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Supplementary Methods, Table 1, Figures 1-8 from Loss of Metallothionein Predisposes Mice to Diethylnitrosamine-Induced Hepatocarcinogenesis by Activating NF-κB Target Genes

We have recently shown that the gene encoding the truncated form of protein tyrosine phosphatase receptor-type O (PTPROt) expressed predominantly in hematopoietic cells, is epigenetically silenced in human primary chronic lymphocytic leukemia (B-CLL). To determine whether increased phosphorylation of the PTPROt substrates following PTPROt suppression alters signal transduction pathway(s) that impart a growth advantage to the leukemic lymphocytes, it is critical to discern the key substrates of PTPROt. Here, we used substrate-trapping assay to identify two novel substrates of PTPROt, the tyrosine kinases Lyn and ZAP70. Both Lyn and ZAP70 were dephosphorylated by wild type PTPROt, but not by its catalytic site (CS) mutant. A critical phosphorylation site in Lyn, Y397, essential for its activity was dephosphorylated by PTPROt. Consequently, the activity of Lyn kinase was compromised when co-expressed with PTPROt-WT compared to vector control or upon co-expression with PTPROt-CS. Ectopic expression of PTPROt in Raji cells reduced phosphorylation of Lyn in the absence of any change in its protein levels. These results have revealed the physiological importance of PTPROt in regulating B-cell receptor signaling at Lyn kinase. Further, ectopic expression of PTPROt also sensitized the cells to the VEGF-R inhibitor Pazopanib.

We have shown earlier that miR-221 and -222 are up-regulated in tamoxifen-resistant MCF-7 (OHT(R)) cells and Her2-positive human breast tumors when compared with Her2 negative tumors. In this study, we report markedly enhanced expression of miR-181b in OHT(R) cells and endocrine-resistant tumors. Further, anti-miR-222 or -181b in combination with tamoxifen suppressed growth of tamoxifen-resistant xenografts in mice. Luciferase reporter assay and expression analysis showed that TIMP3, a tissue metalloproteinase inhibitor, is a common target of miR-221/222 and -181b. In situ hybridization and immunohistochemical analysis demonstrated reciprocal relationships between TIMP3 and miR-221/222/181b expression in primary human breast carcinomas. Ectopic expression of TIMP3 inhibited growth of the OHT(R) cells, and its depletion in MCF-7 cells reduced sensitivity to tamoxifen in vitro and in vivo. EGF-induced MAPK and AKT phosphorylation were significantly higher in OHT(R) cells and miR-221/222-overexpressing MCF-7 cells than in control cells, which suggests modulation of mitogenic signaling by TIMP3 and the miRs. On the contrary, phosphoMAPK and phosphoAKT levels were diminished in TIMP3-overexpressing OHT(R) cells and increased in TIMP3-depleted MCF-7 cells. Low levels of estrogen or tamoxifen elicited similar differences in phosphoMAPK levels in these cells. Reduced levels of TIMP3 facilitated growth of tamoxifen-resistant cells by alleviating its inhibitory effect on ADAM10 and ADAM17, which are critical for OHT(R) cell growth. In conclusion, miR-221/222 and -181b facilitate growth factor signaling in tamoxifen-resistant breast cancer by down-regulating TIMP3, and corresponding anti-miRs can be used to render these tumors responsive to tamoxifen.

We have previously demonstrated the tumor suppressor characteristics of protein tyrosine phosphatase receptor-type O (PTPRO) in leukemia and lung cancer, including its suppression by promoter methylation. Here, we show tumor-specific methylation of the PTPRO CpG island in primary human breast cancer. PTPRO expression was significantly reduced in established breast cancer cell lines MCF-7 and MDA-MB-231 due to promoter methylation compared with its expression in normal human mammary epithelial cells (48R and 184). Further, the silenced gene could be demethylated and reactivated in MCF-7 and MDA-MB-231 cells upon treatment with 5-Azacytidine, a DNA hypomethylating agent. Because PTPRO promoter harbors estrogen-responsive elements and 17beta-estradiol (E2) plays a role in breast carcinogenesis, we examined the effect of E2 and its antagonist tamoxifen on PTPRO expression in human mammary epithelial cells and PTPRO-expressing breast cancer cell line Hs578t. Treatment with E2 significantly curtailed PTPRO expression in 48R and Hs578t cells, which was facilitated by ectopic expression of estrogen receptor (ER)beta but not ERalpha. On the contrary, treatment with tamoxifen increased PTPRO expression. Further, knockdown of ERbeta by small interfering RNA abolished these effects of E2 and tamoxifen. Chromatin immunoprecipitation assay showed association of c-Fos and c-Jun with PTPRO promoter in untreated cells, which was augmented by tamoxifen-mediated recruitment of ERbeta to the promoter. Estradiol treatment resulted in dissociation of c-Fos and c-Jun from the promoter. Ectopic expression of PTPRO in the nonexpressing MCF-7 cells sensitized them to growth-suppressive effects of tamoxifen. These data suggest that estrogen-mediated suppression of PTPRO is probably one of the early events in estrogen-induced tumorigenesis and that expression of PTPRO could facilitate endocrine therapy of breast cancer.

Epigenetic events that are essential drivers of lymphocyte transformation remain incompletely characterized. We used models of Epstein-Barr virus (EBV)–induced B-cell transformation to document the relevance of protein arginine methyltransferase 5 (PRMT5) to regulation of epigenetic-repressive marks during lymphomagenesis. EBV+ lymphomas and transformed cell lines exhibited abundant expression of PRMT5, a type II PRMT enzyme that promotes transcriptional silencing of target genes by methylating arginine residues on histone tails. PRMT5 expression was limited to EBV-transformed cells, not resting or activated B lymphocytes, validating it as an ideal therapeutic target. We developed a first-in-class, small-molecule PRMT5 inhibitor that blocked EBV-driven B-lymphocyte transformation and survival while leaving normal B cells unaffected. Inhibition of PRMT5 led to lost recruitment of a PRMT5/p65/HDAC3-repressive complex on the miR96 promoter, restored miR96 expression, and PRMT5 downregulation. RNA-sequencing and chromatin immunoprecipitation experiments identified several tumor suppressor genes, including the protein tyrosine phosphatase gene PTPROt, which became silenced during EBV-driven B-cell transformation. Enhanced PTPROt expression following PRMT5 inhibition led to dephosphorylation of kinases that regulate B-cell receptor signaling. We conclude that PRMT5 is critical to EBV-driven B-cell transformation and maintenance of the malignant phenotype, and that PRMT5 inhibition shows promise as a novel therapeutic approach for B-cell lymphomas. This work was supported by grants from the American Society of Hematology/European Hematology Association (L.Alinari), the Leukemia & Lymphoma Society Translational Research Project (LLS TRP) (R.A.B.), Friends of Jason Gould Foundation (R.A.B., P.L.S., J.T.P.), The Ohio State University Drug Development Institute (R.A.B., C.L.), National Institutes of Health, National Institute of Neurological Disorders and Stroke grant R21NS071346 (R.A.B., C.L.) and National Cancer Institute grant R01CA116093 (S.S., R.A.B.).

We have previously reported that the gene encoding protein tyrosine phosphatase receptor type-O (PTPRO) is suppressed by promoter methylation in a rat model of hepatocellular carcinoma (HCC) and it functions as tumor suppressor in leukemia and lung cancer. Here, we explored the methylation and expression of PTPRO as well as its function in human HCC. MassARRAY analysis of primary human HCC and matching liver samples (n = 24) revealed significantly higher (P = 0.004) methylation density at the promoter CGI in tumors. Combined bisulfite restriction analysis (COBRA) of another set of human HCC samples (n = 17) demonstrated that the CGI was methylated in 29% of tumors where expression of PTPRO was lower than that in corresponding matching livers. A substrate-trapping mutant of PTPRO that stabilizes the bound substrates was used to identify its novel substrate(s). VCP/p97 was found to be a PTPRO substrate by mass spectrometry of the peptides pulled down by the substrate-trapping mutant of PTPRO. Tyrosyl dephosphorylation of VCP following ectopic expression of wild-type PTPRO in H293T and HepG2 cells confirmed that it is a bona fide substrate of PTPRO. Treatment of PTPRO overexpressing HepG2 cells with Doxorubicin, a DNA damaging drug commonly used in therapy of primary HCC, sensitized these cells to this potent anticancer drug that correlated with dephosphorylation of VCP. Taken together, these results demonstrate methylation and downregulation of PTPRO in a subset of primary human HCC and establish VCP as a novel functionally important substrate of this tyrosine phosphatase that could be a potential molecular target for HCC therapy.

We previously demonstrated that the gene encoding PTPROt, the truncated form of protein tyrosine phosphatase receptor type O expressed predominantly in hematopoietic cells, is a candidate tumor suppressor and is down-regulated in chronic lymphocytic leukemia (CLL). Here, we show that PTPROt expression is significantly reduced in CD19+ spleen B cells from Eμ-T cell leukemia 1 (TCL1) transgenic mice relative to the wild-type mice. Strikingly, as much as a 60% decrease in PTPROt expression occurs at 7 weeks independently of promoter methylation. To elucidate the potential mechanism for this early suppression of PTPROt in these mice, we explored the role of activating protein-1 (AP-1) in its expression. We first demonstrate that AP-1 activation by 12-O-tetradecanoylphorbol-13-acetate induces PTPROt expression with concurrent recruitment of c-fos and c-jun to its promoter. The PTPROt promoter is also responsive to over- and underexpression of AP-1, confirming the role of AP-1 in PTPROt expression. Next, we demonstrate that TCL1 can repress the PTPROt promoter by altering c-fos expression and c-jun activation state. Finally, using primary CLL cells we have shown an inverse relationship between TCL1 and PTPROt expression. These findings further substantiate the role of TCL1 in PTPROt suppression and its importance in the pathogenesis of CLL.

Metallothioneins (MT) are potent scavengers of free radicals that are silenced in primary hepatocellular carcinomas (HCC) of human and rodent origin. To examine whether loss of MT promotes hepatocarcinogenesis, male Mt-1 and Mt-2 double knockout (MTKO) and wild-type (WT) mice were exposed to diethylnitrosamine (DEN) and induction of HCC was monitored at 23 and 33 weeks. The size and number of liver tumors, the ratio between liver and body weight, and liver damage were markedly elevated in the MTKO mice at both time points compared with the WT mice. At 23 weeks, MTKO mice developed HCC whereas WT mice developed only preneoplastic nodules suggesting that loss of MT accelerates hepatocarcinogenesis. MTKO tumors also exhibited higher superoxide anion levels. Although NF-κB activity increased in the liver nuclear extracts of both genotypes after DEN exposure, the complex formed in MTKO mice was predominantly p50/65 heterodimer (transcriptional activator) as opposed to p50 homodimer (transcriptional repressor) in WT mice. Phosphorylation of p65 at Ser276 causing its activation was also significantly augmented in DEN-exposed MTKO livers. NF-κB targets that include early growth response genes and proinflammatory cytokines were significantly upregulated in MTKO mice. Concurrently, there was a remarkable increase (∼100-fold) in Pai-1 expression; significant increase in c-Jun, c-Fos, c-Myc, Ets2, and ATF3 expressions; and growth factor signaling that probably contributed to the increased tumor growth in MTKO mice. Taken together, these results demonstrate that MTs protect mice from hepatocarcinogen-induced liver damage and carcinogenesis, underscoring their potential therapeutic application against hepatocellular cancer. Cancer Res; 70(24); 10265–76. ©2010 AACR.

In an effort to understand the epigenetic regulation of ribosomal RNA gene (rDNA) expression we have previously demonstrated the role of DNA methyltransferases and methyl CpG binding proteins in rRNA synthesis. Here, we studied the role of protein arginine methyltransferase PRMT5 and the two methylated histones H3R8Me2 and H4R3Me2, in rDNA expression in Epstein Barr virus- transformed primary B-cells (LCLs) and in HeLa cells responding to serum-regulated growth. Chromatin immunoprecipitation assay showed that histones H3 and H4 associated with rRNA promoters were differentially methylated at arginine residues 8 and 3, respectively, depending on its transcriptional activity. Association of PRMT5 and methylated H3 with the unmethylated promoters in resting B-cells was significantly reduced in rapidly growing LCLs. Unlike PRMT5 and H3R8Me2, histone H4 associated with both methylated and unmethylated rRNA promoters in resting B-cells was methylated at the R3 residue. However, a dramatic decrease in R3 methylation of H4 recruited to the unmethylated rRNA promoters was observed in LCLs while it remained unaltered in the fraction bound to the methylated promoters. Differential interaction of PRMT5 and methylation of H3 and H4 associated with the rRNA promoters was also observed when serum starved HeLa cells were allowed to grow in serum replenished media. Ectopic expression of PRMT5 suppressed activity of both unmethylated and methylated rRNA promoter in transient transfection assay whereas siRNA mediated knockdown of PRMT5 increased rRNA synthesis in HeLa cells. These data suggest a key role of PRMT5 and the two methylated histones in regulating rRNA promoter activity.

Chronic myelogenous leukemia is typified by constitutive activation of the c-abl kinase as a result of its fusion to the breakpoint cluster region (BCR). Because the truncated isoform of protein-tyrosine phosphatase receptor-type O (PTPROt) is specifically expressed in hematopoietic cells, we tested the possibility that it could potentially dephosphorylate and inactivate the fusion protein bcr/abl. Ectopic expression of PTPROt in the chronic myelogenous leukemia cell line K562 indeed resulted in hypophosphorylation of bcr/abl and reduced phosphorylation of its downstream targets CrkL and Stat5, confirming that PTPROt could inactivate the function of bcr/abl. Furthermore, the expression of catalytically active PTPROt in K562 cells caused reduced proliferation, delayed transition from G0/G1 to S phase, loss of anchorage independent growth, inhibition of ex vivo tumor growth, and increased their susceptibility to apoptosis, affirming that this tyrosine phosphatase can revert the transformation potential of bcr/abl. Additionally, the catalytically inactive PTPROt acted as a trapping mutant that was also able to inhibit anchorage independence and facilitate apoptosis of K562 cells. The inhibitory action of PTPROt on bcr/abl was also confirmed in a murine myeloid cell line overexpressing bcr/abl. PTPROt expression was suppressed in K562 cells and was relieved upon treatment of the cells with 5-azacytidine, an inhibitor of DNA methyltransferase, with concomitant hypomethylation of the PTPRO CpG island. These data demonstrate that suppression of PTPROt by promoter methylation could contribute to the augmented phosphorylation and constitutive activity of its substrate bcr/abl and provide a potentially significant molecular therapeutic target for bcr/abl-positive leukemia.

Background It has been an open question how similar human and canine lung cancers are. This has major implications in availability of human treatments for dogs and in establishing translational models to test new therapies in pet dogs. The prognosis for canine advanced lung cancer is poor and new treatments are needed. Heat shock protein 90 (HSP90) is an ATPase-dependent molecular chaperone ubiquitously expressed in eukaryotic cells. HSP90 is essential for posttranslational conformational maturation and stability of client proteins including protein kinases and transcription factors, many of which are important for the proliferation and survival of cancer cells. We investigated the activity of STA-1474, a HSP90 inhibitor, in two canine lung cancer cell lines, BACA and CLAC. Results Comparative genomic hybridization analysis of both cell lines revealed genetic relevance to human non-small cell lung cancer. STA-1474 inhibited growth and induced apoptosis of both cell lines in a dose- and time-dependent manner. The ICs50 after 72 h treatment with STA-1474 were 0.08 and 0.11 μM for BACA and CLAC, respectively. When grown as spheroids, the IC50 of STA-1474 for BACA cells was approximately two-fold higher than when grown as a monolayer (0.348 μM vs. 0.168 μM), whereas CLAC spheroids were relatively drug resistant. Treatment of tumor-stromal fibroblasts with STA-1474 resulted in a dose-dependent decrease in their relative cell viability with a low IC50 of 0.28 μM. Conclusions Here we first established that lung adenocarcinoma in people and dogs are genetically and biochemically similar. STA1474 demonstrated biological activity in both canine lung cancer cell lines and tumor-stromal fibroblasts. As significant decreases in relative cell viability can be achieved with nanomolar concentrations of STA-1474, investigation into the clinical efficacy of this drug in canine lung cancer patients is warranted.

Abstract 1322 Recent studies have suggested that deregulated expression of tyrosine phosphatases and the resulting alteration in the phosphorylation status of their substrates play a significant role in the oncogenic function of tyrosine-phosphorylated proteins. Over the past decade we have been studying the tumor suppressive function of protein tyrosine phosphatase receptor-type O (PTPRO). The gene for PTPRO encodes two functional isoforms, full-length form (PTPRO-FL) and a truncated form (PTPROt) that are expressed in a tissue-specific manner. PTPROt is primarily expressed in hematopoietic cells. It is, however, transcriptionally and epigenetically suppressed in CLL-like cell lines and primary CLL [Clin Cancer Res. 2007 Jun 1;13(11):3174–81, Blood. 2011 Dec 1;118(23):6132–40]. We have now shown that PTPROt is also hypermethylated in a discovery set of primary AML samples (n=77) provided by the University hospital Ulm biobank relative to bone marrow from normal controls. Analysis of the AML cell lines Kasumi-1 and ME-1 cells also showed dramatically reduced PTPROt expression relative to THP-1 and MV4-11 cells. Treatment of Kasumi-1 cells with the hypomethylating agent decitabine led to re-activation of PTPROt at both RNA and protein levels. PTPRO CpG island (CGI), methylated in Kasumi-1 cells, became hypomethylated following treatment with decitabine. Similarly, bone marrow samples from elderly AML patients who received decitabine 20 mg/m2/day – 10 days on the OSU 07017 study exhibited hypomethylation and upregulation of PTPROt. To further evaluate the functional significance of hypermethylation and silencing of PTPROt in AML, we then searched for potential kinase substrates of the protein. Kasumi-1 and ME-1 cells (where PTPROt is suppressed) are both CBF cell lines characterized by RUNX1/RUNX1T1 [(8;21) translocation] and CBFB/MYH11 [inv(16)], respectively. While CBF AML patients are generally classified within a more favorable cytogenetic group, those cases harboring mutation in the KIT gene that results in constitutively active receptor tyrosine kinase have a poor outcome. In addition, increased expression of the encoded kinase protein occurs in ∼80% of CBF AML, regardless of KIT mutational status. Kasumi-1 and ME-1 cells are also characterized by mutated (constitutively active) and over-expressed KIT, respectively. Since tyrosine phosphorylation regulates the enzymatic activity and oncogenic function of KIT protein we hypothesized that this kinase could be a substrate of PTPROt and that expression of PTPROt would be critical to maintain control of its activity. Indeed, using in vitro substrate-trapping assay we demonstrated that KIT is a direct substrate of PTPROt. Further, in vivo studies conducted by co-transfecting KITD816V and PTPROt (wild type or catalytic site mutant) in H293T cells showed that phosphorylation of KIT at Y719, a read-out for KIT activity, was reduced when KIT was co-expressed with PTPROt-WT but not with vector control or catalytic site mutant of PTPROt. These observations suggest that suppression of PTPROt in CBF AML with over-expressed or mutated KIT could contribute to the leukemogeneic function of KIT. Given that the epigenetic suppression of PTPROt can be reversed by hypomethylating agent decitabine, it is possible that combination of decitabine and tyrosine kinase inhibitors with or without chemotherapy may represent a novel therapeutic approach in CBF AML. [Supported by grant CA101956] Disclosures: No relevant conflicts of interest to declare.

Protein tyrosine phosphatases (PTPs) and kinases (PTKs) and their corresponding substrates are integrated within elaborate signal transduction networks essential for regulating several important cellular functions including growth, differentiation, cell cycle, gene transcription, the immune response and survival. Defective operation of these networks leads to aberrant tyrosine phosphorylation that contributes to the development of many human diseases including cancer. Importantly, as observed with PTKs, deregulation of PTPs plays a key role in the pathogenesis of these diseases. Thus, PTPs and their substrates represent novel molecular targets for the development of agents in the treatment of these diseases. We have previously demonstrated that the gene encoding the hematopoietic-specific truncated isoform of protein tyrosine phosphatase receptor-type O (PTPROt), a phosphatase demonstrating growth suppressor characteristics, is suppressed in chronic lymphocytic leukemia (Clin Cancer Res. 2007 Jun 1;13(11):3174-81). We have further shown that expression of PTPROt is severely impeded by T-cell leukemia 1 (TCL1), an oncoprotein aberrantly expressed in CLL B-cells (Blood. 2011 Oct 14. [Epub ahead of print]). Additionally, PTPROt plays an important role in regulating B-cell receptor (BCR) signaling (J Cell Biochem. 2010 Jul 1;110(4):846-56; Blood. 2006 Nov 15;108(10):3428-33). To comprehend the physiological relevance of PTPROt we have now generated transgenic (Tg) mice expressing PTPROt in B-cells. These mice live their normal life span and exhibit normal B- and T-cell development. Lyn and SYK kinases, the established substrates of PTPROt, are hypo-phosphorylated in B-lymphocytes from the spleen of PTPROt Tg mice relative to those from NTg mice. Because expression of PTPROt is significantly lower in splenic B-lymphocytes from TCL1 Tg mouse model of CLL relative to splenic B-cells from non-transgenic (NTg) mice (Blood. 2011 Oct 14. [Epub ahead of print]), we crossed the TCL1 Tg and PTPROt Tg mice to determine whether expression of PTPROt will inhibit/delay TCL1-mediated leukemogenesis and utilize this model to elucidate the mechanism of PTPROt function in maintaining normal cell physiology. Preliminary gene expression analysis performed on splenic B-cells from NTg, TCL1 Tg and PTPROt/TCL1 double Tg mice demonstrated that the key positive regulators of cell cycle elevated in the TCL1 Tg mice (relative to the NTg mice) are downregulated in the PTPROt/TCL1 double Tg mice (relative to the TCL1 Tg mice). Further, the spleen size as well as the total number of spleen cells was significantly lower in the PTPROt/TCL1 double Tg group relative to the TCL1 Tg group. We are currently in the process of assessing the function of the altered genes in the TCL1 Tg mouse model as well as in human CLL. [Supported by grant CA101956] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1351. doi:1538-7445.AM2012-1351

We have shown earlier that microRNA (miR)-221 and -222 are upregulated in tamoxifen (TAM) resistant breast cancer cell line MCF-7 (OHTR), and in Her2 positive primary human breast tumors compared to the Her2 negative tumors1. In addition, expression of miR-181b was 3.5-fold higher in OHTR cells compared to the MCF-7 cells and endocrine resistant primary tumors. To determine the underlying mechanism of miR-mediated regulation of TAM resistance, we focused on identification of target genes regulated by these miRs. Bioinformatics search revealed presence of seed sequences for all three miRs on 3’-UTR of TIMP3, a tissue metalloprotease inhibitor. Luciferase assay confirmed TIMP3 to be a direct target of these miRs and was found to be regulated at both RNA and protein levels. In situ hybridization and immunohistochemical analysis demonstrated reciprocal relationship between TIMP3 and miR 221/222 expression in primary human tumors, where invasive ductal carcinomas expressed high level of the miRs and normal or non-invasive cancers expressed TIMP3, but not the miRs. Cell proliferation and wound healing assay demonstrated increased sensitization of the TAM resistant cells to the drug upon ectopic expression of TIMP3. In addition, mouse mammary tumors induced with TIMP3 overexpressing cells were found to be more sensitive to TAM compared to the tumors initiated with control cells. Conversely, knocking down TIMP3 by siRNA reduced sensitivity to TAM, both in cell cultures and in mouse mammary tumors. As TIMP3 is an inhibitor of matrix metalloproteases involved in positive regulation of growth factor signaling, we speculated that reduction in TIMP3 due to high miR-221/222/181 expression could facilitate growth factor signaling in OHTR cells. Indeed, EGF-induced MAPK phosphorylation was significantly higher in OHTR cells compared to MCF-7 cells and miR-221/222 overexpressing MCF-7 cells mimicked this phenomenon. On the contrary, phosphMAPK level was reduced when TIMP3 was overexpressed in OHTR cells and increased upon knocking down of TIMP3 in MCF-7 cells. Similar differences in MAPK phosphorylation was also observed in cells treated with low level estrogen or TAM, agonists for TAM resistant cells. Based on this data we conclude that overexpression of miR-221/222 and -181b facilitates growth factor signaling in TAM resistant cells by downregulating TIMP3. 1. Miller et. al. J. Biol. Chem 2008; 283 (44): 29897 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1154. doi:10.1158/1538-7445.AM2011-1154

The gene for protein tyrosine phosphatase receptor-type O encodes two functional isoforms in a tissue-specific manner. The full-length form is expressed in epithelial cells, particularly in the brain and kidney, whereas the truncated form (PTPROt) is primarily expressed in hematopoietic cells. We have previously demonstrated that PTPROt is a bona fide tumor suppressor (J Biol Chem. 2009 Jan 2;284(1):455-64) and is down-regulated in primary CLL samples (Clin Cancer Res. 2007 Jun 1;13(11):3174-81). Further, studies from two laboratories including ours have shown that Syk and Lyn, two key protein tyrosine kinases involved in B-cell receptor signaling, are substrates of PTPROt, igniting interest in understanding deregulation of this tyrosine phosphatase in CLL. While the PTPRO promoter is methylated, the mechanism of its silencing in the pathogenesis of CLL is unexplored. Here, we utilized the Tcl1 transgenic (Tg) mouse model of CLL and a cell line with TPA-inducible expression of PTPROt to elucidate the mechanism of PTPROt silencing. This study demonstrates that PTPROt is transcriptionally silenced in CD19+ spleen B cells from Tcl1 transgenic (Tg) mouse when compared to wild type mice. As observed with primary human CLL, the methylation of PTPRO CGI (CpG island) is significantly higher in Tcl1 Tg CLL B-lymphocytes than in normal B lymphocytes. Significant suppression of PTPROt is also observed at very early time-point (∼7 wks) in CD19+ spleen B-cells from Tcl1 Tg mice compared to normal spleen B-lymphocytes from age-matched WT mice in the absence of detectable methylation at CGI. This data suggests that transcriptional silencing occurs prior to methylation. To understand further the potential transcriptional mechanism of PTPROt suppression in CLL, the role of AP1 in the expression of this gene was explored. We first demonstrate that AP-1 activation by TPA in U937 cells induces PTPROt expression with concurrent recruitment of c-fos and c-jun to its promoter. PTPROt promoter is also responsive to over- and under-expression of AP-1 confirming the role of AP-1 in PTPROt expression. Next, we demonstrate that Tcl1 can repress PTPROt promoter by altering c-fos expression and c-jun activation state. These findings further substantiate the importance of TCL1 in the pathogenesis of CLL. [Supported, in part, by grants CA101956 and CA086978]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 276. doi:10.1158/1538-7445.AM2011-276

Hepatocellular Carcinoma (HCC) is one of the most prevalent cancers in the world and the third leading cause of cancer-related death. Although hepatitis C and fatty liver disease appear to be the main risk factors in most western countries other factors including alcohol, tobacco, diabetes, and other metabolic disorders can also increase the likelihood of HCC development. HCC is believed to develop through a tumor promoting mechanism involving inflammation and oxidative stress. We have previously shown that metallothionein (MT), a ubiquitously expressed free radical scavenger, is dramatically downregulated in human and rodent HCCs. To test the hypothesis that MT protects the liver from carcinogenesis by relieving oxidative stress, MT knockout (MTKO) mice were injected with Diethylnitrosamine (DEN), a potent liver carcinogen. When compared to wild-type DEN treated mice, the MTKO groups exhibited significantly higher (5-fold) incidence and an accelerated rate of hepatocarcinogenesis. Gene expression analysis at the preneoplastic stage revealed a dramatic increase in the levels of Serpine-1 in the MTKO mice, which was validated by real-time PCR. Serpine-1, a tissue glycoprotein and an active suppressor of fibrinolysis through the inhibition of tissue plasminogen activator and urokinase plasminogen activator, is also believed to increase cell migration and angiogenesis. Real-time PCR analysis of the pro-inflammatory cytokines known to induce Serpine-1 revealed substantial upregulation of TNF-α, IL-6, and IL-1β in the MTKO/DEN mice compared to the WT/DEN and untreated controls. Electrophoretic mobility shift assay confirmed differential activation of NF-κB, a downstream effector of pro-inflammatory cytokines and inducer of Serpine-1 expression, in the MTKO/DEN mice at the preneoplastic stage. These data demonstrate that Serpine-1 and NF- κB act in concert at the early stages of hepatocarcinogenesis when free radical scavenging is compromised. These data show the protective role of MT against HCC induction in a mouse model and provide the rationale for an alternate therapeutic strategy in HCC therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 909. doi:10.1158/1538-7445.AM2011-909

Abstract #3023 Background: Breast cancer is the most common malignancy in women, accounting for 31% of all female cancers. Over two-thirds of breast cancers exhibit high concentrations of estrogen receptor, which contribute to tumor growth and progression. Blocking the steroid hormone pathway with tamoxifen and/or oophorectomy has been shown to be effective in this patient population. However, approximately 30% of the breast cancer is resistant to tamoxifen. Recent studies have highlighted the key regulatory roles of microRNAs (miR) in all fundamental cellular processes in animals and plants including primary human cancers. We hypothesized that alteration in the expression of specific miRs in breast cancer could contribute to tamoxifen resistance. Methods: To test this hypothesis, we performed microRNA microarray analysis using MCF-7 cell lines that are either sensitive (parental) or resistant to tamoxifen (4-hydroxy tamoxifen resistant-OHTR). Using Real-time RT-PCR we validated altered expression of the miRs in both the cell culture model and the primary human breast cancer tissues. Cells overexpressing miR-221/222 or p27(kip1) were created by transfection of mammalian expression vectors using Lipofectamine2000 followed by G418 selection. Cell viability upon tamoxifen treatment was measured by MTT assay, extent of apoptosis was monitored by Western blot analysis of PAPR and Caspase cleavage and cell cycle profile was studied using Flow cytometry. Results: Eight miRs were found to be significantly upregulated while seven miRs were significantly down-regulated in the OHTR cells compared to parental MCF-7 cells. The increased expression of three upregulated (miR-221, miR-222 and miR-181) and three downregulated (miR-21, miR-342 and miR-489) miRs was later validated in the cell lines by real-time RT-PCR. In addition, the level of miR-221 and miR-222 was significantly elevated in Her2/neu(+) primary human breast cancer tissues, compared to Her2/neu(-) tissue samples. The Her2/neu expressing tumors are known to be relatively resistant to endocrine therapy. Ectopic expression of miR-221/miR-222 in parental MCF-7 cells rendered the cells more tolerant to tamoxifen than the control cells. The cell cycle inhibitor p27/Kip1, a known target of miR-221/miR-222 was significantly reduced in both OHTR cells and miR-221/222 overexpressing MCF-7 cells, which was consistent with the upregulation of the miRs. Ectopic expression of p27/Kip1 in the resistant OHTR cells enhanced cell death when exposed to tamoxifen. Conclusion: This study has revealed a specific miR signature for the tamoxifen-resistant breast cancer and an important cell cycle inhibitor target of the altered miR, which could be used as a prognostic marker for the drug-resistant breast cancer. Further studies of other miRs differentially expressed in tamoxifen resistant cell lines, will help us not only in identifying such patients but also may serve as a therapeutic target in the future. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3023.