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Low molecular weight protein tyrosine phosphatase (LMWPTP) upregulation mediates malignant potential in colorectal cancer.

Hoekstra E, Kodach LL, Das AM, Ruela-de-Sousa RR, Ferreira CV, Hardwick JC, van der Woude CJ, Peppelenbosch MP, Ten Hagen TL, Fuhler GM - Oncotarget (2015)

Bottom Line: In the present study, we show that LMWPTP expression is not only significantly increased in colorectal cancer (CRC), but also follows a step-wise increase in different levels of dysplasia.Chemical inhibition of LMWPTP significantly reduces CRC growth.In conclusion, this study shows that LMWPTP is not only overexpressed in colorectal cancer, but it is correlated with the malignant potential of this cancer, suggesting that this phosphatase may act as a predictive biomaker of CRC stage and represents a rational novel target in the treatment of this disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology and Hepatology, Erasmus MC, University Medical Center Rotterdam, 's Gravendijkwa, Rotterdam, The Netherlands.

ABSTRACT
Phosphatases have long been regarded as tumor suppressors, however there is emerging evidence for a tumor initiating role for some phosphatases in several forms of cancer. Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP; acid phosphatase 1 [ACP1]) is an 18 kDa enzyme that influences the phosphorylation of signaling pathway mediators involved in cancer and is thus postulated to be a tumor-promoting enzyme, but neither unequivocal clinical evidence nor convincing mechanistic actions for a role of LMWPTP have been identified. In the present study, we show that LMWPTP expression is not only significantly increased in colorectal cancer (CRC), but also follows a step-wise increase in different levels of dysplasia. Chemical inhibition of LMWPTP significantly reduces CRC growth. Furthermore, downregulation of LMWPTP in CRC leads to a reduced migration ability in both 2D- and 3D-migration assays, and sensitizes tumor cells to the chemotherapeutic agent 5-FU. In conclusion, this study shows that LMWPTP is not only overexpressed in colorectal cancer, but it is correlated with the malignant potential of this cancer, suggesting that this phosphatase may act as a predictive biomaker of CRC stage and represents a rational novel target in the treatment of this disease.

No MeSH data available.


Related in: MedlinePlus

Effects of chemical inhibition and knockdown of LMWPTP on the oncogenic potential of colorectal cancer cells(A) Immunoprecipitated phosphatases (LMWPTP, PTP1B and SHP-1) from HCT116 lysates were incubated with the only known inhibitor of LMWPTP, PLP, resulting in reduction of LMWPTP phosphatase activity in LMWPTP precipitates, while enzymatic activity of the two other PNPP phosphatases remain unaffected upon PLP treatment. (B) Treatment of colorectal cancer cell lines (HCT116 and CACO-2) with PLP dose-dependently reduced viable cell numbers as determined by MTT assay, while non-transformed cell lines (EPC2-hTERT and PBMCs) are hardly effected. (C) Propidium-iodine staining of CACO-2 cells followed by FACS analysis shows that PLP treatment induces a G0/G1 cell cycle arrest. (D) PLP treatment of CRC cells results in apoptosis, as shown by FACS analysis with Annexin V/PI staining on CACO-2 cells treated either with 500 uM PLP or vehicle. (E) Stably transfected cell lines were created, by lentiviral transfection of HCT116 and CACO-2 cells with shRNA against LMWPTP. This resulted in cell lines harboring a knockdown of approximately 50% as compared to non-target transfected control cells. (F) LMWPTP knockdown does not affect overall cell proliferation as shown by MTT assay.
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Figure 3: Effects of chemical inhibition and knockdown of LMWPTP on the oncogenic potential of colorectal cancer cells(A) Immunoprecipitated phosphatases (LMWPTP, PTP1B and SHP-1) from HCT116 lysates were incubated with the only known inhibitor of LMWPTP, PLP, resulting in reduction of LMWPTP phosphatase activity in LMWPTP precipitates, while enzymatic activity of the two other PNPP phosphatases remain unaffected upon PLP treatment. (B) Treatment of colorectal cancer cell lines (HCT116 and CACO-2) with PLP dose-dependently reduced viable cell numbers as determined by MTT assay, while non-transformed cell lines (EPC2-hTERT and PBMCs) are hardly effected. (C) Propidium-iodine staining of CACO-2 cells followed by FACS analysis shows that PLP treatment induces a G0/G1 cell cycle arrest. (D) PLP treatment of CRC cells results in apoptosis, as shown by FACS analysis with Annexin V/PI staining on CACO-2 cells treated either with 500 uM PLP or vehicle. (E) Stably transfected cell lines were created, by lentiviral transfection of HCT116 and CACO-2 cells with shRNA against LMWPTP. This resulted in cell lines harboring a knockdown of approximately 50% as compared to non-target transfected control cells. (F) LMWPTP knockdown does not affect overall cell proliferation as shown by MTT assay.

Mentions: As our data in primary CRC indicates that an increased LMWPTP expression may contribute to tumor progression, we wondered whether inhibition of LMWPTP might reverse any of the oncogenic processes involved. The only LMWPTP inhibitor available to date is PLP, an active derivative of Vitamin B6, which has been shown to inhibit LMWPTP activity by interacting with the Asp129 site [16]. To confirm the effectiveness and selectivity of this compound, we precipitated LMWPTP and two other phosphatases with activity towards the substrate PNPP (SHP-1 and PTP1B) from CRC cells and demonstrated that the phosphatase activity of LMWPTP was indeed decreased in the presence of PLP, while the activity of SHP-1 and PTP1B were unaffected (Figure 3A and Supplementary Figure S2A). Next, we treated CRC cells (CACO-2 and HCT116), as well as the non-transformed human gastrointestinal epithelial cell line EPC2-hTERT and freshly isolated PBMCs, with this compound and assessed cell viability. As shown in Figure 3B, PLP dose-dependently reduced viable cell numbers of the CRC lines, while non-transformed cells are hardly affected by this treatment. PLP induced a G0/G1 cell cycle arrest in the cancer cells (Figure 3C and Supplementary Figure S2C). Furthermore, PLP treatment concomitantly caused apoptosis in CRC cells, as shown by an increased Annexin V staining (Figure 3D and Supplementary Figure S2B). Thus, these data suggest that chemical inhibition of LMWPTP may reduce CRC growth.


Low molecular weight protein tyrosine phosphatase (LMWPTP) upregulation mediates malignant potential in colorectal cancer.

Hoekstra E, Kodach LL, Das AM, Ruela-de-Sousa RR, Ferreira CV, Hardwick JC, van der Woude CJ, Peppelenbosch MP, Ten Hagen TL, Fuhler GM - Oncotarget (2015)

Effects of chemical inhibition and knockdown of LMWPTP on the oncogenic potential of colorectal cancer cells(A) Immunoprecipitated phosphatases (LMWPTP, PTP1B and SHP-1) from HCT116 lysates were incubated with the only known inhibitor of LMWPTP, PLP, resulting in reduction of LMWPTP phosphatase activity in LMWPTP precipitates, while enzymatic activity of the two other PNPP phosphatases remain unaffected upon PLP treatment. (B) Treatment of colorectal cancer cell lines (HCT116 and CACO-2) with PLP dose-dependently reduced viable cell numbers as determined by MTT assay, while non-transformed cell lines (EPC2-hTERT and PBMCs) are hardly effected. (C) Propidium-iodine staining of CACO-2 cells followed by FACS analysis shows that PLP treatment induces a G0/G1 cell cycle arrest. (D) PLP treatment of CRC cells results in apoptosis, as shown by FACS analysis with Annexin V/PI staining on CACO-2 cells treated either with 500 uM PLP or vehicle. (E) Stably transfected cell lines were created, by lentiviral transfection of HCT116 and CACO-2 cells with shRNA against LMWPTP. This resulted in cell lines harboring a knockdown of approximately 50% as compared to non-target transfected control cells. (F) LMWPTP knockdown does not affect overall cell proliferation as shown by MTT assay.
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Related In: Results  -  Collection

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Figure 3: Effects of chemical inhibition and knockdown of LMWPTP on the oncogenic potential of colorectal cancer cells(A) Immunoprecipitated phosphatases (LMWPTP, PTP1B and SHP-1) from HCT116 lysates were incubated with the only known inhibitor of LMWPTP, PLP, resulting in reduction of LMWPTP phosphatase activity in LMWPTP precipitates, while enzymatic activity of the two other PNPP phosphatases remain unaffected upon PLP treatment. (B) Treatment of colorectal cancer cell lines (HCT116 and CACO-2) with PLP dose-dependently reduced viable cell numbers as determined by MTT assay, while non-transformed cell lines (EPC2-hTERT and PBMCs) are hardly effected. (C) Propidium-iodine staining of CACO-2 cells followed by FACS analysis shows that PLP treatment induces a G0/G1 cell cycle arrest. (D) PLP treatment of CRC cells results in apoptosis, as shown by FACS analysis with Annexin V/PI staining on CACO-2 cells treated either with 500 uM PLP or vehicle. (E) Stably transfected cell lines were created, by lentiviral transfection of HCT116 and CACO-2 cells with shRNA against LMWPTP. This resulted in cell lines harboring a knockdown of approximately 50% as compared to non-target transfected control cells. (F) LMWPTP knockdown does not affect overall cell proliferation as shown by MTT assay.
Mentions: As our data in primary CRC indicates that an increased LMWPTP expression may contribute to tumor progression, we wondered whether inhibition of LMWPTP might reverse any of the oncogenic processes involved. The only LMWPTP inhibitor available to date is PLP, an active derivative of Vitamin B6, which has been shown to inhibit LMWPTP activity by interacting with the Asp129 site [16]. To confirm the effectiveness and selectivity of this compound, we precipitated LMWPTP and two other phosphatases with activity towards the substrate PNPP (SHP-1 and PTP1B) from CRC cells and demonstrated that the phosphatase activity of LMWPTP was indeed decreased in the presence of PLP, while the activity of SHP-1 and PTP1B were unaffected (Figure 3A and Supplementary Figure S2A). Next, we treated CRC cells (CACO-2 and HCT116), as well as the non-transformed human gastrointestinal epithelial cell line EPC2-hTERT and freshly isolated PBMCs, with this compound and assessed cell viability. As shown in Figure 3B, PLP dose-dependently reduced viable cell numbers of the CRC lines, while non-transformed cells are hardly affected by this treatment. PLP induced a G0/G1 cell cycle arrest in the cancer cells (Figure 3C and Supplementary Figure S2C). Furthermore, PLP treatment concomitantly caused apoptosis in CRC cells, as shown by an increased Annexin V staining (Figure 3D and Supplementary Figure S2B). Thus, these data suggest that chemical inhibition of LMWPTP may reduce CRC growth.

Bottom Line: In the present study, we show that LMWPTP expression is not only significantly increased in colorectal cancer (CRC), but also follows a step-wise increase in different levels of dysplasia.Chemical inhibition of LMWPTP significantly reduces CRC growth.In conclusion, this study shows that LMWPTP is not only overexpressed in colorectal cancer, but it is correlated with the malignant potential of this cancer, suggesting that this phosphatase may act as a predictive biomaker of CRC stage and represents a rational novel target in the treatment of this disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology and Hepatology, Erasmus MC, University Medical Center Rotterdam, 's Gravendijkwa, Rotterdam, The Netherlands.

ABSTRACT
Phosphatases have long been regarded as tumor suppressors, however there is emerging evidence for a tumor initiating role for some phosphatases in several forms of cancer. Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP; acid phosphatase 1 [ACP1]) is an 18 kDa enzyme that influences the phosphorylation of signaling pathway mediators involved in cancer and is thus postulated to be a tumor-promoting enzyme, but neither unequivocal clinical evidence nor convincing mechanistic actions for a role of LMWPTP have been identified. In the present study, we show that LMWPTP expression is not only significantly increased in colorectal cancer (CRC), but also follows a step-wise increase in different levels of dysplasia. Chemical inhibition of LMWPTP significantly reduces CRC growth. Furthermore, downregulation of LMWPTP in CRC leads to a reduced migration ability in both 2D- and 3D-migration assays, and sensitizes tumor cells to the chemotherapeutic agent 5-FU. In conclusion, this study shows that LMWPTP is not only overexpressed in colorectal cancer, but it is correlated with the malignant potential of this cancer, suggesting that this phosphatase may act as a predictive biomaker of CRC stage and represents a rational novel target in the treatment of this disease.

No MeSH data available.


Related in: MedlinePlus