Elmer Hoekstra

The role of protein tyrosine phosphatases in colorectal cancer

Colorectal cancer is one of the most common oncogenic diseases in the Western world. Several cancer associated cellular pathways have been identified, in which protein phosphorylation and dephosphorylation, especially on tyrosine residues, are one of most abundant regulatory mechanisms. The balance between these processes is under tight control by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Aberrant activity of oncogenic PTKs is present in a large portion of human cancers. Because of the counteracting role of PTPs on phosphorylation-based activation of signal pathways, it has long been thought that PTPs must act as tumor suppressors. This dogma is now being challenged, with recent evidence showing that dephosphorylation events induced by some PTPs may actually stimulate tumor formation. As such, PTPs might form a novel attractive target for anticancer therapy. In this review, we summarize the action of different PTPs, the consequences of their altered expression in colorectal cancer, and their potential as target for the treatment of this deadly disease.

β-Catenin signaling dosage dictates tissue-specific tumor predisposition in Apc-driven cancer

Apc-driven tumor formation in patients and Apc-mutant mouse models is generally attributed to increased levels of β-catenin signaling. We and others have proposed that a specific level of β-catenin signaling is required to successfully initiate tumor formation, and that each tissue prefers different dosages of signaling. This is illustrated by APC genotype−tumor phenotype correlations in cancer patients, and by the different tumor phenotypes displayed by different Apc-mutant mouse models. Apc1638N mice, associated with intermediate β-catenin signaling, characteristically develop intestinal tumors (<10) and extra-intestinal tumors, including cysts and desmoids. Apc1572T mice associated with lower levels of β-catenin signaling are free of intestinal tumors, but instead develop mammary tumors. Although the concept of β-catenin signaling dosage and its impact on tumor growth among tissues is gaining acceptance, it has not been formally proven. Additionally, alternative explanations for Apc-driven tumor formation have been proposed. To obtain direct evidence for the dominant role of β-catenin dosage in tumor formation and tissue-specific tumor predisposition, we crossed Apc1638N mice with heterozygous β-catenin knockout mice, thereby reducing β-catenin levels. Whereas all the Apc1638N;Ctnnb1+/+ mice developed gastrointestinal tumors, none were present in the Apc1638N;Ctnnb1−/+ mice. Incidence of other Apc1638N-associated lesions, including desmoids and cysts, was strongly reduced as well. Interestingly, Apc1638N;Ctnnb1−/+ females showed an increased incidence of mammary tumors, which are normally rarely observed in Apc1638N mice, and the histological composition of the tumors resembled that of Apc1572T-related tumors. Hereby, we provide in vivo genetic evidence confirming the dominant role of β-catenin dosage in tumor formation and in dictating tumor predisposition among tissues in Apc-driven cancer.

Increased PTP1B expression and phosphatase activity in colorectal cancer results in a more invasive phenotype and worse patient outcome

Cell signaling is dependent on the balance between phosphorylation of proteins by kinases and dephosphorylation by phosphatases. This balance if often disrupted in colorectal cancer (CRC), leading to increased cell proliferation and invasion. For many years research has focused on the role of kinases as potential oncogenes in cancer, while phosphatases were commonly assumed to be tumor suppressive. However, this dogma is currently changing as phosphatases have also been shown to induce cancer growth. One of these phosphatases is protein tyrosine phosphatase 1B (PTP1B). Here we report that the expression of PTP1B is increased in colorectal cancer as compared to normal tissue, and that the intrinsic enzymatic activity of the protein is also enhanced. This suggests a role for PTP1B phosphatase activity in CRC formation and progression. Furthermore, we found that increased PTP1B expression is correlated to a worse patient survival and is an independent prognostic marker for overall survival and disease free survival. Knocking down PTP1B in CRC cell lines results in a less invasive phenotype with lower adhesion, migration and proliferation capabilities. Together, these results suggest that inhibition of PTP1B activity is a promising new target in the treatment of colorectal cancer and the prevention of metastasis.

Dichotomal effect of space flight-associated microgravity on stress-activated protein kinases in innate immunity

Space flight strongly moderates human immunity but is in general well tolerated. Elucidation of the mechanisms by which zero gravity interacts with human immunity may provide clues for developing rational avenues to deal with exaggerated immune responses, e.g. as in autoimmune disease. Using two sounding rockets and one manned Soyuz launch, the influence of space flight on immunological signal transduction provoked by lipopolysaccharide (LPS) stimulation was investigated in freshly isolated peripheral blood monocytes and was compared to samples obtained from on-board centrifuge-loaded 1 g controls. The effect of microgravity on immunological signal transduction is highly specific, since LPS dependent Jun-N-terminal kinase activation is impaired in the 0 g condition, while the corresponding LPS dependent activation of p38 MAP kinase remains unaffected. Thus our results identify Jun-N-terminal kinase as a relevant target in immunity for microgravity and support using Jun-N-terminal kinase specific inhibitors for combating autoimmune disease.

Lipid phosphatase SHIP2 functions as oncogene in colorectal cancer by regulating PKB activation

Colorectal cancer (CRC) is the second most common cause of cancer-related death, encouraging the search for novel therapeutic targets affecting tumor cell proliferation and migration. These cellular processes are under tight control of two opposing groups of enzymes; kinases and phosphatases. Aberrant activity of kinases is observed in many forms of cancer and as phosphatases counteract such “oncogenic” kinases, it is generally assumed that phosphatases function as tumor suppressors. However, emerging evidence suggests that the lipid phosphatase SH2-domain-containing 5 inositol phosphatase (SHIP2), encoded by the INPPL1 gene, may act as an oncogene. Just like the well-known tumor suppressor gene Phosphatase and Tensin Homolog (PTEN) it hydrolyses phosphatidylinositol (3,4,5) triphosphate (PI(3,4,5)P3). However, unlike PTEN, the reaction product is PI(3,4)P2, which is required for full activation of the downstream protein kinase B (PKB/Akt), suggesting that SHIP2, in contrast to PTEN, could have a tumor initiating role through PKB activation. In this work, we investigated the role of SHIP2 in colorectal cancer. We found that SHIP2 and INPPL1 expression is increased in colorectal cancer tissue in comparison to adjacent normal tissue, and this is correlated with decreased patient survival. Moreover, SHIP2 is more active in colorectal cancer tissue, suggesting that SHIP2 can induce oncogenesis in colonic epithelial cells. Furthermore, in vitro experiments performed on colorectal cancer cell lines shows an oncogenic role for SHIP2, by enhancing chemoresistance, cell migration, and cell invasion. Together, these data indicate that SHIP2 expression contributes to the malignant potential of colorectal cancer, providing a possible target in the fight against this devastating disease.

Sa1987 Lipid Phosphatase SHIP2 Functions As Oncogene in Colorectal Cancer by Regulating PKB Activation

Colorectal cancer (CRC) is the second most common cause of cancer-related death, encouraging the search for novel therapeutic targets affecting tumor cell proliferation and migration. These cellular processes are under tight control of two opposing groups of enzymes; kinases and phosphatases. Aberrant activity of kinases is observed in many forms of cancer and as phosphatases counteract such “oncogenic” kinases, it is generally assumed that phosphatases function as tumor suppressors. However, emerging evidence suggests that the lipid phosphatase SH2-domain-containing 5 inositol phosphatase (SHIP2), encoded by the INPPL1 gene, may act as an oncogene. Just like the well-known tumor suppressor gene Phosphatase and Tensin Homolog (PTEN) it hydrolyses phosphatidylinositol (3,4,5) triphosphate (PI(3,4,5)P3). However, unlike PTEN, the reaction product is PI(3,4)P2, which is required for full activation of the downstream protein kinase B (PKB/Akt), suggesting that SHIP2, in contrast to PTEN, could have a tumor initiating role through PKB activation. In this work, we investigated the role of SHIP2 in colorectal cancer. We found that SHIP2 and INPPL1 expression is increased in colorectal cancer tissue in comparison to adjacent normal tissue, and this is correlated with decreased patient survival. Moreover, SHIP2 is more active in colorectal cancer tissue, suggesting that SHIP2 can induce oncogenesis in colonic epithelial cells. Furthermore, in vitro experiments performed on colorectal cancer cell lines shows an oncogenic role for SHIP2, by enhancing chemoresistance, cell migration, and cell invasion. Together, these data indicate that SHIP2 expression contributes to the malignant potential of colorectal cancer, providing a possible target in the fight against this devastating disease.

Tu1663 Protein Tyrosine Phosphatase 1B (PTP1B) Expression and Phosphatase Activity Are Increased in Primary Colorectal Cancer Which Leads to a More Invasive Phenotype in CRC Cells

Background: Cell signaling is dependent on the balance between phosphorylation of proteins by kinases and dephosphorylation by phosphatases. This balance if often disrupted in colorectal cancer (CRC), leading to increased cell proliferation and invasion. For many years research has focused on the role of kinases as potential oncogenes in cancer, while phosphatases were commonly assumed to be tumor suppressive. However, this dogma is currently changing as phosphatases have also been shown to stimulate cancer growth. One of these phosphatases is protein tyrosine phosphatase 1B (PTP1B). The aim of this study was to investigate the expression and phosphatase activity of PTP1B in CRC, and elucidate its effects on cellular functions and signaling. Methods: PTP1B expression was analysed by immunohistochemistry on microsections from biopsies of dysplastic polyps (n=6), adenocarcinoma (n=9) and control (inactive ulcerative colitis, n=5), as well as by western blotting of paired freshly frozen CRC and normal adjacent tissue (n=11). Phosphatase activity was also assessed in these latter samples by immunoprecipitating PTP1B under saturating conditions, followed by a phosphatase activity assay using PNPP as substrate. To investigate the effects of PTP1B on proliferation, adhesion, migration, and elucidate its downstream targets, we manipulated the PTP1B expression in vitro by lentiviral transduction of HCT116 and Caco2 cells with 2 different shRNAs against PTP1B. Results: PTP1B expression in intestinal epithelial cells (IECs) is low in normal colon (14% positive; mean intensity 0.2±0.1) and increases from dysplasia to carcinoma (100% positive IECs; with mean intensity rising from 1.4±0.3 to 1.8±0.3 respectively). These results were confirmed by western blot analysis. The intrinsic enzymatic activity of the PTP1B protein is significantly increased in cancer compared to adjacent normal tissue (mean OD 1.0 in CRC compared to 0.2 in normal tissue) (p=0.001). Knocking down PTP1B in CRC cells reduced the phosphorylation of the mitogenic kinase ERK by approximately 50%, and decreased mRNA levels of downstream targets involved in proliferation; i.e. c-MYC and CyclinD1. Furthermore, adhesion, migration, and proliferation were significantly reduced in PTP1B knockdown cells. Conclusion: Not only is the expression of PTP1B is increased in colorectal cancer as compared to normal tissue, but strikingly, the intrinsic enzymatic activity of the protein is also enhanced, suggesting a role for PTP1B phosphatase activity in CRC progression. Knocking down PTP1B in CRC cell lines results in a less invasive phenotype with lower adhesion, migration and proliferation capabilities, by interfering in the RAS-RAF-ERK pathway. Together these results suggest that inhibition of PTP1B activity is a promising new target in the treatment of colorectal cancer and the prevention of metastasis.