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Expression of HER-2 affects patient survival and paclitaxel sensitivity in endometrial cancer.

Mori N, Kyo S, Nakamura M, Hashimoto M, Maida Y, Mizumoto Y, Takakura M, Ohno S, Kiyono T, Inoue M - Br. J. Cancer (2010)

Bottom Line: Kaplan-Meier analysis revealed that high HER-2 expression was a factor that negatively influenced the progression-free and overall survival rate (P<0.05), and multivariate analysis showed high HER-2 expression to be an independent prognostic factor.Subsequently, we performed in vitro knockdown analysis to investigate the linkage between HER-2 expression and PI3K-AKT pathways.As the PI3K-AKT pathway is known to have crucial roles in anticancer drug sensitivity, we examined the involvement of HER-2 in sensitivity to paclitaxel.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Kanazawa University Graduate School of Medical Science, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8641, Japan.

ABSTRACT

Background: Disabled phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen-activated protein kinase/extracellular signal-regulated kinase signalling is involved in endometrial carcinogenesis, and there is evidence that expression of epidermal growth factor receptor (EGFR) family members has a role in such intracellular signalling pathways. This study analysed the prognostic impact of EGFR family expression in endometrial cancer in relation to PI3K-AKT and MAPK-ERK signalling, as well as drug sensitivity.

Methods and results: Immunohistochemical analysis using 63 surgical specimens of endometrioid-type endometrial cancers revealed that EGFR, human epidermal growth factor receptor (HER)-2 and HER-4 were expressed in 25 (39.7%) of 63, 26 (41.3%) of 63 and 31 (49.2%) of 63 tumours, respectively. Gene amplification of HER-2 was observed in 2 of 26 patients with high HER-2 expression. Kaplan-Meier analysis revealed that high HER-2 expression was a factor that negatively influenced the progression-free and overall survival rate (P<0.05), and multivariate analysis showed high HER-2 expression to be an independent prognostic factor. Subsequently, we performed in vitro knockdown analysis to investigate the linkage between HER-2 expression and PI3K-AKT pathways. Short interfering RNA (siRNA)-based knockdown of HER-2 in endometrial cancer cells led to a significant reduction in phosphorylated AKT (p-AKT) expression, indicating the existence of a HER-2/PI3K-AKT axis. As the PI3K-AKT pathway is known to have crucial roles in anticancer drug sensitivity, we examined the involvement of HER-2 in sensitivity to paclitaxel. Short interfering RNA-based knockdown of HER-2 conferred increased sensitivity to paclitaxel in endometrial cancer cells, attenuating the induction of p-AKT on paclitaxel stimulation, which was cancelled by inactivating AKT by the introduction of a dominant-negative form.

Conclusion: HER-2 is a significant prognostic factor of endometrioid-type endometrial cancer, as well as a key molecule that affects paclitaxel sensitivity by HER-2 interaction with the PI3K-AKT pathway.

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Inactivation of AKT cancels the effect of HER-2 on paclitaxel sensitivity. Endometrial immortalised EM-E6/E7/TERT cells (A) or those with the dominant-negative form of AKT (B) were incubated with siRNA against HER-2 (siHER-2) or scrambled siRNA (siNC) for 48 h. Cells were then treated with 10 n of paclitaxel for an additional 48 h, followed by the WST-1 assay to examine cell viability. The viability of untreated cells incubated with siNC was set as the control level (100%) and the percentage of cell viability in each cell type was normalised relative to the untreated control. Each experiment was performed in triplicate in three independent experiments. Columns, mean; bars, ±s.d. *P<0.05.
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fig5: Inactivation of AKT cancels the effect of HER-2 on paclitaxel sensitivity. Endometrial immortalised EM-E6/E7/TERT cells (A) or those with the dominant-negative form of AKT (B) were incubated with siRNA against HER-2 (siHER-2) or scrambled siRNA (siNC) for 48 h. Cells were then treated with 10 n of paclitaxel for an additional 48 h, followed by the WST-1 assay to examine cell viability. The viability of untreated cells incubated with siNC was set as the control level (100%) and the percentage of cell viability in each cell type was normalised relative to the untreated control. Each experiment was performed in triplicate in three independent experiments. Columns, mean; bars, ±s.d. *P<0.05.

Mentions: There is accumulating evidence that the PI3K–AKT pathway is critically involved in drug resistance to chemotherapies of various types of cancers by activation of survival signals. Linkage of HER-2 to p-AKT expression prompted us to examine the involvement of HER-2 expression in the chemosensitivity of endometrial cancer cells. We therefore sought to test the sensitivity to paclitaxel, a key drug for endometrial cancer cells. First, preparatory experiments were conducted to test the efficacy of paclitaxel against Ishikawa, HEC1A and immortalised endometrial epithelial cells, in which cells were treated with 1–1000 n of paclitaxel and cell viability was measured by WST-1 assay. We found that 10 or 100 n was an ideal concentration to elicit specific effects of paclitaxel in each cell type examined (data not shown). On the basis of these findings, all three cell lines were treated with or without HER-2 siRNA and exposed to 10 n of paclitaxel for 48 h. Cell viability was then examined by WST-1 assay. As shown in Figures 4 and 5A, HER-2 knockdown significantly augmented the cytotoxic effect of paclitaxel in each cell type.


Expression of HER-2 affects patient survival and paclitaxel sensitivity in endometrial cancer.

Mori N, Kyo S, Nakamura M, Hashimoto M, Maida Y, Mizumoto Y, Takakura M, Ohno S, Kiyono T, Inoue M - Br. J. Cancer (2010)

Inactivation of AKT cancels the effect of HER-2 on paclitaxel sensitivity. Endometrial immortalised EM-E6/E7/TERT cells (A) or those with the dominant-negative form of AKT (B) were incubated with siRNA against HER-2 (siHER-2) or scrambled siRNA (siNC) for 48 h. Cells were then treated with 10 n of paclitaxel for an additional 48 h, followed by the WST-1 assay to examine cell viability. The viability of untreated cells incubated with siNC was set as the control level (100%) and the percentage of cell viability in each cell type was normalised relative to the untreated control. Each experiment was performed in triplicate in three independent experiments. Columns, mean; bars, ±s.d. *P<0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2966616&req=5

fig5: Inactivation of AKT cancels the effect of HER-2 on paclitaxel sensitivity. Endometrial immortalised EM-E6/E7/TERT cells (A) or those with the dominant-negative form of AKT (B) were incubated with siRNA against HER-2 (siHER-2) or scrambled siRNA (siNC) for 48 h. Cells were then treated with 10 n of paclitaxel for an additional 48 h, followed by the WST-1 assay to examine cell viability. The viability of untreated cells incubated with siNC was set as the control level (100%) and the percentage of cell viability in each cell type was normalised relative to the untreated control. Each experiment was performed in triplicate in three independent experiments. Columns, mean; bars, ±s.d. *P<0.05.
Mentions: There is accumulating evidence that the PI3K–AKT pathway is critically involved in drug resistance to chemotherapies of various types of cancers by activation of survival signals. Linkage of HER-2 to p-AKT expression prompted us to examine the involvement of HER-2 expression in the chemosensitivity of endometrial cancer cells. We therefore sought to test the sensitivity to paclitaxel, a key drug for endometrial cancer cells. First, preparatory experiments were conducted to test the efficacy of paclitaxel against Ishikawa, HEC1A and immortalised endometrial epithelial cells, in which cells were treated with 1–1000 n of paclitaxel and cell viability was measured by WST-1 assay. We found that 10 or 100 n was an ideal concentration to elicit specific effects of paclitaxel in each cell type examined (data not shown). On the basis of these findings, all three cell lines were treated with or without HER-2 siRNA and exposed to 10 n of paclitaxel for 48 h. Cell viability was then examined by WST-1 assay. As shown in Figures 4 and 5A, HER-2 knockdown significantly augmented the cytotoxic effect of paclitaxel in each cell type.

Bottom Line: Kaplan-Meier analysis revealed that high HER-2 expression was a factor that negatively influenced the progression-free and overall survival rate (P<0.05), and multivariate analysis showed high HER-2 expression to be an independent prognostic factor.Subsequently, we performed in vitro knockdown analysis to investigate the linkage between HER-2 expression and PI3K-AKT pathways.As the PI3K-AKT pathway is known to have crucial roles in anticancer drug sensitivity, we examined the involvement of HER-2 in sensitivity to paclitaxel.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Kanazawa University Graduate School of Medical Science, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8641, Japan.

ABSTRACT

Background: Disabled phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen-activated protein kinase/extracellular signal-regulated kinase signalling is involved in endometrial carcinogenesis, and there is evidence that expression of epidermal growth factor receptor (EGFR) family members has a role in such intracellular signalling pathways. This study analysed the prognostic impact of EGFR family expression in endometrial cancer in relation to PI3K-AKT and MAPK-ERK signalling, as well as drug sensitivity.

Methods and results: Immunohistochemical analysis using 63 surgical specimens of endometrioid-type endometrial cancers revealed that EGFR, human epidermal growth factor receptor (HER)-2 and HER-4 were expressed in 25 (39.7%) of 63, 26 (41.3%) of 63 and 31 (49.2%) of 63 tumours, respectively. Gene amplification of HER-2 was observed in 2 of 26 patients with high HER-2 expression. Kaplan-Meier analysis revealed that high HER-2 expression was a factor that negatively influenced the progression-free and overall survival rate (P<0.05), and multivariate analysis showed high HER-2 expression to be an independent prognostic factor. Subsequently, we performed in vitro knockdown analysis to investigate the linkage between HER-2 expression and PI3K-AKT pathways. Short interfering RNA (siRNA)-based knockdown of HER-2 in endometrial cancer cells led to a significant reduction in phosphorylated AKT (p-AKT) expression, indicating the existence of a HER-2/PI3K-AKT axis. As the PI3K-AKT pathway is known to have crucial roles in anticancer drug sensitivity, we examined the involvement of HER-2 in sensitivity to paclitaxel. Short interfering RNA-based knockdown of HER-2 conferred increased sensitivity to paclitaxel in endometrial cancer cells, attenuating the induction of p-AKT on paclitaxel stimulation, which was cancelled by inactivating AKT by the introduction of a dominant-negative form.

Conclusion: HER-2 is a significant prognostic factor of endometrioid-type endometrial cancer, as well as a key molecule that affects paclitaxel sensitivity by HER-2 interaction with the PI3K-AKT pathway.

Show MeSH
Related in: MedlinePlus