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HOXA1 is overexpressed in oral squamous cell carcinomas and its expression is correlated with poor prognosis.

Bitu CC, Destro MF, Carrera M, da Silva SD, Graner E, Kowalski LP, Soares FA, Coletta RD - BMC Cancer (2012)

Bottom Line: In particular, HOXA1, which has been described as one of the HOX members that plays an important role in tumorigenesis, was significantly more expressed in OSCCs compared to healthy oral mucosas.Further analysis demonstrated that overexpression of HOXA1 in HaCAT human epithelial cells promotes proliferation, whereas downregulation of HOXA1 in human OSCC cells (SCC9 cells) decreases it.A high number of HOXA1-positive cells was significantly associated with T stage, N stage, tumor differentiation and proliferative potential of the tumors, and was predictive of poor survival.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oral Diagnosis, School of Dentistry, State University of Campinas, CP 52, CEP 13414-018 Piracicaba, São Paulo, Brazil. coletta@fop.unicamp.br

ABSTRACT

Background: HOX genes encode homeodomain-containing transcription factors involved in the regulation of cellular proliferation and differentiation during embryogenesis. However, members of this family demonstrated oncogenic properties in some malignancies. The present study investigated whether genes of the HOXA cluster play a role in oral cancer.

Methods: In order to identify differentially expressed HOXA genes, duplex RT-PCR in oral samples from healthy mucosa and squamous cell carcinoma was used. The effects of HOXA1 on proliferation, apoptosis, adhesion, invasion, epithelial-mesenchymal transition (EMT) and anchorage-independent growth were assessed in cells with up- and down-regulation of HOXA1. Immunohistochemical analysis using a tissue microarray (TMA) containing 127 oral squamous cell carcinomas (OSCC) was performed to determine the prognostic role of HOXA1 expression.

Results: We showed that transcripts of HOXA genes are more abundant in OSCC than in healthy oral mucosa. In particular, HOXA1, which has been described as one of the HOX members that plays an important role in tumorigenesis, was significantly more expressed in OSCCs compared to healthy oral mucosas. Further analysis demonstrated that overexpression of HOXA1 in HaCAT human epithelial cells promotes proliferation, whereas downregulation of HOXA1 in human OSCC cells (SCC9 cells) decreases it. Enforced HOXA1 expression in HaCAT cells was not capable of modulating other events related to tumorigenesis, including apoptosis, adhesion, invasion, EMT and anchorage-independent growth. A high number of HOXA1-positive cells was significantly associated with T stage, N stage, tumor differentiation and proliferative potential of the tumors, and was predictive of poor survival. In multivariate analysis, HOXA1 was an independent prognostic factor for OSCC patients (HR: 2.68; 95% CI: 1.59-2.97; p = 0.026).

Conclusion: Our findings indicate that HOXA1 may contribute to oral carcinogenesis by increasing tumor cell proliferation, and suggest that HOXA1 expression might be helpful as a prognostic marker for patients with OSCC.

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Overexpression of HOXA1 induces cellular proliferation. (A) Representative duplex RT-PCR and western blot analysis of HOXA1 in HaCAT-Control and HaCAT-HOXA1 transfectants, revealing an increase in HOXA1 levels in overexpressing clones. Assays measuring BrdU incorporation (B) and Ki67 expression (C) demonstrated that HOXA1 overexpressing cell lines have a statistically significant increase in proliferation as compared to control cells (for BrdU index p < 0.01 between groups, and for Ki67 index p < 0.05 between groups). The labeling index of BrdU and Ki67 corresponds to the mean percentage of positive cells of 3 experiments for each cell line.
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Figure 4: Overexpression of HOXA1 induces cellular proliferation. (A) Representative duplex RT-PCR and western blot analysis of HOXA1 in HaCAT-Control and HaCAT-HOXA1 transfectants, revealing an increase in HOXA1 levels in overexpressing clones. Assays measuring BrdU incorporation (B) and Ki67 expression (C) demonstrated that HOXA1 overexpressing cell lines have a statistically significant increase in proliferation as compared to control cells (for BrdU index p < 0.01 between groups, and for Ki67 index p < 0.05 between groups). The labeling index of BrdU and Ki67 corresponds to the mean percentage of positive cells of 3 experiments for each cell line.

Mentions: To better understand the role of HOXA1 in the events that control oral tumorigenesis, we overexpressed HOXA1 in the HaCAT epithelial cell line, which shows very low levels of its expression. Stable HOXA1 and control transfectants were generated and examined for HOXA1 mRNA and protein levels. Three stable HOXA1-overexpressing clones (HaCAT-HOXA1) and 3 control clones (HaCAT-Control) were chosen for further analyses (Figure 4A). HOXA1-overexpressing cells showed a statistically significant increase in proliferation when compared to HaCAT-Control cells, as assessed by both BrdU incorporation (p < 0.01) and Ki67 expression (p < 0.05) indexes (Figure 4B and 4C). In order to confirm these findings, we next knocked down HOXA1 levels in SCC9 cells, which have high endogenous levels of HOXA1. Either specific-stranded RNA oligonucleotides against HOXA1 or negative RNA control were transfected into SCC9 cells. When HOXA1-specific oligonucleotides were used, a rapid downregulation of HOXA1 mRNA was detected (Figure 5A). The decrease in HOXA1 levels resulted in a concomitant decrease in proliferation. Both cell proliferation assays (BrdU incorporation and Ki67 expression) showed a statistically significant decrease in proliferation when HOXA1 was downregulated with siRNA (p < 0.01 for BrdU index and p < 0.05 for Ki67 index; Figure 5B and 5C).


HOXA1 is overexpressed in oral squamous cell carcinomas and its expression is correlated with poor prognosis.

Bitu CC, Destro MF, Carrera M, da Silva SD, Graner E, Kowalski LP, Soares FA, Coletta RD - BMC Cancer (2012)

Overexpression of HOXA1 induces cellular proliferation. (A) Representative duplex RT-PCR and western blot analysis of HOXA1 in HaCAT-Control and HaCAT-HOXA1 transfectants, revealing an increase in HOXA1 levels in overexpressing clones. Assays measuring BrdU incorporation (B) and Ki67 expression (C) demonstrated that HOXA1 overexpressing cell lines have a statistically significant increase in proliferation as compared to control cells (for BrdU index p < 0.01 between groups, and for Ki67 index p < 0.05 between groups). The labeling index of BrdU and Ki67 corresponds to the mean percentage of positive cells of 3 experiments for each cell line.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351375&req=5

Figure 4: Overexpression of HOXA1 induces cellular proliferation. (A) Representative duplex RT-PCR and western blot analysis of HOXA1 in HaCAT-Control and HaCAT-HOXA1 transfectants, revealing an increase in HOXA1 levels in overexpressing clones. Assays measuring BrdU incorporation (B) and Ki67 expression (C) demonstrated that HOXA1 overexpressing cell lines have a statistically significant increase in proliferation as compared to control cells (for BrdU index p < 0.01 between groups, and for Ki67 index p < 0.05 between groups). The labeling index of BrdU and Ki67 corresponds to the mean percentage of positive cells of 3 experiments for each cell line.
Mentions: To better understand the role of HOXA1 in the events that control oral tumorigenesis, we overexpressed HOXA1 in the HaCAT epithelial cell line, which shows very low levels of its expression. Stable HOXA1 and control transfectants were generated and examined for HOXA1 mRNA and protein levels. Three stable HOXA1-overexpressing clones (HaCAT-HOXA1) and 3 control clones (HaCAT-Control) were chosen for further analyses (Figure 4A). HOXA1-overexpressing cells showed a statistically significant increase in proliferation when compared to HaCAT-Control cells, as assessed by both BrdU incorporation (p < 0.01) and Ki67 expression (p < 0.05) indexes (Figure 4B and 4C). In order to confirm these findings, we next knocked down HOXA1 levels in SCC9 cells, which have high endogenous levels of HOXA1. Either specific-stranded RNA oligonucleotides against HOXA1 or negative RNA control were transfected into SCC9 cells. When HOXA1-specific oligonucleotides were used, a rapid downregulation of HOXA1 mRNA was detected (Figure 5A). The decrease in HOXA1 levels resulted in a concomitant decrease in proliferation. Both cell proliferation assays (BrdU incorporation and Ki67 expression) showed a statistically significant decrease in proliferation when HOXA1 was downregulated with siRNA (p < 0.01 for BrdU index and p < 0.05 for Ki67 index; Figure 5B and 5C).

Bottom Line: In particular, HOXA1, which has been described as one of the HOX members that plays an important role in tumorigenesis, was significantly more expressed in OSCCs compared to healthy oral mucosas.Further analysis demonstrated that overexpression of HOXA1 in HaCAT human epithelial cells promotes proliferation, whereas downregulation of HOXA1 in human OSCC cells (SCC9 cells) decreases it.A high number of HOXA1-positive cells was significantly associated with T stage, N stage, tumor differentiation and proliferative potential of the tumors, and was predictive of poor survival.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oral Diagnosis, School of Dentistry, State University of Campinas, CP 52, CEP 13414-018 Piracicaba, São Paulo, Brazil. coletta@fop.unicamp.br

ABSTRACT

Background: HOX genes encode homeodomain-containing transcription factors involved in the regulation of cellular proliferation and differentiation during embryogenesis. However, members of this family demonstrated oncogenic properties in some malignancies. The present study investigated whether genes of the HOXA cluster play a role in oral cancer.

Methods: In order to identify differentially expressed HOXA genes, duplex RT-PCR in oral samples from healthy mucosa and squamous cell carcinoma was used. The effects of HOXA1 on proliferation, apoptosis, adhesion, invasion, epithelial-mesenchymal transition (EMT) and anchorage-independent growth were assessed in cells with up- and down-regulation of HOXA1. Immunohistochemical analysis using a tissue microarray (TMA) containing 127 oral squamous cell carcinomas (OSCC) was performed to determine the prognostic role of HOXA1 expression.

Results: We showed that transcripts of HOXA genes are more abundant in OSCC than in healthy oral mucosa. In particular, HOXA1, which has been described as one of the HOX members that plays an important role in tumorigenesis, was significantly more expressed in OSCCs compared to healthy oral mucosas. Further analysis demonstrated that overexpression of HOXA1 in HaCAT human epithelial cells promotes proliferation, whereas downregulation of HOXA1 in human OSCC cells (SCC9 cells) decreases it. Enforced HOXA1 expression in HaCAT cells was not capable of modulating other events related to tumorigenesis, including apoptosis, adhesion, invasion, EMT and anchorage-independent growth. A high number of HOXA1-positive cells was significantly associated with T stage, N stage, tumor differentiation and proliferative potential of the tumors, and was predictive of poor survival. In multivariate analysis, HOXA1 was an independent prognostic factor for OSCC patients (HR: 2.68; 95% CI: 1.59-2.97; p = 0.026).

Conclusion: Our findings indicate that HOXA1 may contribute to oral carcinogenesis by increasing tumor cell proliferation, and suggest that HOXA1 expression might be helpful as a prognostic marker for patients with OSCC.

Show MeSH
Related in: MedlinePlus