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XPC inhibits NSCLC cell proliferation and migration by enhancing E-Cadherin expression.

Cui T, Srivastava AK, Han C, Yang L, Zhao R, Zou N, Qu M, Duan W, Zhang X, Wang QE - Oncotarget (2015)

Bottom Line: Deletion of XPC is associated with early stages of human lung carcinogenesis, and reduced XPC mRNA levels predict poor patient outcome for non-small cell lung cancer (NSCLC).Restoration of E-Cadherin in these cells suppressed XPC knockdown-induced cell growth both in vitro and in vivo.Mechanistic studies showed that the loss of XPC repressed E-Cadherin expression by activating the ERK pathway and upregulating Snail expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology, The Ohio State University Wexner Medical Center, Columbus, OH, USA.

ABSTRACT
Xeroderma pigmentosum complementation group C (XPC) protein is an important DNA damage recognition factor in nucleotide excision repair. Deletion of XPC is associated with early stages of human lung carcinogenesis, and reduced XPC mRNA levels predict poor patient outcome for non-small cell lung cancer (NSCLC). However, the mechanisms linking loss of XPC expression and poor prognosis in lung cancer are still unclear. Here, we report evidence that XPC silencing drives proliferation and migration of NSCLC cells by down-regulating E-Cadherin. XPC knockdown enhanced proliferation and migration while decreasing E-Cadherin expression in NSCLC cells with an epithelial phenotype. Restoration of E-Cadherin in these cells suppressed XPC knockdown-induced cell growth both in vitro and in vivo. Mechanistic studies showed that the loss of XPC repressed E-Cadherin expression by activating the ERK pathway and upregulating Snail expression. Our findings indicate that XPC silencing-induced reduction of E-Cadherin expression contributes, at least in part, to the poor outcome of NSCLC patients with low XPC expression.

No MeSH data available.


Related in: MedlinePlus

E-Cadherin is able to reverse the effect of XPC downregulation on cell growth(A-C) A549 cells were transiently transfected with siXPC alone, or simultaneously with E-Cadherin expressing vectors. The protein levels of XPC and E-Cadherin were determined using immunoblotting. The intensity of each band was quantified using ImageJ and normalized to Actin and then to their corresponding siCtrl-transfected cells (A). Cell growth was determined using methylene blue staining. n = 5, bar: SD, **, P < 0.01 compared with A549-Ctrl cells; ##, P < 0.01 compared with A549-siXPC cells (B). The transwell migration assay was conducted to quantify the migrated cells. n = 3, bar: SD, **, P < 0.01 compared with A549-Ctrl cells; ##, P < 0.01 compared with A549-siXPC cells (C). (D,E) E-Cadherin expressing vectors were stably transfected into A549-shXPC-c2 cells, and two cell clones were selected (shXPC-c2+E-Cad-c1, and shXPC-c2+E-Cad-c1). The protein level of E-Cadherin was determined using immunoblotting. The intensity of each band was quantified using ImageJ and normalized to Lamin B and then to shCtrl-transfected cells (D). Cell growth was determined using methylene blue staining. n = 5, bar: SD, *, P < 0.05 compared with A549-shCtrl cells (E). (F-H) Xenografts were generated by A549 cells (shCtrl), A549 cells with stable XPC knockdown (shXPC-c2), and two clones of A549-shXPC-c2 cells with stable overexpression of E-Cadherin (E-Cad-c1, and E-Cad-c2). Tumor sizes were measured every two days (F). Tumors were removed from mice after 22 days (G), and weighed (H). n = 10, bar: SD, **, P < 0.01 compared with A549-shCtrl; #, P < 0.05, ##, P < 0.01 compared with A549-shXPC-c2.
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Figure 3: E-Cadherin is able to reverse the effect of XPC downregulation on cell growth(A-C) A549 cells were transiently transfected with siXPC alone, or simultaneously with E-Cadherin expressing vectors. The protein levels of XPC and E-Cadherin were determined using immunoblotting. The intensity of each band was quantified using ImageJ and normalized to Actin and then to their corresponding siCtrl-transfected cells (A). Cell growth was determined using methylene blue staining. n = 5, bar: SD, **, P < 0.01 compared with A549-Ctrl cells; ##, P < 0.01 compared with A549-siXPC cells (B). The transwell migration assay was conducted to quantify the migrated cells. n = 3, bar: SD, **, P < 0.01 compared with A549-Ctrl cells; ##, P < 0.01 compared with A549-siXPC cells (C). (D,E) E-Cadherin expressing vectors were stably transfected into A549-shXPC-c2 cells, and two cell clones were selected (shXPC-c2+E-Cad-c1, and shXPC-c2+E-Cad-c1). The protein level of E-Cadherin was determined using immunoblotting. The intensity of each band was quantified using ImageJ and normalized to Lamin B and then to shCtrl-transfected cells (D). Cell growth was determined using methylene blue staining. n = 5, bar: SD, *, P < 0.05 compared with A549-shCtrl cells (E). (F-H) Xenografts were generated by A549 cells (shCtrl), A549 cells with stable XPC knockdown (shXPC-c2), and two clones of A549-shXPC-c2 cells with stable overexpression of E-Cadherin (E-Cad-c1, and E-Cad-c2). Tumor sizes were measured every two days (F). Tumors were removed from mice after 22 days (G), and weighed (H). n = 10, bar: SD, **, P < 0.01 compared with A549-shCtrl; #, P < 0.05, ##, P < 0.01 compared with A549-shXPC-c2.

Mentions: Downregulation of E-Cadherin is regarded as a trigger for cancer invasion and metastasis [24, 16]. Therefore, we sought to determine whether reduced expression of E-Cadherin contributes to XPC deficiency-promoted NSCLC cell proliferation. We transfected siXPC alone or together with E-Cadherin expressing vectors into A549 cells, in which XPC was knocked down, and E-Cadherin was either downregulated, or upregulated (Figure 3A). The siXPC-transfected A549 cells with re-expression of E-Cadherin exhibited decreased cell proliferation and migration compared to those transfected with XPC siRNA alone (Figures 3B-C), indicating that E-Cadherin is able to reverse the effect of XPC downregulation on cell growth. To examine the role of E-Cadherin in XPC-mediated cell growth inhibition in vivo, we transfected E-Cadherin into A549 cells that already had stable XPC knockdown (A549-shXPC-c2), generated E-Cadherin stably expressing A549-shXPC cell lines (A549-shXPC-c2-E-Cad-c1 and A549-shXPC-c2-E-Cad-c2) (Figure 3D), and confirmed the function of E-Cadherin overexpression in the inhibition of cell growth in A549-shXPC-c2 cells (Figure 3E). These cells were used to generate xenografts in Athymic nude mice and tumor growth dynamics were recorded. Histological analysis showed typical epithelial cancer cell morphology, and no difference was noted among the xenografts generated by different cell lines (Supplementary Figure 6). Tumor xenografts initiated with A549-shXPC cells grew faster than those derived from XPC-proficient A549-shCtrl cells. However, overexpression of E-Cadherin in A549-shXPC cells significantly retarded the growth of these cells in vivo (Figure 3F). The tumor volumes and weights at the end of the experiments also significantly decreased in two E-Cadherin overexpressing groups compared to the A549-shXPC group (Figures 3G-H). These data suggest that E-Cadherin is a downstream effector in the process of XPC-induced inhibition of NSCLC cell proliferation.


XPC inhibits NSCLC cell proliferation and migration by enhancing E-Cadherin expression.

Cui T, Srivastava AK, Han C, Yang L, Zhao R, Zou N, Qu M, Duan W, Zhang X, Wang QE - Oncotarget (2015)

E-Cadherin is able to reverse the effect of XPC downregulation on cell growth(A-C) A549 cells were transiently transfected with siXPC alone, or simultaneously with E-Cadherin expressing vectors. The protein levels of XPC and E-Cadherin were determined using immunoblotting. The intensity of each band was quantified using ImageJ and normalized to Actin and then to their corresponding siCtrl-transfected cells (A). Cell growth was determined using methylene blue staining. n = 5, bar: SD, **, P < 0.01 compared with A549-Ctrl cells; ##, P < 0.01 compared with A549-siXPC cells (B). The transwell migration assay was conducted to quantify the migrated cells. n = 3, bar: SD, **, P < 0.01 compared with A549-Ctrl cells; ##, P < 0.01 compared with A549-siXPC cells (C). (D,E) E-Cadherin expressing vectors were stably transfected into A549-shXPC-c2 cells, and two cell clones were selected (shXPC-c2+E-Cad-c1, and shXPC-c2+E-Cad-c1). The protein level of E-Cadherin was determined using immunoblotting. The intensity of each band was quantified using ImageJ and normalized to Lamin B and then to shCtrl-transfected cells (D). Cell growth was determined using methylene blue staining. n = 5, bar: SD, *, P < 0.05 compared with A549-shCtrl cells (E). (F-H) Xenografts were generated by A549 cells (shCtrl), A549 cells with stable XPC knockdown (shXPC-c2), and two clones of A549-shXPC-c2 cells with stable overexpression of E-Cadherin (E-Cad-c1, and E-Cad-c2). Tumor sizes were measured every two days (F). Tumors were removed from mice after 22 days (G), and weighed (H). n = 10, bar: SD, **, P < 0.01 compared with A549-shCtrl; #, P < 0.05, ##, P < 0.01 compared with A549-shXPC-c2.
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Figure 3: E-Cadherin is able to reverse the effect of XPC downregulation on cell growth(A-C) A549 cells were transiently transfected with siXPC alone, or simultaneously with E-Cadherin expressing vectors. The protein levels of XPC and E-Cadherin were determined using immunoblotting. The intensity of each band was quantified using ImageJ and normalized to Actin and then to their corresponding siCtrl-transfected cells (A). Cell growth was determined using methylene blue staining. n = 5, bar: SD, **, P < 0.01 compared with A549-Ctrl cells; ##, P < 0.01 compared with A549-siXPC cells (B). The transwell migration assay was conducted to quantify the migrated cells. n = 3, bar: SD, **, P < 0.01 compared with A549-Ctrl cells; ##, P < 0.01 compared with A549-siXPC cells (C). (D,E) E-Cadherin expressing vectors were stably transfected into A549-shXPC-c2 cells, and two cell clones were selected (shXPC-c2+E-Cad-c1, and shXPC-c2+E-Cad-c1). The protein level of E-Cadherin was determined using immunoblotting. The intensity of each band was quantified using ImageJ and normalized to Lamin B and then to shCtrl-transfected cells (D). Cell growth was determined using methylene blue staining. n = 5, bar: SD, *, P < 0.05 compared with A549-shCtrl cells (E). (F-H) Xenografts were generated by A549 cells (shCtrl), A549 cells with stable XPC knockdown (shXPC-c2), and two clones of A549-shXPC-c2 cells with stable overexpression of E-Cadherin (E-Cad-c1, and E-Cad-c2). Tumor sizes were measured every two days (F). Tumors were removed from mice after 22 days (G), and weighed (H). n = 10, bar: SD, **, P < 0.01 compared with A549-shCtrl; #, P < 0.05, ##, P < 0.01 compared with A549-shXPC-c2.
Mentions: Downregulation of E-Cadherin is regarded as a trigger for cancer invasion and metastasis [24, 16]. Therefore, we sought to determine whether reduced expression of E-Cadherin contributes to XPC deficiency-promoted NSCLC cell proliferation. We transfected siXPC alone or together with E-Cadherin expressing vectors into A549 cells, in which XPC was knocked down, and E-Cadherin was either downregulated, or upregulated (Figure 3A). The siXPC-transfected A549 cells with re-expression of E-Cadherin exhibited decreased cell proliferation and migration compared to those transfected with XPC siRNA alone (Figures 3B-C), indicating that E-Cadherin is able to reverse the effect of XPC downregulation on cell growth. To examine the role of E-Cadherin in XPC-mediated cell growth inhibition in vivo, we transfected E-Cadherin into A549 cells that already had stable XPC knockdown (A549-shXPC-c2), generated E-Cadherin stably expressing A549-shXPC cell lines (A549-shXPC-c2-E-Cad-c1 and A549-shXPC-c2-E-Cad-c2) (Figure 3D), and confirmed the function of E-Cadherin overexpression in the inhibition of cell growth in A549-shXPC-c2 cells (Figure 3E). These cells were used to generate xenografts in Athymic nude mice and tumor growth dynamics were recorded. Histological analysis showed typical epithelial cancer cell morphology, and no difference was noted among the xenografts generated by different cell lines (Supplementary Figure 6). Tumor xenografts initiated with A549-shXPC cells grew faster than those derived from XPC-proficient A549-shCtrl cells. However, overexpression of E-Cadherin in A549-shXPC cells significantly retarded the growth of these cells in vivo (Figure 3F). The tumor volumes and weights at the end of the experiments also significantly decreased in two E-Cadherin overexpressing groups compared to the A549-shXPC group (Figures 3G-H). These data suggest that E-Cadherin is a downstream effector in the process of XPC-induced inhibition of NSCLC cell proliferation.

Bottom Line: Deletion of XPC is associated with early stages of human lung carcinogenesis, and reduced XPC mRNA levels predict poor patient outcome for non-small cell lung cancer (NSCLC).Restoration of E-Cadherin in these cells suppressed XPC knockdown-induced cell growth both in vitro and in vivo.Mechanistic studies showed that the loss of XPC repressed E-Cadherin expression by activating the ERK pathway and upregulating Snail expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology, The Ohio State University Wexner Medical Center, Columbus, OH, USA.

ABSTRACT
Xeroderma pigmentosum complementation group C (XPC) protein is an important DNA damage recognition factor in nucleotide excision repair. Deletion of XPC is associated with early stages of human lung carcinogenesis, and reduced XPC mRNA levels predict poor patient outcome for non-small cell lung cancer (NSCLC). However, the mechanisms linking loss of XPC expression and poor prognosis in lung cancer are still unclear. Here, we report evidence that XPC silencing drives proliferation and migration of NSCLC cells by down-regulating E-Cadherin. XPC knockdown enhanced proliferation and migration while decreasing E-Cadherin expression in NSCLC cells with an epithelial phenotype. Restoration of E-Cadherin in these cells suppressed XPC knockdown-induced cell growth both in vitro and in vivo. Mechanistic studies showed that the loss of XPC repressed E-Cadherin expression by activating the ERK pathway and upregulating Snail expression. Our findings indicate that XPC silencing-induced reduction of E-Cadherin expression contributes, at least in part, to the poor outcome of NSCLC patients with low XPC expression.

No MeSH data available.


Related in: MedlinePlus