Cameron N Johnstone

Publications of Cameron N Johnstone

• IMP-1 displays cross-talk with K-Ras and modulates colon cancer cell survival through the novel proapoptotic protein CYFIP2.

  Authors: Perry S Mongroo, Felicite K Noubissi, Miriam Cuatrecasas, Jiri Kalabis, Catrina E King, Cameron N Johnstone, Mark J Bowser, Antoni Castells, Vladimir S Spiegelman, Anil K Rustgi

  Cancer research. 01/2011; 71(6):2172-82.

  Insulin-like growth factor 2 mRNA-binding protein-1 (IMP-1) is an oncofetal protein that binds directly to and stabilizes oncogenic c-Myc and regulates, in turn, its posttranscriptional expressionInsulin-like growth factor 2 mRNA-binding protein-1 (IMP-1) is an oncofetal protein that binds directly to and stabilizes oncogenic c-Myc and regulates, in turn, its posttranscriptional expression and translation. In contrast to normal adult tissue, IMP-1 is reexpressed and/or overexpressed in human cancers. We show that knockdown of c-Myc in human colon cancer cell lines increases the expression of mature let-7 miRNA family members and downregulates several of its mRNA targets: IMP-1, Cdc34, and K-Ras. We further show that loss of IMP-1 inhibits Cdc34, Lin-28B, and K-Ras, suppresses SW-480 cell proliferation and anchorage-independent growth, and promotes caspase- and lamin-mediated cell death. We also found that IMP-1 binds to the coding region and 3'UTR of K-Ras mRNA. RNA microarray profiling and validation by reverse transcription PCR reveals that the p53-inducible proapoptotic protein CYFIP2 is upregulated in IMP-1 knockdown SW480 cells, a novel finding. We also show that overexpression of IMP-1 increases c-Myc and K-Ras expression and LIM2405 cell proliferation. Furthermore, we show that loss of IMP-1 induces Caspase-3- and PARP-mediated apoptosis, and inhibits K-Ras expression in SW480 cells, which is rescued by CYFIP2 knockdown. Importantly, analysis of 228 patients with colon cancers reveals that IMP-1 is significantly upregulated in differentiated colon tumors (P ≤ 0.0001) and correlates with K-Ras expression (r = 0.35, P ≤ 0.0001) relative to adjacent normal mucosa. These findings indicate that IMP-1, interrelated with c-Myc, acts upstream of K-Ras to promote survival through a novel mechanism that may be important in colon cancer pathogenesis.

• Deregulation of MYCN, LIN28B and LET7 in a molecular subtype of aggressive high-grade serous ovarian cancers.

  Authors: Åslaug Helland, Michael S Anglesio, Joshy George, Prue A Cowin, Cameron N Johnstone, Colin M House, Karen E Sheppard, Dariush Etemadmoghadam, Nataliya Melnyk, Anil K Rustgi [......] Ruth Holm, Gunnar B Kristensen, Michael J Birrer, Richard B Pearson, Anne-Lise Børresen-Dale, David G Huntsman, Anna deFazio, Chad J Creighton, Gordon K Smyth, David D L Bowtell

  PloS one. 01/2011; 6(4):e18064.

  Molecular subtypes of serous ovarian cancer have been recently described. Using data from independent datasets including over 900 primary tumour samples, we show that deregulation of the Let-7Molecular subtypes of serous ovarian cancer have been recently described. Using data from independent datasets including over 900 primary tumour samples, we show that deregulation of the Let-7 pathway is specifically associated with the C5 molecular subtype of serous ovarian cancer. DNA copy number and gene expression of HMGA2, alleles of Let-7, LIN28, LIN28B, MYC, MYCN, DICER1, and RNASEN were measured using microarray and quantitative reverse transcriptase PCR. Immunohistochemistry was performed on 127 samples using tissue microarrays and anti-HMGA2 antibodies. Fluorescence in situ hybridisation of bacterial artificial chromosomes hybridized to 239 ovarian tumours was used to measure translocation at the LIN28B locus. Short interfering RNA knockdown in ovarian cell lines was used to test the functionality of associations observed. Four molecular subtypes (C1, C2, C4, C5) of high-grade serous ovarian cancers were robustly represented in each dataset and showed similar pattern of patient survival. We found highly specific activation of a pathway involving MYCN, LIN28B, Let-7 and HMGA2 in the C5 molecular subtype defined by MYCN amplification and over-expression, over-expression of MYCN targets including the Let-7 repressor LIN28B, loss of Let-7 expression and HMGA2 amplification and over-expression. DICER1, a known Let-7 target, and RNASEN were over-expressed in C5 tumours. We saw no evidence of translocation at the LIN28B locus in C5 tumours. The reported interaction between LIN28B and Let-7 was recapitulated by siRNA knockdown in ovarian cancer cell lines. Our results associate deregulation of MYCN and downstream targets, including Let-7 and oncofetal genes, with serous ovarian cancer. We define for the first time how elements of an oncogenic pathway, involving multiple genes that contribute to stem cell renewal, is specifically altered in a molecular subtype of serous ovarian cancer. By defining the drivers of a molecular subtype of serous ovarian cancers we provide a novel strategy for targeted therapeutic intervention.

• Annexin-1 signals mitogen-stimulated breast tumor cell proliferation by activation of the formyl peptide receptors (FPRs) 1 and 2.

  Authors: Thippadey Khau, Shenna Y Langenbach, Michael Schuliga, Trudi Harris, Cameron N Johnstone, Robin L Anderson, Alastair G Stewart

  FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 10/2010; 25(2):483-96.

  The role of the calcium- and phospholipid-binding protein annexin I (ANXA1) in cell cycle regulation has been investigated in estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 breastThe role of the calcium- and phospholipid-binding protein annexin I (ANXA1) in cell cycle regulation has been investigated in estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 breast tumor cell lines. In MCF-7 cells, ANXA1-targeting small interfering RNA (siRNA) reduced ANXA1 mRNA and protein levels and attenuated cell proliferation induced by FCS, estradiol, or epidermal growth factor. Well-characterized agonists for the known ANXA1 receptor, FPR2, including the ANXA1 N-terminal proteolytic product ANXA1(2-26), lipoxin A(4) (LXA(4)), and the synthetic peptide, Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), stimulated proliferation of MCF-7 and MDA-MB-231 cells that was attenuated by incubation with FPR2 antagonists WRW(4) (1 μM) or Boc2 (100 nM) or by siRNA against FPR2. FCS-induced mitogenic responses were attenuated by each of the FPR antagonists and by siRNA against FPR2 and, to a lesser extent, FPR1. LXA(4) increased phosphorylation of Akt, p70(S6K) but not ERK1/2. Increases in cyclin D1 protein induced by FCS or LXA(4) were blocked by the PI3 kinase inhibitor, LY294002, and attenuated by FPR2 antagonism using Boc2. In invasive breast cancer, immunohistochemistry revealed the presence of ANXA1 and its receptor, FPR2, in both tumor epithelium and stromal cells. These observations suggest a novel signaling role for ANXA1 in mitogen-activated proliferation of breast tumor epithelial cells that is mediated via activation of FPR1 and FPR2.

• Low frequency of MLL3 mutations in colorectal carcinoma.

  Authors: Christopher R Vakoc, Gerd A Blobel, Yu-Ye Wen, Richard A Gibbs, Cameron N Johnstone, Anil K Rustgi

  Cancer genetics and cytogenetics. 04/2009; 189(2):140-1.

• MicroRNA Microarray Identifies Let-7i as a Novel Biomarker and Therapeutic Target in Human Epithelial Ovarian Cancer.

  Authors: Nuo Yang, Sippy Kaur, Stefano Volinia, Joel Greshock, Heini Lassus, Kosei Hasegawa, Shun Liang, Arto Leminen, Shan Deng, Lori Smith [......] Xian-Ming Chen, Chang-Gong Liu, Qihong Huang, Dionyssios Katsaros, George Adrian Calin, Barbara L Weber, Ralf Bützow, Carlo M Croce, George Coukos, Lin Zhang

  Cancer research. 01/2009; 68(24):10307-14.

  MicroRNAs (miRNA) are approximately 22-nucleotide noncoding RNAs that negatively regulate protein-coding gene expression in a sequence-specific manner via translational inhibition or mRNAMicroRNAs (miRNA) are approximately 22-nucleotide noncoding RNAs that negatively regulate protein-coding gene expression in a sequence-specific manner via translational inhibition or mRNA degradation. Our recent studies showed that miRNAs exhibit genomic alterations at a high frequency and their expression is remarkably deregulated in ovarian cancer, strongly suggesting that miRNAs are involved in the initiation and progression of this disease. In the present study, we performed miRNA microarray to identify the miRNAs associated with chemotherapy response in ovarian cancer and found that let-7i expression was significantly reduced in chemotherapy-resistant patients (n = 69, P = 0.003). This result was further validated by stem-loop real-time reverse transcription-PCR (n = 62, P = 0.015). Both loss-of-function (by synthetic let-7i inhibitor) and gain-of-function (by retroviral overexpression of let-7i) studies showed that reduced let-7i expression significantly increased the resistance of ovarian and breast cancer cells to the chemotherapy drug, cis-platinum. Finally, using miRNA microarray, we found that decreased let-7i expression was significantly associated with the shorter progression-free survival of patients with late-stage ovarian cancer (n = 72, P = 0.042). This finding was further validated in the same sample set by stem-loop real-time reverse transcription-PCR (n = 62, P = 0.001) and in an independent sample set by in situ hybridization (n = 53, P = 0.049). Taken together, our results strongly suggest that let-7i might be used as a therapeutic target to modulate platinum-based chemotherapy and as a biomarker to predict chemotherapy response and survival in patients with ovarian cancer. [Cancer Res 2008;68(24):10307-14].

• Genomic and epigenetic alterations deregulate microRNA expression in human epithelial ovarian cancer.

  Authors: Lin Zhang, Stefano Volinia, Tomas Bonome, George Adrian Calin, Joel Greshock, Nuo Yang, Chang-Gong Liu, Antonis Giannakakis, Pangiotis Alexiou, Kosei Hasegawa [......] Phyllis A Gimotty, Angela DeMichele, Qihong Huang, Ralf Bützow, Anil K Rustgi, Barbara L Weber, Michael J Birrer, Artemis G Hatzigeorgiou, Carlo M Croce, George Coukos

  Proceedings of the National Academy of Sciences of the United States of America. 06/2008; 105(19):7004-9.

  MicroRNAs (miRNAs) are an abundant class of small noncoding RNAs that function as negative gene regulators. miRNA deregulation is involved in the initiation and progression of human cancer; however,MicroRNAs (miRNAs) are an abundant class of small noncoding RNAs that function as negative gene regulators. miRNA deregulation is involved in the initiation and progression of human cancer; however, the underlying mechanism and its contributions to genome-wide transcriptional changes in cancer are still largely unknown. We studied miRNA deregulation in human epithelial ovarian cancer by integrative genomic approach, including miRNA microarray (n = 106), array-based comparative genomic hybridization (n = 109), cDNA microarray (n = 76), and tissue array (n = 504). miRNA expression is markedly down-regulated in malignant transformation and tumor progression. Genomic copy number loss and epigenetic silencing, respectively, may account for the down-regulation of approximately 15% and at least approximately 36% of miRNAs in advanced ovarian tumors and miRNA down-regulation contributes to a genome-wide transcriptional deregulation. Last, eight miRNAs located in the chromosome 14 miRNA cluster (Dlk1-Gtl2 domain) were identified as potential tumor suppressor genes. Therefore, our results suggest that miRNAs may offer new biomarkers and therapeutic targets in epithelial ovarian cancer.

• Parvin-beta inhibits breast cancer tumorigenicity and promotes CDK9-mediated peroxisome proliferator-activated receptor gamma 1 phosphorylation.

  Authors: Cameron N Johnstone, Perry S Mongroo, A. Sophie Rich, Michael Schupp, Mark J Bowser, Andrew S. deLemos, John W Tobias, Yingqiu Liu, Gregory E Hannigan, Anil K Rustgi

  Molecular and cellular biology. 02/2008; 28(2):687-704.

  Parvin-beta is a focal adhesion protein downregulated in human breast cancer cells. Loss of Parvin-beta contributes to increased integrin-linked kinase activity, cell-matrix adhesion, and invasionParvin-beta is a focal adhesion protein downregulated in human breast cancer cells. Loss of Parvin-beta contributes to increased integrin-linked kinase activity, cell-matrix adhesion, and invasion through the extracellular matrix in vitro. The effect of ectopic Parvin-beta expression on the transcriptional profile of MDA-MB-231 breast cancer cells, which normally do not express Parvin-beta, was evaluated. Particular emphasis was placed upon propagating MDA-MB-231 breast cancer cells in three-dimensional culture matrices. Interestingly, Parvin-beta reexpression in MDA-MB-231 cells increased the mRNA expression, serine 82 phosphorylation (mediated by CDK9), and activity of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARgamma), and there was a concomitant increase in lipogenic gene expression as a downstream effector of PPARgamma. Importantly, Parvin-beta suppressed breast cancer growth in vivo, with associated decreased proliferation. These data suggest that Parvin-beta might influence breast cancer progression.

• The functional interplay between EGFR overexpression, hTERT activation, and p53 mutation in esophageal epithelial cells with activation of stromal fibroblasts induces tumor development, invasion, and differentiation.

  Authors: Takaomi Okawa, Carmen Z Michaylira, Jiri Kalabis, Douglas B Stairs, Hiroshi Nakagawa, Claudia D Andl, Cameron N Johnstone, Andres J Klein-Szanto, Wafik S El-Deiry, Edna Cukierman, Meenhard Herlyn, Anil K Rustgi

  Genes & development. 12/2007; 21(21):2788-803.

  Esophageal cancer is a prototypic squamous cell cancer that carries a poor prognosis, primarily due to presentation at advanced stages. We used human esophageal epithelial cells as a platform toEsophageal cancer is a prototypic squamous cell cancer that carries a poor prognosis, primarily due to presentation at advanced stages. We used human esophageal epithelial cells as a platform to recapitulate esophageal squamous cell cancer, thereby providing insights into the molecular pathogenesis of squamous cell cancers in general. This was achieved through the retroviral-mediated transduction into normal, primary human esophageal epithelial cells of epidermal growth factor receptor (EGFR), the catalytic subunit of human telomerase (hTERT), and p53(R175H), genes that are frequently altered in human esophageal squamous cell cancer. These cells demonstrated increased migration and invasion when compared with control cells. When these genetically altered cells were placed within the in vivo-like context of an organotypic three-dimensional (3D) culture system, the cells formed a high-grade dysplastic epithelium with malignant cells invading into the stromal extracellular matrix (ECM). The invasive phenotype was in part modulated by the activation of matrix metalloproteinase-9 (MMP-9). Using pharmacological and genetic approaches to decrease MMP-9, invasion into the underlying ECM could be suppressed partially. In addition, tumor differentiation was influenced by the type of fibroblasts within the stromal ECM. To that end, fetal esophageal fibroblasts fostered a microenvironment conducive to poorly differentiated invading tumor cells, whereas fetal skin fibroblasts supported a well-differentiated tumor as illustrated by keratin "pearl" formation, a hallmark feature of well-differentiated squamous cell cancers. When inducible AKT was introduced into fetal skin esophageal fibroblasts, a more invasive, less-differentiated esophageal cancer phenotype was achieved. Invasion into the stromal ECM was attenuated by genetic knockdown of AKT1 as well as AKT2. Taken together, alterations in key oncogenes and tumor suppressor genes in esophageal epithelial cells, the composition and activation of fibroblasts, and the components of the ECM conspire to regulate the physical and biological properties of the stroma.

• IGFBP-3 regulates esophageal tumor growth through IGF-dependent and independent mechanisms.

  Authors: Munenori Takaoka, Seok Hyun Kim, Takaomi Okawa, Carmen Z Michaylira, Douglas B Stairs, Cameron N Johnstone, Claudia D Andl, Ben Rhoades, James J Lee, Andres J P Klein-Szanto, Wafik S El-Deiry, Hiroshi Nakagawa

  Cancer biology & therapy. 05/2007; 6(4):534-40.

  Insulin-like growth factor binding protein (IGFBP)-3 exerts either proapoptotic or growth stimulatory effects depending upon the cellular context. IGFBP-3 is overexpressed frequently in esophagealInsulin-like growth factor binding protein (IGFBP)-3 exerts either proapoptotic or growth stimulatory effects depending upon the cellular context. IGFBP-3 is overexpressed frequently in esophageal cancer. Yet, the role of IGFBP-3 in esophageal tumor biology remains elusive. To delineate the functional consequences of IGFBP-3 overexpression, we stably transduced Ha-Ras(V12)-transformed human esophageal cells with either wild-type or mutant IGFBP-3, the latter incapable of binding Insulin-like growth factor (IGFs) as a result of substitution of amino-terminal Ile56, Leu80, and Leu81 residues with Glycine residues. Wild-type, but not mutant, IGFBP-3 prevented IGF-1 from activating the IGF-1 receptor and AKT, and suppressed anchorage-independent cell growth. When xenografted in nude mice, in vivo bioluminescence imaging demonstrated that wild-type, but not mutant IGFBP-3, abrogated tumor formation by the Ras-transformed cells with concurrent induction of apoptosis, implying a prosurvival effect of IGF in cancer cell adaptation to the microenvironment. Moreover, there was more aggressive tumor growth by mutant IGFBP-3 overexpressing cells than control cell tumors, without detectable caspase-3 cleavage in tumor tissues, indicating an IGF-independent growth stimulatory effect of mutant IGFBP-3. In aggregate, these data suggest that IGFBP-3 contributes to esophageal tumor development and progression through IGF-dependent and independent mechanisms.

• TGF-betaRII rescues development of small intestinal epithelial cells in Elf3-deficient mice.

  Authors: Nicole Flentjar, Po-Yin Chu, Annie Y-N Ng, Cameron N Johnstone, Joan K Heath, Matthias Ernst, Paul J Hertzog, Melanie A Pritchard

  Gastroenterology. 04/2007; 132(4):1410-9.

  BACKGROUND & AIMS: ELF3, a member of the ETS transcription factor family, has been shown to transactivate the transforming growth factor beta type II receptor (TGF-betaRII) promoter. Previously weBACKGROUND & AIMS: ELF3, a member of the ETS transcription factor family, has been shown to transactivate the transforming growth factor beta type II receptor (TGF-betaRII) promoter. Previously we showed that Elf3-null mice have a defect in the small intestine caused by a failure of small intestinal epithelial cells to differentiate and that these cells produced significantly lower levels of Tgf-betaRII. To prove that the defect observed in Elf3-null mice resulted from the lack of Elf3-dependent activation of Tgf-betaRII expression, we performed a genetic rescue. METHODS: We generated transgenic mice that express human TGF-betaRII specifically in the intestinal epithelium under the control of the mouse A33 antigen promoter. Mice expressing the A33-TGF-betaRII transgene were mated with Elf3(+/-) mice, and double heterozygous offspring harboring both the transgene and one mutant Elf3 allele were intercrossed. RESULTS: The resultant A33-TGF-betaRII transgenic Elf3(-/-) pups displayed normal small intestinal morphology, while the characteristic abnormality was retained in all Elf3(-/-) mice that did not express the transgene. This phenotypic rescue shows for the first time in vivo that a single gene, Elf3, is the critical upstream regulator of Tgf-betaRII in mouse small intestinal epithelium. CONCLUSIONS: This has important implications for our understanding of tissue-specific gene regulation and further strengthens the potential clinical connection between ELF3 and colorectal cancer involving transforming growth factor beta insensitivity.

• N-cadherin and keratinocyte growth factor receptor mediate the functional interplay between Ki-RASG12V and p53V143A in promoting pancreatic cell migration, invasion, and tissue architecture disruption.

  Authors: Therese B Deramaudt, Munenori Takaoka, Rabi Upadhyay, Mark J Bowser, Jess Porter, Amy Lee, Ben Rhoades, Cameron N Johnstone, Ralph Weissleder, Sunil R Hingorani, Umar Mahmood, Anil K Rustgi

  Molecular and cellular biology. 07/2006; 26(11):4185-200.

  The genetic basis of pancreatic ductal adenocarcinoma, which constitutes the most common type of pancreatic malignancy, involves the sequential activation of oncogenes and inactivation of tumorThe genetic basis of pancreatic ductal adenocarcinoma, which constitutes the most common type of pancreatic malignancy, involves the sequential activation of oncogenes and inactivation of tumor suppressor genes. Among the pivotal genetic alterations are Ki-RAS oncogene activation and p53 tumor suppressor gene inactivation. We explain that the combination of these genetic events facilitates pancreatic carcinogenesis as revealed in novel three-dimensional cell (spheroid cyst) culture and in vivo subcutaneous and orthotopic xenotransplantation models. N-cadherin, a member of the classic cadherins important in the regulation of cell-cell adhesion, is induced in the presence of Ki-RAS mutation but subsequently downregulated with the acquisition of p53 mutation as revealed by gene microarrays and corroborated by reverse transcription-PCR and Western blotting. N-cadherin modulates the capacity of pancreatic ductal cells to migrate and invade, in part via complex formation with keratinocyte growth factor receptor and neural cell adhesion molecule and in part via interaction with p120-catenin. However, modulation of these complexes by Ki-RAS and p53 leads to enhanced cell migration and invasion. This preferentially induces the downstream effector AKT over mitogen-activated protein kinase to execute changes in cellular behavior. Thus, we are able to define molecules that in part are directly affected by Ki-RAS and p53 during pancreatic ductal carcinogenesis, and this provides a platform for potential new molecularly based therapeutic interventions.

• PRR5 encodes a conserved proline-rich protein predominant in kidney: analysis of genomic organization, expression, and mutation status in breast and colorectal carcinomas.

  Authors: Cameron N Johnstone, Sergi Castellví-Bel, Laura M Chang, Raphael K Sung, Mark J Bowser, Josep M Piqué, Antoni Castells, Anil K Rustgi

  Genomics. 04/2005; 85(3):338-51.

  Loss of heterozygosity on chromosome 22q13.31 is a frequent event during human breast and colorectal carcinogenesis. Herein we characterize a novel gene at chromosome 22q13.31 designated PRR5.Loss of heterozygosity on chromosome 22q13.31 is a frequent event during human breast and colorectal carcinogenesis. Herein we characterize a novel gene at chromosome 22q13.31 designated PRR5. Alternative promoter usage and splicing converge to generate five PRR5 transcript variants with maximum mRNA expression in kidney. In vitro transcription/translation demonstrated that the five variants generate three protein isoforms differing in their N-terminal length. Mutational analysis of PRR5 in human breast and colorectal tumors did not reveal somatic mutations. However, mRNA expression analyses revealed PRR5 overexpression in a majority of colorectal tumors but substantial downregulation of PRR5 expression in a subset of breast tumors and reduced expression in two breast cancer cell lines. Treatment with trichostatin A increased PRR5 mRNA levels in BT549 and MDA-MB-231 cells, whereas 5'-aza-2'-deoxycytidine induced expression in MDA-MB-231 cells only. Thus, PRR5 may represent a potential candidate tumor suppressor gene in breast cancer.

• Beta-parvin inhibits integrin-linked kinase signaling and is downregulated in breast cancer.

  Authors: Perry S Mongroo, Cameron N Johnstone, Izabela Naruszewicz, Chungyee Leung-Hagesteijn, Raphael K Sung, Leanne Carnio, Anil K Rustgi, Gregory E Hannigan

  Oncogene. 11/2004; 23(55):8959-70.

  We analysed breast tumors and breast cancer cell lines for the expression of beta-parvin (ParvB), an adaptor protein that binds to the integrin-linked kinase (ILK). Quantitative RT-PCR indicated thatWe analysed breast tumors and breast cancer cell lines for the expression of beta-parvin (ParvB), an adaptor protein that binds to the integrin-linked kinase (ILK). Quantitative RT-PCR indicated that ParvB mRNA was downregulated, by at least 60%, in four of nine breast tumors, relative to patient-matched normal mammary gland tissue. We also found that ParvB protein levels were reduced by > or =90% in five of seven advanced tumors, relative to matched normal breast tissue. Conversely, ILK protein and kinase activity levels were elevated in these tumors, suggesting that downregulation of ParvB stimulates ILK signaling. Western blot analyses indicated very low levels of ParvB protein in MDA-MB-231 and MCF7 breast cancer cells, facilitating functional studies of the effects of ParvB on ILK signaling. Expression of ParvB in MDA-MB-231 and MCF7 cells increased cell adhesion to collagen. ParvB inhibited ILK kinase activity, anchorage-independent cell growth and in vitro matrigel invasion by MDA-MB-231 cells. EGF-induced phosphorylation of two ILK targets, PKB (Ser473) and glycogen synthase kinase 3beta (Ser9), was also inhibited by ParvB. These results indicated that ParvB inhibits ILK signaling downstream of receptor tyrosine kinases. Our results suggest that loss of ParvB expression is a novel mechanism for upregulating ILK activity in tumors.

• Ha-Ras(G12V) induces senescence in primary and immortalized human esophageal keratinocytes with p53 dysfunction.

  Authors: Munenori Takaoka, Hideki Harada, Therese B Deramaudt, Kenji Oyama, Claudia D Andl, Cameron N Johnstone, Ben Rhoades, Gregory H Enders, Oliver G Opitz, Hiroshi Nakagawa

  Oncogene. 10/2004; 23(40):6760-8.

  Oncogenic Ras induces premature senescence in primary cells. Such an oncogene-induced senescence involves activation of tumor suppressor genes that provide a checkpoint mechanism against malignantOncogenic Ras induces premature senescence in primary cells. Such an oncogene-induced senescence involves activation of tumor suppressor genes that provide a checkpoint mechanism against malignant transformation. In mouse, the ARF-p53 pathway mediates Ha-Ras(G12V)-induced senescence, and p19(ARF-/-) and p53(-/-) cells undergo transformation upon Ras activation. In addition, mouse cells, unlike human cells, express constitutively active telomerase and have long telomeres. However, it is unclear how Ras activation affects human cells of epithelial origin with p53 mutation and/or telomerase activation. In order to address this question, Ha-Ras(G12V) was expressed ectopically in primary as well as hTERT-immortalized human esophageal keratinocytes stably expressing dominant-negative p53 mutants. In human esophageal keratinocytes, we found that Ha-Ras(G12V) induced senescence regardless of p53 status and telomerase activation. Ras activation resulted in changes of cellular morphology, activation of senescence-associated beta-galactosidase, and suppression of cell proliferation, all coupled with reduction in the hyperphosphorylated form of the retinoblastoma protein (pRb). Furthermore, Ha-Ras(G12V) upregulated p16(INK4a) and downregulated cyclin-dependent kinase Cdk4 in human esophageal keratinocytes. Thus, Ras-mediated senescence may involve distinct mechanisms between human and mouse cells. Inactivation of the pRb pathway may be necessary for Ras to overcome senescence and transform human esophageal epithelial cells.

• ARHGAP8 is a novel member of the RHOGAP family related to ARHGAP1/CDC42GAP/p50RHOGAP: mutation and expression analyses in colorectal and breast cancers.

  Authors: Cameron N Johnstone, Sergi Castellví-Bel, Laura M Chang, Xavier Bessa, Hiroshi Nakagawa, Hideki Harada, Raphael K Sung, Josep M Piqué, Antoni Castells, Anil K Rustgi

  Gene. 08/2004; 336(1):59-71.

  The RHO family of small GTPases has multiple functions, including regulation of cytoskeletal organization, cell cycle progression and cell migration, among others. The key members of this family areThe RHO family of small GTPases has multiple functions, including regulation of cytoskeletal organization, cell cycle progression and cell migration, among others. The key members of this family are RHO, RAC and CDC42. Active GTP-bound RHO proteins are down-regulated by RHO GTPase-activating proteins (RHOGAPs). Herein, we describe the identification, characterization and mutational analysis of a novel RHOGAP designated as ARHGAP8, which is located within a critical region of loss-of-heterozygosity on chromosome 22q13.31 in breast and colorectal carcinomas. ARHGAP8 shares an identical genomic organization with ARHGAP1/CDC42GAP/p50RHOGAP and the corresponding proteins share the same functional domains that distinguish them from other ARHGAP members. These domains include the C-terminal RHOGAP domain, a central SH3-binding motif, and an N-terminal BNIP-2/CDC42GAP homology (BCH)/Sec14p-like domain. Three alternatively spliced ARHGAP8 transcripts were expressed in normal mammary gland and colon, which differed in the size of the BCH/Sec14p-like domain only. PCR-SSCP analyses revealed a total of six germline missense variants in individuals with colorectal or breast cancer; however, somatic mutations were not identified. Surprisingly, ARHGAP8 expression was up-regulated in the majority of primary colorectal tumors analyzed. Taken together, ARHGAP8 encodes a novel RHOGAP with unique functional domains that is highly homologous to ARHGAP1/CDC42GAP/p50RHOGAP.

• Evaluation of PARVG located on 22q13 as a candidate tumor suppressor gene for colorectal and breast cancer.

  Authors: Sergi Castellví-Bel, Antoni Castells, Cameron N Johnstone, Virgínia Piñol, Maria Pellisé, Josep I Elizalde, Neus Romo, Anil K Rustgi, Josep M Piqué

  Cancer genetics and cytogenetics. 08/2003; 144(1):80-2.

  Colorectal cancer (CRC) and breast cancer constitute common neoplasms in Western countries and leading causes of cancer-related death. Development and progression of both malignancies occur as aColorectal cancer (CRC) and breast cancer constitute common neoplasms in Western countries and leading causes of cancer-related death. Development and progression of both malignancies occur as a multistep process, requiring the activation of oncogenes and the inactivation of several tumor suppressor genes. Our group has recently identified a minimal region of deletion on 22q13 involved in CRC and breast cancer patients, which is highly indicative of the existence of a tumor suppressor gene (or genes). We performed mutation analysis of the PARVG gene, one of the genes present on the 22q13 region of interest, which has been previously demonstrated to have a reduced expression in some cancer cell lines. We have identified several DNA variants that are not compatible with pathogenic mutations. Accordingly, PARVG appears not to be a tumor suppressor gene involved in CRC and breast cancer development and progression.

• AACR special meeting in cancer research: colon cancer--genetics to prevention.

  Authors: Cameron N Johnstone, Laura M Chang, Matthias Ernst

  Cancer research. 12/2002; 62(22):6779-83.

• Analysis of the regulation of the A33 antigen gene reveals intestine-specific mechanisms of gene expression.

  Authors: Cameron N Johnstone, Sara J White, Niall C Tebbutt, Fiona J Clay, Matthias Ernst, William H Biggs, Carrie S Viars, Suzanne Czekay, Karen C Arden, Joan K Heath

  The Journal of biological chemistry. 10/2002; 277(37):34531-9.

  The A33 antigen is a transmembrane protein expressed almost exclusively by intestinal epithelial cells. The level of its expression is robust and uniform throughout the rostrocaudal axis of the humanThe A33 antigen is a transmembrane protein expressed almost exclusively by intestinal epithelial cells. The level of its expression is robust and uniform throughout the rostrocaudal axis of the human and mouse intestines. In the colon, strong expression is found in the basolateral membranes of both the proliferating cells in the lower regions of the crypts and the differentiating cells in the upper regions of crypts. Similarly, in the small intestine, the protein is highly expressed by all the epithelial cells in the crypts and by the differentiated cells migrating over the villi. Thus, the A33 antigen has emerged as a definitive marker for all intestinal epithelial cells, irrespective of cell lineage and differentiation status. To understand the molecular mechanisms mediating this rare tissue-specific expression pattern, we undertook a comprehensive analysis of the 5'-regulatory region of the human A33 antigen gene. This allowed us to point to positive cis-regulatory elements incorporating consensus Krüppel-like factor and caudal-related homeobox (CDX)-binding sites, located just upstream from the human A33 antigen transcription start site, as being important for the intestine-specific expression pattern of this gene. Further analysis provided evidence that the A33 antigen gene may be one of only a few target genes to be described thus far for the intestine-specific homeobox transcription factor, CDX1. Taken together, our data lead us to propose that the activity of CDX1 is pivotal in mediating the exquisite, intestine-specific expression pattern of the A33 antigen gene.

• ARHGAP8 is a novel member of the RHOGAP family related to ARHGAP1/CDC42GAP/p50RHOGAP: mutation and expression analyses in colorectal and breast cancers

  Authors: Cameron N. Johnstone, Sergi Castellvı́-Bel, Laura M. Chang, Xavier Bessa, Hiroshi Nakagawa, Hideki Harada, Raphael K. Sung, Josep M. Piqué, Antoni Castells, Anil K. Rustgi

  Gene.

  The RHO family of small GTPases has multiple functions, including regulation of cytoskeletal organization, cell cycle progression and cell migration, among others. The key members of this family areThe RHO family of small GTPases has multiple functions, including regulation of cytoskeletal organization, cell cycle progression and cell migration, among others. The key members of this family are RHO, RAC and CDC42. Active GTP-bound RHO proteins are down-regulated by RHO GTPase-activating proteins (RHOGAPs). Herein, we describe the identification, characterization and mutational analysis of a novel RHOGAP designated as ARHGAP8, which is located within a critical region of loss-of-heterozygosity on chromosome 22q13.31 in breast and colorectal carcinomas. ARHGAP8 shares an identical genomic organization with ARHGAP1/CDC42GAP/p50RHOGAP and the corresponding proteins share the same functional domains that distinguish them from other ARHGAP members. These domains include the C-terminal RHOGAP domain, a central SH3-binding motif, and an N-terminal BNIP-2/CDC42GAP homology (BCH)/Sec14p-like domain. Three alternatively spliced ARHGAP8 transcripts were expressed in normal mammary gland and colon, which differed in the size of the BCH/Sec14p-like domain only. PCR-SSCP analyses revealed a total of six germline missense variants in individuals with colorectal or breast cancer; however, somatic mutations were not identified. Surprisingly, ARHGAP8 expression was up-regulated in the majority of primary colorectal tumors analyzed. Taken together, ARHGAP8 encodes a novel RHOGAP with unique functional domains that is highly homologous to ARHGAP1/CDC42GAP/p50RHOGAP.