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Upregulation of KLHDC4 Predicts a Poor Prognosis in Human Nasopharyngeal Carcinoma.

Lian YF, Yuan J, Cui Q, Feng QS, Xu M, Bei JX, Zeng YX, Feng L - PLoS ONE (2016)

Bottom Line: Consistently, KLHDC4 knockout cell-derived xenografts also showed elevated cleaved caspase-3 and PARP but reduced Ki-67 staining.In conclusion, our results suggest that KLHDC4 promotes NPC oncogenesis by suppressing cellular apoptosis.Thus, KLHDC4 may serve as a prognosis biomarker and a potential therapeutic target for NPC.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Research, Sun Yat-sen University Cancer Center, State Key Laboratory Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, China.

ABSTRACT
Kelch proteins are implicated in the pathogenesis of many human diseases, including cancer. Nasopharyngeal carcinoma (NPC) is a rare malignancy in most countries, but prevalent in southern China and certain areas of Southeast Asia. In this study, we identified Kelch Domain Containing 4 (KLHDC4), an orphan member of the kelch repeat superfamily, as a prognosis marker for NPC. We examined the expression of KLHDC4 in 168 NPC cases by immunohistochemical staining and found a substantially higher level of KLHDC4 in NPC biopsies compared to adjacent normal nasopharyngeal mucosa. KLHDC4 expression was significantly related to the T classification (P <0.05), N classification (P <0.05) and total staging (P <0.01) in NPC, and patients with higher KLHDC4 expression had poorer overall (P <0.01) and metastasis-free survival (P <0.05) rates. Knockout (KO) of KLHDC4 via CRISPR/Cas9-mediated gene editing in NPC cell line dramatically inhibited cell proliferation, colony formation in soft agar and tumor formation in nude mice. In addition, cell migration and invasion were also impaired by KLHDC4 depletion as revealed by wound healing and Transwell assay. Mechanically, loss of KLHDC4 markedly induced spontaneous apoptosis in NPC cells, as evidenced by increased levels of cleaved caspase-3 and cleaved PARP. Consistently, KLHDC4 knockout cell-derived xenografts also showed elevated cleaved caspase-3 and PARP but reduced Ki-67 staining. In conclusion, our results suggest that KLHDC4 promotes NPC oncogenesis by suppressing cellular apoptosis. Thus, KLHDC4 may serve as a prognosis biomarker and a potential therapeutic target for NPC.

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Loss of KLHDC4 induces apoptosis in NPC cells.(A) Control and KLHDC4 KO cells were stained with DAPI and photographed by fluorescence microscopy under fluorescence and light fields. Left panel: representative images; the arrows indicated the condensed or fragmented nuclear and multiblebbing cells. Right panels: quantification of cells with condensed nuclear per field. **P <0.01. (B) Representative flow cytometric analysis of apoptotic CNE2 control and KLHDC4 KO cells stained for annexin V-FITC/PI under both non-treated and cis-platin treated conditions. The results are summarized in the right panel. Bars denote the S.E.M. (C) Western blot analysis of the expression of cleaved caspase-3, cleaved PARP and GAPDH, as the loading control, under both non-treated and cis-platin treated conditions. (D) Immunohistochemical analysis of KLHDC4, cleaved caspase-3, cleaved PARP, and Ki-67 expression in xenografts generated from CNE2 control and KLHDC4 KO cells. Left panels: representative images; Scale bars: 100 μm. Right panels: quantification of average percentage of cells with positive staining per field; CC3: cleaved caspase-3; CP: cleaved PARP. ***P <0.001.
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pone.0152820.g005: Loss of KLHDC4 induces apoptosis in NPC cells.(A) Control and KLHDC4 KO cells were stained with DAPI and photographed by fluorescence microscopy under fluorescence and light fields. Left panel: representative images; the arrows indicated the condensed or fragmented nuclear and multiblebbing cells. Right panels: quantification of cells with condensed nuclear per field. **P <0.01. (B) Representative flow cytometric analysis of apoptotic CNE2 control and KLHDC4 KO cells stained for annexin V-FITC/PI under both non-treated and cis-platin treated conditions. The results are summarized in the right panel. Bars denote the S.E.M. (C) Western blot analysis of the expression of cleaved caspase-3, cleaved PARP and GAPDH, as the loading control, under both non-treated and cis-platin treated conditions. (D) Immunohistochemical analysis of KLHDC4, cleaved caspase-3, cleaved PARP, and Ki-67 expression in xenografts generated from CNE2 control and KLHDC4 KO cells. Left panels: representative images; Scale bars: 100 μm. Right panels: quantification of average percentage of cells with positive staining per field; CC3: cleaved caspase-3; CP: cleaved PARP. ***P <0.001.

Mentions: Apoptosis has long been known to be associated with tumor growth and metastasis. Induction of apoptosis is also a therapeutic strategy for the treatment of NPC. To define the underlying molecular mechanism by which KLHDC4 inhibits NPC progression, we first examined morphological and biochemical changes in cells after loss of KLHDC4. The control CNE2 cells exhibited a normal cobblestone epithelial morphology and apoptotic cells were hardly observed. In contrast, around 10% of the KLHDC4 KO cells displayed the typical characteristics of apoptosis, such as cell shrinkage, membrane blebbing, and nuclear condensation (Fig 5A). Compared to the control cells, annexin V/PI staining showed a significant increase in apoptotic cells (annexin V positive/PI negative and annexin V positive/PI positive) among the KLHDC4 KO population under both non-treated and cis-platin treated conditions (Fig 5B). In addition, the activity of caspase-3 and PARP, as demonstrated by the appearance of their cleaved forms, were markedly elevated in KLHDC4 KO cells, indicating that the cell death induced by KLHDC4 deficiency via a route of apoptosis (Fig 5C). In agreement with the aforementioned in vitro results, IHC of the KLHDC4 knockout xenograft biopsies showed a substantial increase in the number of cleaved caspase-3 and cleaved PARP-positive cells and decreased Ki-67 positive cells (Fig 5D), further supporting the role of KLHDC4 in promoting NPC progression through regulating apoptosis.


Upregulation of KLHDC4 Predicts a Poor Prognosis in Human Nasopharyngeal Carcinoma.

Lian YF, Yuan J, Cui Q, Feng QS, Xu M, Bei JX, Zeng YX, Feng L - PLoS ONE (2016)

Loss of KLHDC4 induces apoptosis in NPC cells.(A) Control and KLHDC4 KO cells were stained with DAPI and photographed by fluorescence microscopy under fluorescence and light fields. Left panel: representative images; the arrows indicated the condensed or fragmented nuclear and multiblebbing cells. Right panels: quantification of cells with condensed nuclear per field. **P <0.01. (B) Representative flow cytometric analysis of apoptotic CNE2 control and KLHDC4 KO cells stained for annexin V-FITC/PI under both non-treated and cis-platin treated conditions. The results are summarized in the right panel. Bars denote the S.E.M. (C) Western blot analysis of the expression of cleaved caspase-3, cleaved PARP and GAPDH, as the loading control, under both non-treated and cis-platin treated conditions. (D) Immunohistochemical analysis of KLHDC4, cleaved caspase-3, cleaved PARP, and Ki-67 expression in xenografts generated from CNE2 control and KLHDC4 KO cells. Left panels: representative images; Scale bars: 100 μm. Right panels: quantification of average percentage of cells with positive staining per field; CC3: cleaved caspase-3; CP: cleaved PARP. ***P <0.001.
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pone.0152820.g005: Loss of KLHDC4 induces apoptosis in NPC cells.(A) Control and KLHDC4 KO cells were stained with DAPI and photographed by fluorescence microscopy under fluorescence and light fields. Left panel: representative images; the arrows indicated the condensed or fragmented nuclear and multiblebbing cells. Right panels: quantification of cells with condensed nuclear per field. **P <0.01. (B) Representative flow cytometric analysis of apoptotic CNE2 control and KLHDC4 KO cells stained for annexin V-FITC/PI under both non-treated and cis-platin treated conditions. The results are summarized in the right panel. Bars denote the S.E.M. (C) Western blot analysis of the expression of cleaved caspase-3, cleaved PARP and GAPDH, as the loading control, under both non-treated and cis-platin treated conditions. (D) Immunohistochemical analysis of KLHDC4, cleaved caspase-3, cleaved PARP, and Ki-67 expression in xenografts generated from CNE2 control and KLHDC4 KO cells. Left panels: representative images; Scale bars: 100 μm. Right panels: quantification of average percentage of cells with positive staining per field; CC3: cleaved caspase-3; CP: cleaved PARP. ***P <0.001.
Mentions: Apoptosis has long been known to be associated with tumor growth and metastasis. Induction of apoptosis is also a therapeutic strategy for the treatment of NPC. To define the underlying molecular mechanism by which KLHDC4 inhibits NPC progression, we first examined morphological and biochemical changes in cells after loss of KLHDC4. The control CNE2 cells exhibited a normal cobblestone epithelial morphology and apoptotic cells were hardly observed. In contrast, around 10% of the KLHDC4 KO cells displayed the typical characteristics of apoptosis, such as cell shrinkage, membrane blebbing, and nuclear condensation (Fig 5A). Compared to the control cells, annexin V/PI staining showed a significant increase in apoptotic cells (annexin V positive/PI negative and annexin V positive/PI positive) among the KLHDC4 KO population under both non-treated and cis-platin treated conditions (Fig 5B). In addition, the activity of caspase-3 and PARP, as demonstrated by the appearance of their cleaved forms, were markedly elevated in KLHDC4 KO cells, indicating that the cell death induced by KLHDC4 deficiency via a route of apoptosis (Fig 5C). In agreement with the aforementioned in vitro results, IHC of the KLHDC4 knockout xenograft biopsies showed a substantial increase in the number of cleaved caspase-3 and cleaved PARP-positive cells and decreased Ki-67 positive cells (Fig 5D), further supporting the role of KLHDC4 in promoting NPC progression through regulating apoptosis.

Bottom Line: Consistently, KLHDC4 knockout cell-derived xenografts also showed elevated cleaved caspase-3 and PARP but reduced Ki-67 staining.In conclusion, our results suggest that KLHDC4 promotes NPC oncogenesis by suppressing cellular apoptosis.Thus, KLHDC4 may serve as a prognosis biomarker and a potential therapeutic target for NPC.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Research, Sun Yat-sen University Cancer Center, State Key Laboratory Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, China.

ABSTRACT
Kelch proteins are implicated in the pathogenesis of many human diseases, including cancer. Nasopharyngeal carcinoma (NPC) is a rare malignancy in most countries, but prevalent in southern China and certain areas of Southeast Asia. In this study, we identified Kelch Domain Containing 4 (KLHDC4), an orphan member of the kelch repeat superfamily, as a prognosis marker for NPC. We examined the expression of KLHDC4 in 168 NPC cases by immunohistochemical staining and found a substantially higher level of KLHDC4 in NPC biopsies compared to adjacent normal nasopharyngeal mucosa. KLHDC4 expression was significantly related to the T classification (P <0.05), N classification (P <0.05) and total staging (P <0.01) in NPC, and patients with higher KLHDC4 expression had poorer overall (P <0.01) and metastasis-free survival (P <0.05) rates. Knockout (KO) of KLHDC4 via CRISPR/Cas9-mediated gene editing in NPC cell line dramatically inhibited cell proliferation, colony formation in soft agar and tumor formation in nude mice. In addition, cell migration and invasion were also impaired by KLHDC4 depletion as revealed by wound healing and Transwell assay. Mechanically, loss of KLHDC4 markedly induced spontaneous apoptosis in NPC cells, as evidenced by increased levels of cleaved caspase-3 and cleaved PARP. Consistently, KLHDC4 knockout cell-derived xenografts also showed elevated cleaved caspase-3 and PARP but reduced Ki-67 staining. In conclusion, our results suggest that KLHDC4 promotes NPC oncogenesis by suppressing cellular apoptosis. Thus, KLHDC4 may serve as a prognosis biomarker and a potential therapeutic target for NPC.

Show MeSH
Related in: MedlinePlus