2&barbelow;, -7&barbelow; or L27 dimerization domain. Amino acids important to L27 domain integrity are described. It is also shown that the mLIN-7 L27 domain binds several membrane associated guanylate kinase proteins (MAGUKs) which themselves have two L27 domains, and the more carboxy terminal (L27C) domain mediates the binding. The mLIN-7 associated MAGUKs are mLIN-2, DLG2, DLG3, and two novel proteins, P&barbelow;roteins A&barbelow;ssociated with mL&barbelow;in-S&barbelow;even-1 and 2 (PALS1 and PALS2). We show that three mLIN-7 binding partners, mLIN-2, DLG3, and DLG2, specifically bind the mammalian ortholog of the Drosophila tumor suppressor Discs Large via L27 heterodimerization. The requirements for binding are analyzed and, surprisingly, two modes of interaction are uncovered. PALS1 was shown by others to have a partner for its more amino terminal L2 7N domain named P&barbelow;ALS1 a&barbelow;ssociated T&barbelow;ight J&barbelow;unction protein, or PATJ. This interaction was further determined to be essential for epithelial polarity. We determined the X-ray structure of the complex between PALS1(L27N) and PATJ(L27) domains. The structure reveals a novel fold with hydrophobic residues from three alpha helices of each monomer contributing to a compact binding interface. Taken together, this work elucidates a molecular basis for L27 domain heterodimerization between proteins important in cell polarity.Ph.D.BiochemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/124447/2/3138190.pd

The Bcl-2-Beclin 1 interaction in (-)-gossypol-induced autophagy versus apoptosis in prostate cancer cells

Bcl-2 is a key dual regulator of autophagy and apoptosis, but how the level of Bcl-2 influences the cellular decision between autophagy and apoptosis is unclear. The natural BH3-mimetic (-)-gossypol preferentially induces autophagy in androgen-independent (AI) prostate cancer cells that have high levels of Bcl-2 and are resistant to apoptosis, whereas apoptosis is preferentially induced in androgen-dependent or -independent cells with low Bcl-2. (-)-Gossypol induces autophagy via blocking Bcl-2-Beclin 1 interaction at the endoplasmic reticulum (ER), together with downregulating Bcl-2, upregulating Beclin 1 and activating the autophagic pathway. Furthermore, (-)-gossypol-induced autophagy is Beclin 1- and Atg5-dependent. These results provide new insights into the mode of cell death induced by Bcl-2 inhibitors, which could facilitate the rational design of clinical trials by selecting patients who are most likely to benefit from the Bcl-2-targeted molecular therapy

No small matter: microRNAs – key regulators of cancer stem cells

Emerging evidence demonstrates that both tumor suppressor and oncogenic miRNAs play an essential role in stem cell self-renewal and differentiation by negatively regulating the expression of certain key genes in stem cells. It seems logical that they may also be critical players in cancer stem cells. Though small in size, miRNAs play a key role in the epigenetic regulation of cancer stem cells. Specifically, the imbalance of oncogenic vs. tumor suppressor miRNAs may lead to dysregulation of cancer stem cells, thus causing excessive self-renewal and survival of cancer stem cells, and resistance to chemo/radiotherapy. We postulate that restoring the balance of miRNAs will correct this dysregulation via the direct and simultaneous modulation of downstream stem cell pathways involved in cancer stem cell self-renewal and/or differentiation. The resultant restoration of key regulatory pathways could improve therapeutic response. Restoring tumor suppressor miRNAs and/or inhibiting oncogenic miRNAs may provide a novel molecular therapy for human cancers, potentially via modulating cancer stem cells