HOXB7 As a Prognostic Factor and Mediator of Colorectal Cancer Progression

Published OnlineFirst April 7, 2011; DOI: 10.1158/1078-0432.CCR-10-2533

Clinical Cancer Human Cancer Biology Research

HOXB7 as a Prognostic Factor and Mediator of Colorectal Cancer Progression

Wen-Ting Liao1,2,3, Dan Jiang1,2,3, Jian Yuan1,2,3, Yan-Mei Cui1,2,3, Xi-Wen Shi1,2,3, Cui-Min Chen1,2,3, Xiu-Wu Bian4, Yong-Jian Deng1,2,3, and Yan-Qing Ding1,2,3

Abstract Purpose: This study was to investigate the clinicopathologic significance and potential role of HOXB7 in the development and progression of colorectal cancer (CRC). Experimental Design: The relationship between HOXB7 expression and clinical characteristics of CRC was analyzed in 224 paraffin-embedded archived CRC specimens by immunohistochemistry (IHC). The effects of HOXB7 on cell growth and proliferation, as well as on tumorigenesis, were examined both in vitro and in vivo, using MTT assay, colony formation assay, cell cycle analysis, soft agar assay, and tumorigenesis in nude mice. Western blotting and real-time reverse transcriptase-PCR were performed to examine the impact of HOXB7 on the PI3K/Akt and MAPK signaling pathways. Results: HOXB7 protein level was significantly correlated with advanced Dukes stage (P < 0.001), T stage (P ¼ 0.012), distant metastasis (P ¼ 0.042), higher proliferation index (P ¼ 0.007) and poor survival of patients (P ¼ 0.005). Enforced expression of HOXB7 in CRC cell lines significantly enhanced cell growth, proliferation and tumorigenesis. Conversely, knockdown of HOXB7 caused an inhibition of cell growth,

proliferation, and tumorigenesis. We also showed that HOXB7 accelerated G0–G1 to S-phase transition concomitantly with upregulation of cyclin D1 and downregulation of p27Kip1. On the contrary, knock-

down of HOXB7 caused G1–S-phase arrest, downregulation of cyclin D1 and upregulation of p27Kip1. Enforced expression of HOXB7 could enhance PI3K/AKT and MAPK pathway activity. Conclusion: Our findings suggest that HOXB7 protein, as a valuable marker of CRC prognosis, plays an important role in the development and progression of human CRC. Clin Cancer Res; 17(11); 3569–78. 2011 AACR.

Introduction epithelial-cell proliferation, apoptosis, differentiation, and survival mechanisms (1–2). About half of the indivi- Colorectal cancer (CRC) is one of the most common duals with locally advanced CRC can be cured by surgery malignancies worldwide. Colorectal carcinogenesis is a and multimodal treatment. Because traditional methods multistep process involving progressive disruption of do not allow precise prediction of prognosis for the patients after surgical removal of the primary tumor, there Authors' Affiliations: 1Department of Pathology, School of Basic Med- is an acute need for biomarkers capable of distinguishing 2 ical Sciences, Southern Medical University; Department of Pathology, patients with poor or good prognosis (3). Nanfang Hospital, Southern Medical University; 3Guangdong Provincial Key Laboratory of Molecular Tumor Pathology, Guangzhou; and 4Insti- The homeobox genes encode a family of transcriptional tute of Pathology and Southwest Cancer Center, Southwest Hospital, factors, which are essential for morphogenesis and differ- Third Military Medical University, Chongqing, People's Republic of China entiation (4, 5). Homeobox genes are categorized into 2 large groups. Homeobox genes of class I, also called HOX Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). genes, contain at least 39 members that are organized in 4 W.-T. Liao, D. Jiang, and Jian Yuan contributed equally to this work. clusters (A, B, C, and D) located on chromosomes 7, 17, 2, and 12, respectively (6). Homeobox genes of class II, Corresponding Authors: Yan-Qing Ding, Department of Pathology and Guangdong Provincial Key Laboratory of Molecular Tumor Pathology, known as divergent homeobox genes (non-HOX), are Southern Medical University, Guangzhou 510515, China; Phone: 86 (20) scattered on different chromosomes (6). Homeobox genes 6164-2148; Fax: 86 (20) 6164-2148; E-mail: [email protected] or Yong-Jian of both classes play key roles in development and carci- Deng, Department of Pathology and Guangdong Provincial Key Labora- tory of Molecular Tumor Pathology, Southern Medical University, Guangz- nogenesis in gastrointestinal tract (7, 8). For instance, Cdx1 hou 510515, China; Phone: 86 (20) 6164-2148; Fax: 86 (20) 6164-2148; and Cdx2, 2 divergent Homeobox genes, are normally E-mail: [email protected] or Xiu-Wu Bian, Institute of Pathology and South- west Cancer Center, Southwest Hospital, Third Military Medical University, expressed in the gut during development and adulthood. Chongqing 400038, People's Republic of China; Phone: 86 (23) 6875- However, expressions of Cdx1 and Cdx2 are often lost in 4431; Fax: 86 (23) 6539-7004; E-mail: [email protected] CRC cell lines and tissues (9). Downregulation of Cdx1 or doi: 10.1158/1078-0432.CCR-10-2533 Cdx2 expression can promote tumor development and 2011 American Association for Cancer Research. invasiveness, whereas overexpression of Cdx2 in CRC cell

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Translational Relevance Vectors construction and retroviral infection The HOXB7 construct was generated by subcloning PCR- HOXB7 is a transcriptional factor that regulates the amplified full-length human HOXB7 cDNA into pLncx2. expression of multiple genes involved in cell growth, For deletion of HOXB7, shRNA sequence (GCTCAG- viability and tissue-specific differentiation. Amplifica- GAACTGACCGCAAAC) was cloned into pSuper-retro- tion or overexpression of HOXB7 is closely associated puro. Retroviral production and infection were performed with transformation, proliferation, and survival of as previously described (26). Stable cell lines expressing tumor cells. However, the biological functions of HOXB7 or shHOXB7 were selected for 10 days with 400 m HOXB7 in the control of colorectal tumorigenesis mg/mL G418 or 0.5 g/mL puromycin, respectively. and tumor progression are largely unknown. This study revealed that expression of HOXB7 was significantly Patient samples correlated with the invasive and aggressive characteris- Paraffin-embedded, archived CRC samples were tics of human CRC and poor survival of patients as well. obtained from 224 patients diagnosed as CRC between We also revealed that HOXB7 overexpression promoted January 2001 and December 2003 at Nanfang Hospital, proliferation and tumorigenic growth of human CRC Southern Medical University. Medical records of the 224 cells, both in vitro and in vivo. The effect of HOXB7 on patients provided information of age, gender, and follow- proliferation and tumorigenic growth of human CRC ing parameters: tumor histology, pathologic stage, Dukes may result from the upregulation of cyclinD1 and the stage, T stage, lymph node metastases, and distant metas- downregulation of p27Kip1 via activation of MAPK and tasis. Survival data were available for all patients. The PI3K-Akt signaling pathways. Our findings suggest that median follow-up time was 57.5 months (range, 2–87 HOXB7 protein, as a valuable marker of CRC prognosis, months). The mean age at diagnosis was 56.4 years (range, plays an important role in the development and pro- 23–86 years). One hundred and twenty-seven patients gression of human CRC. (56.7%) were male. At the end of the follow-up period, distant metastasis was noted in 72 patients. Eight biopsies of CRC tissues and the matched adjacent noncancerous lines can inhibit cell growth and tumorigenesis (10–12). mucosa tissues were frozen and stored in liquid nitrogen On the contrary, HOXB6, B8, C8, and C9 transcripts were until further use. upregulated at various stages of CRC (13). Other researchers found that expressions of HOXA9, HOXB7, and HOXD11 were altered in primary colon cancers and their RNA extraction, RT-PCR and real-time PCR hepatic metastases (14). In short, these findings suggest an Total RNA extraction, RT-PCR and real-time PCR were association between deregulation of HOX gene expression performed as previously described (27). See Supplemen- and CRC progression. tary Methods for details. It has been well documented that HOXB7, a member of class I homeobox, plays an important role in tumorigen- Western blotting esis. First, deregulation of HOXB7 was observed in several Western blotting was performed according to standard kinds of tumors (15–19). Overexpression of HOXB7 was methods as described previously (27), using anti-HOXB7 closely associated with poor prognosis of breast cancer and (Sigma-Aldrich), anti-phospho-ERK, anti-ERK, anti-phos- oral cancer patients (18, 20). Second, HOXB7 facilitated pho-AKT, anti-AKT, anti-phospho-GSK3b, anti-GSK3b, transforming ability in NIH3T3 fibroblasts and in immor- anti-Cyclin D1, and anti-p27 antibodies ((Bioworld Tech- talized breast epithelial cells MCF10A (21, 22). Enforced nology). A mouse monoclonal anti-a-Tubulin antibody HOXB7 promoted invasion, metastasis, angiogenesis, and (Sigma-Aldrich) was used as inner control to confirm equal cell proliferation (17, 18, 20, 23–25). However, biological loading of proteins. functions of HOXB7 in the control of colorectal tumorigenesis and tumor progression have not been well char- acterized. Here, we sought to investigate the potential role Immunohistochemistry of HOXB7 in the development and progression of CRC. Immunohistochemistry (IHC) staining and scoring were done as previously described (27). For details, please see Materials and Methods Supplementary Methods.

Cell cultures Colony formation assays The human CRC cell lines Caco2, Colo205, Ls174t, Cells were trypsinized and plated on 6-well plates (200 DLD-1, HT-29, HCT116, SW480, and SW620 were pur- cells/well) and cultured for 2 weeks. The colonies were chased from American Type Culture Collection. SW620 stained with 1% crystal violet for 30s after fixation with 4% and HT29 were cultured in DMEM medium (Gibco) sup- paraformaldehyde for 5 minutes. The number of colonies, plemented with 10% FBS (PAA). Caco2, Colo205, Ls174t, defined as >50 cells/colony were counted. Three indepen- DLD-1, HT-29, HCT116, and SW480 were cultured in dent experiments were performed. The data was calculated RPMI 1640 medium (Gibco) with 10% FBS (PAA). using paired t test.

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HOXB7 Accelerates Progression of Colorectal Cancer

AB3.0 Figure 1. Expression of HOXB7 2.5 was elevated in CRC. A, 2.0

expression of HOXB7 protein in Colo205 Caco-2 Ls174t DLD1 HT29 HCT116 SW480 SW620 1.5 CRC cell lines. Expression levels HOXB7 1.0 were normalized with a-Tubulin. 0.5 B, expression of HOXB7 mRNA in α-Tubulin expresstion Relative 0 CRC cell lines by Real-time PCR. mRNA of HOXB7 level Expression levels were normalized DLD1 HT29 Caco-2Ls174t for GAPDH. Error bars represent Colo205 HCT116SW480SW620 mean SD calculated from 3 C parallel experiments. C and D, 1N 1T 2N 2T 3N 3T 4N 4T 5N 5T 6N 6T 7N 7T 8N 8T expression of HOXB7 protein (C) and mRNA (D) in each of the HOXB7 primary CRC (T) and adjacent α-Tubulin noncancerous tissues (N) paired from the same patient by western blotting (C) and RT-PCR (D). E, DE real-time PCR analysis of HOXB7 25 expression in each of the T and N 20 tissues. GADPH was used as an 1N 1T 2N 2T 3N 3T 4N 4T 5N 5T 6N 6T 7N 7T 8N 8T 15 internal control. Columns, mean HOXB7 10 5 from 3 parallel experiments; GAPDH Fold Increase Fold 0 bars, SD. 12 34 5 678 Samples

Soft agar assays and eosin according to standard protocols. Sections were Cells (1 104) were resuspended in RPMI 1640 contain- further under IHC staining using antibodies against ing 10% fetal bovine serum with 0.3% agarose and layered HOXB7 and Ki-67. on top of 0.6% agar in medium supplemented with 20% fetal bovine serum on 60-mm plates. The plates were Statistical analysis incubated at 37 C in a humid atmosphere of 5% CO2. All statistical analyses were performed using the SPSS After 2 to 3 weeks, cell colony numbers were counted under 13.0 statistical software package. Comparisons between microscope and cell colonies were photographed at an groups for statistical significance were performed with a original magnification of 100. Only cell colonies contain- 2-tailed paired Student’s t test. The relationships between ing more than 50 cells were counted. The experiment was HOXB7 expression and clinicopathologic characteristics performed for 3 independent times for each cell line. were tested using Chi-square test. Survival curves were plotted by Kaplan–Meier method and compared by log- MTT assays and cell cycle analysis rank test. The significance of various survival-related vari- See Supplementary Methods for details. ables was assessed by Cox regression model in the multivariate analysis. P < 0.05 was considered statistically Tumorigenesis in nude mice significant. Xenograft tumors were generated by subcutaneous injection of cells (2 106 for SW480/Vector and SW480/ Results HOXB7, 5 105 for SW620/Scramble and SW620/ shHOXB7, n ¼ 5) on the hindlimbs of each 4- to 6- HOXB7 was frequently upregulated in CRC week-old Balb/C athymic nude mouse (nu/nu) obtained Real-time PCR and western blotting analysis revealed from the Animal Center of Southern Medical University, that all 8 CRC cell lines, including Colo205, Caco2, Ls174t, Guangzhou, China. All mice were housed and maintained DLD1, HT29, HCT116, SW480, and SW620, exhibited under specific pathogen-free conditions and used in accor- different levels of HOXB7 expression. HOXB7 expression dance with institutional guidelines and approved by the was relatively lower in Ls174t and SW480 than that in other Use Committee for Animal Care. Tumor size was measured cell lines (Fig. 1A and B). Comparative analysis indicated by a slide caliper and tumor volume was determined by the that HOXB7 was significantly upregulated in 8 examined formula 0.44 A B2 (A indicates tumor base diameter tumor samples paired with adjacent noncancerous tissues one direction and B the corresponding perpendicular from the same patients (Fig. 1C and D). The tumor/normal value). The tumor were rapidly taken out and fixed in (T/N) ratio of HOXB7 mRNA expression was >2-fold in all 10% neutral buffered formalin, embedded in paraffin. samples, and the highest ratio was up to 16.4-fold, as Sections of 4 mm were cut and stained with haematoxylin analysis by Real-time PCR (Fig. 1E).

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ABFigure 2. HOXB7 expression in 200× 400× 200× 400× paraffin-embedded CRC a bcd Overall Survival specimens. A, representative 100 images of HOXB7 expression in normal intestinal epithelium and 90 CRC specimens examined by IHC. HOXB7 was only negatively 80 low HOXB7 (n = 103) (a, b) or weakly (c, d) detected in 70 normal intestinal epithelial cells, whereas it was positively detected e f g h 60 high HOXB7 (n = 121) in CRC cells (e and f display weak 50 signal, whereas g and h display P = 0.005 strong signals). B, Kaplan–Meier Cumulative Survival Rate (%) Cumulative 40 analysis of survival in patients with 0 20 40 60 80 100 CRC. Green, patients with high n ¼ Survival Time (months) HOXB7 expression ( 121, 5- year survival rate 51.1%, median survival 57 months); blue, patients C n 200× 400× 200× 400× with low expression of HOXB7 ( ¼ 103, 5-year survival rate 70.6%, a b e f median survival 64 months; P ¼ 0.005, log-rank test). C, HOXB7 expression in CRC tissues was positively associated with the HOXB7 expression of Ki-67. IHC staining for HOXB7 and Ki-67 in a series of sections from paraffin blocks of c d g h CRC reveals weak expression of HOXB7 (a, b), weak expression of Ki-67 (c, d), strong expression Ki-67 of HOXB7 (e, f), and strong expression of Ki-67 (g, h). The arrows indicate the stroma cells positively stained with HOXB7.

HOXB7 overexpression was associated with HOXB7 promoted human CRC cell growth and progression and poor survival in CRC proliferation Expression of HOXB7 protein was determined by IHC in ToevaluatethepossibleroleofHOXB7intheprolif- 224 paraffin-embedded, archived CRC tissues. HOXB7 eration of human CRC cells, stable HOXB7 expressed cell protein was detected in 179 of 224 (79.9%) cases of tissue lines SW480/HOXB7 and Ls174t/HOXB7 were made samples (Fig. 2A, e–h), whereas there was no or weak signal (Fig. 3A). We chose SW480 and Ls174t because these 2 in adjacent noncancerous areas in all sections detected cell lines were detected to have relatively low endogenous (Fig. 2A, a–d). In addition, HOXB7 was differently HOXB7 expression (Fig. 1A). MTT assay showed that expressed in tumor stroma cells (mainly in fibroblasts, HOXB7 overexpression increased the proliferation of Fig. 2A f, A h, C f, arrows) in some samples. SW480 as compared with the control cells (Fig. 3B; P < Chi-square test showed that the levels of HOXB7 protein 0.05). The population doubling time cells of SW480/ significantly correlated with Dukes stage (P < 0.001), T stage HOXB7 are significantly longer as compared with (P ¼ 0.012), distant metastasis (P ¼ 0.042), and Ki-67 control. This observation was further confirmed by a labeling index (P ¼ 0.007, Supplementary Table S2). cell growth assay (data not shown). Colony formation Kaplan–Meier survival analysis indicated that patients assay revealed that SW480/HOXB7 cells formed much who had low HOXB7 expression levels had a better outcome more and bigger colonies than that of control cells (Fig. 2B). Multivariate survival analysis indicated that the (Fig. 3C; P < 0.01). Similar results were observed in HOXB7 expression level, T stage and Pathologic stage were 3 Ls174t cells (Fig. 3B and C; P < 0.01). To further inves- independent prognostic factors for outcomes in patients tigate the impact of HOXB7 on CRC proliferation, we with CRC (Supplementary Table S3). Chi-square test also knockdown endogenous HOXB7 in SW620 and HCT116 indicated a significant correlation between the Ki-67 labeling CRC cells by expressing short hairpin RNAs (shRNA; index and HOXB7 expression in CRC (P ¼ 0.007, Supple- Fig. 3D). MTT assay and colony formation assay mentary Table S2). Samples that had lower level of HOXB7 (Fig. 3E and F; P < 0.05), and cell growth assay (data expression also had a lower Ki-67 labeling index (Fig. 2C, a– not shown) showed that knockdown of HOXB7 expres- d), whereas samples that had higher level of HOXB7 expression caused evident compromised viability in SW620 and sion had a higher Ki-67 labeling index (Fig. 2C, e–h). DLD1 cells.

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AC SW480 Ls174t SW480 Ls174t

Vector HOXB7 Vector HOXB7

Vector HOXB7 Vector HOXB7 HOXB7 α-Tubulin

Figure 3. HOXB7 promotes B SW480 Ls174t 100 25 human CRC cell growth and 1.2 0.5 Vector 80 20 proliferation. A, ectopic 1.0 0.4 Vector HOXB7 60 15 expression of HOXB7 in SW480 0.8 0.3 10 and Ls174t cells analyzed by 0.6 HOXB7 40 western blotting. B, ectopic 0.2 20 5 0.4 Number Colony Number Colony 0 expression of HOXB7 stimulates 570nm OD value 0.2 570nm OD value 0.1 0 SW480 and Ls174t cell 0 0 proliferation as determined by Days 1 234567 Days 12 3 4 5 6 7 Vector HOXB7 Vector HOXB7 MTT assays (B) and colony formation assays (C). D, Knockdown of endogenous DFHCT116 SW620 HOXB7 in specific shRNA- HCT116 SW620 transduced stable HCT116 and Scramble shHOXB7 Scramble shHOXB7 SW620 cells. E and F, knockdown of HOXB7 inhibits cell growth as ScrambleshHOXB7 ScrambleshHOXB7 determined by MTT assays (E) and HOXB7 colony formation assays (F). Error α-Tubulin bars represent mean SD from 3 independent experiments. 50 120 P < E HCT116 SW620 *, 0.01. 2.5 0.8 40 100 80 2.0 Scramble 30 Scramble 0.6 60 20 1.5 shHOXB7 40 shHOXB7 0.4 10 Colony Number Colony

Colony Number Colony 20 1.0 0 0 0.2

570nm OD value 0.5 570nm OD value 0 0 Scramble Days 1 234567 Days 1 234567 Scramble shHOXB7 shHOXB7

HOXB7 promoted tumorigenesis of CRC in vitro and in SW480/HOXB7 group was 712.7 123.4 mm3, com- in vivo pared with 363.6 203.5 mm3 in the SW480/Vector We next examined the effect of HOXB7 on the tumori- inoculating group (t-test, P < 0.05.). In addition to the genic activity of CRC cells using an anchorage-independent difference of tumor volume, we also found that the tumors growth assay. The results showed that overexpression of formed by SW480/HOXB7 cells displayed much stronger HOXB7 in SW480 caused significant promotion of its HOXB7 staining and higher Ki-67 index than that in anchorage-independent growth, as indicated by increasing tumors formed by SW480/Vector cells, as detected by in colony number and colony size on soft agar (Fig. 4A; IHC analysis of HOXB7 and Ki-67 (Fig. 4D). However, P < 0.01). Although depletion of endogenous HOXB7 in depletion of endogenous HOXB7 in SW620 cells caused SW620 cells caused significant reduction in colony number significant inhibition of tumor growth in terms of tumor and colony size on soft agar (Fig. 4B; P < 0.01). Therefore, volume (Fig. 4E; n ¼ 5; P<0.01). The average final tumor we determined that HOXB7 is essential for tumorigenesis volume in control group was 115.6 40.1 mm3, whereas it of CRC cells. was only 41.5 20.1 mm3 in the SW620/shHOXB7 To confirm this effect in vivo, we performed tumorigen- inoculating group. Difference in the final tumor volume esis assays in nude mice. SW480/Vector, SW480/HOXB7, between these 2 groups was significant (t-test, P < 0.05). SW620/Scramble, and SW620/shHOXB7 cells were inocu- IHC staining showed that the tumors of control group lated in nude mice. All mice developed Xenograft tumors at displayed much stronger HOXB7 staining and higher Ki-67 the injection site. As shown in Figure 4C, tumor growth in index than that in SW620/shHOXB7 group (Fig. 4F). the SW480/HOXB7 group was significantly more rapid than that in the SW480/Vector group. The volumes of HOXB7 accelerated cell cycle progression tumors formed by the SW480/HOXB7 cells were signifi- We further measured the cell cycle distribution by flow cantly larger than those of vector-control cells-formed cytometry to explore the possible mechanism of HOXB7 in tumors (n ¼ 5; P < 0.05). The average final tumor volume controlling CRC cell proliferation. As shown in Figure 5,

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A B 100 SW480 100 SW620 80 80 Vector HOXB7 Scramble shHOXB7 60 60 40 40 20 20 0 0 Colony forming Rate (%) forming Colony Colony forming Rate (%) forming Colony Vector HOXB7 Scramble shHOXB7 ) 3 CD1000 HE HOXB7 Ki-67 800 SW480/Vector 600 SW480/HOXB7 400

200 Vector

tumor volume (mm tumor volume 0 Days 10 12 14 16 18 20 22 24 26 28

Vector

HOXB7 HOXB7 ) E 3 F 200 HE HOXB7 Ki-67 160 SW620/Scramble 120 SW620/shHOXB7 80 40 Scramble 0 Tumor volume (mm volume Tumor Days 6 8 10 12 14 16 18 20

Scramble

shHOXB7 shHOXB7

Figure 4. The impact of HOXB7 expression on tumorigenesis in vitro and in vivo. A and B, overexpression of HOXB7 promotes cell growth of SW480 (A) and silencing endogenous HOXB7 inhibited cell growth of SW620 (B) as determined by anchorage-independent growth ability assays. Only cell colonies containing more than 50 cells were counted. Error bars represent mean SD from 3 independent experiments. *, P < 0.01. C and E, SW480/Vector and SW480/HOXB7 cells (2 106), and SW620/Scramble and SW620/shHOXB7 cells (5 105) were injected in the hindlimbs of nude mice (n ¼ 5). Tumor volumes were measured on the indicated days. Data points are presented as the mean tumor volume SD. D and F, histopathology of xenograft tumors. The tumor sections were under H&E staining and IHC staining using antibodies against HOXB7 and Ki-67.

decrease in the cell number at the G1–G0 phase and contrary, increase in the cell number at the G1–G0 phase increase in the cell number at S phase was observed in and decrease in the cell number at S phase was observed HOXB7 overexpressed cells. The percentage of cells reen- after endogenous HOXB7 was knocking down. The per- tering into S phase after serum starvation in SW480/ centage of S-phase cells in HCT116/shHOXB7 (30.7% HOXB7 (37.83% 3.42) was significantly higher than 1.95) and SW620/shHOXB7 (27.21% 4.47) were sig- that in SW480/Vector cells (26.89% 0.12; Fig. 5A a and B nificantly less than in HCT116/Sramble (38.86% 3.65) a, P < 0.01). The percentage of S-phase cells in Ls174t/ and SW620/shHOXB7 (40.77% 2.17) cells, respectively HOXB7 was 31.96% 2.15, whereas it was only 22.66% (Fig. 5Ac-d and B c-d, P < 0.01). These results indicated that 2.11 in control cells (Fig. 5A b and B b, P < 0.01). On the overexpression of HOXB7 accelerated the G1 to S-phase

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A ab SW480/Vector SW480/HOXB7 Ls174t/Vector Ls174t/HOXB7

800 200 G1–G0:60% 320 G1–G0:55.3% 320 G1–G0:60.04% G1–G0:39.41% S:22.66% S:29.96% 600 S:26.89% S:37.83% 150 240 240 G2:17.34% G2:15.74% G2:13.06% G2:22.77% 400 160 100 160 Number Number Number Number 200 80 50 80 0 0 0 0 0 20 40 60 80 100 120 0 30 60 90 120 0 30 60 90 120 150 0 30 60 90 120 150 Channels (PI-A) Channels (PI-A) Channels (PI-A) Channels (PI-A)

cd HCT116/Scramble HCT116/shHOXB7 SW620/Scramble SW620/shHOXB7

G1–G0:31.6% G –G :45.07% G –G :44.38% 240 S:38.86% 320 1 0 1 0 1000 G –G :54.76% S:30.7% S:40.77% 1 0 G2:29.61% 600 S:27.21% 180 240 G2:24.24% G2:14.86% 800 G2:18.03% 400 600 Number 120 Number 160 Number Number 400 60 80 200 200 0 0 0 0 0 20 40 60 80 100120 0 30 60 90 120 150 0306090120 150 0 20 40 60 80 100120 Channels (PI-A) Channels (PI-A) Channels (PI-A) Channels (PI-A)

B abcd SW480 G1 LS174T G HCT116 G SW620 G 70 70 1 60 1 60 1 S S S S 60 60 50 50 G G G G 50 2 50 2 40 2 40 2 40 40 30 30 30 30 20 20

20 20 (%) Cell ratio Cell ratio (%) Cell ratio (%) Cell ratio Cell ratio (%) Cell ratio 10 10 10 10 0 0 0 0 tor tor Vec Vec HOXB7 HOXB7 Scramble shHOXB7 Scramble shHOXB7

Figure 5. The effect of HOXB7 on cell cycle distribution of CRC cells. A, representative histograms depicting cell cycle profiles of indicated cells. Cells were stained with PI and analyzed by flow cytometry. B, proportion of cells in various phases of the cell cycle. The results are means of 3 independent experiments SD from 3 independent experiments. *, P < 0.01. transition in CRC cell lines, which contributes to the were shown in HOXB7 knockdown CRC cells (Fig. 6A). growth promotion properties of HOXB7. Moreover, the modulation of p27Kip1 and cyclin D1 by HOXB7 were regulated at the transcriptional level (Fig. 6B). HOXB7 regulated cell cycle factors cyclin D1 and Further more, phosphorylated levels of ERK, AKT and p27Kip1: MAPK and PI3K/Akt activations in CRC cells GSK3b were increased in HOXB7-overexpressed CRC cells, were involved whereas they were significantly decreased in HOXB7- To better understand the mechanisms that facilitate the knockdown CRC cells than in control cells (Fig. 6A). G1 to S-cell cycle transition mediated by HOXB7, the expression levels of some of the cell cycle regulators, Discussion including CDK4, CDK2, cyclin D1, cyclin E, p21Cip1/ WAF1, and p27Kip1 were detected. Overexpression of Although altered HOXB7 mRNA level was observed in HOXB7 did not affect the expression of CDK4, CDK2, CRC (14), this study was the first that reported the upre- cyclin E, and p21Cip1/WAF1 (data not shown), whereas gulation of both HOXB7 protein and mRNA in this disease. the expression cyclin D1 was upregulated and p27Kip1 was The current study has revealed that expression of HOXB7 is downregulated (Fig. 6A). In contrast, significant increases significantly correlated with the invasive and aggressive in the expression of p27Kip1 and decreases of cyclin D1 characteristics of human CRC (high Dukes stage, T stage,

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A SW480 Ls174t HCT116 SW620

tor amble Vector HOXB7 Vec HOXB7 Scramble shHOXB7 Scr shHOXB7 p-ERK

ERK

p-AKT

AKT Figure 6. HOXB7 regulates cell proliferation factors cyclin D1 and p-GSK3β p27Kip1 through the MAPK and AKT/GSK3b pathways in CRC GSK3β cells. A, Western blotting analysis of the expression of p-ERK, total cyclinD1 ERK, phosphorylated AKT, total AKT, p-GSK-3b, total GSK-3b, p27 cyclin D1and p27Kip1 proteins in α indicated vector-infected and -Tubulin HOXB7 infected CRC cell lines, and vector-infected and HOXB7 shRNA-infected CRC cell lines. B, B C relative mRNA expression of p27Kip1 and cyclin D1 in indicated 0.030 0.25 CRC cell lines were determined by real-time RT-PCR. Error bars 0.025 0.20 represent mean SD from 3 0.020 0.15 independent experiments. 0.015 0.10 0.010

of p27 mRNA 0.005 0.05 of cyclin D1 mRNA Relative expression Relative 0 expression Relative 0 Vector Vector Vector Vector HOXB7 HOXB7 HOXB7 HOXB7 Scramble Scramble Scramble Scramble shHOXB7 shHOXB7 shHOXB7 shHOXB7 SW480 Ls174t HCT116 SW620 SW480 Ls174t HCT116 SW620

distant metastasis-positive tumors, and high proliferation and multipotent mesenchymal cells (24, 28). Our results index) and with poor survival of patients as well. These suggested that elevated HOXB7 might be associated with implicate that overexpression of HOXB7 protein may be a higher proliferation activity in CRC cells, which contrib- common feature in CRC and can serve as an independent uted to malignant transforming and tumorigenesis. Thus, prognostic marker to identify patients with poor clinical our data mainly support the tumorigenesis promotion outcome. Similar to our results, HOXB7 overexpression function of HOXB7. Interestingly, we found that HOXB7 was reported in association with the clinical progression was not only expressed in cancerous epithelial cells, but and poor outcome of patients with breast cancer and oral also differently expressed in tumor stroma cells (mainly in cancer (17, 18, 20). Upregulation of HOXB7 mRNA and/or fibroblasts, Fig. 2). Similarly, it has been reported that protein level was also observed in melanoma, ovarian HOXB7 is expressed in fibroblasts (29, 30). These observa- cancer, and esophageal squamous cell carcinoma (15, tions suggested that HOXB7 may be involved in a stroma- 16, 19). However, whether HOXB7 can be used as a specific signaling pathway that promotes initiation and universal biomarker or prognostic predictor for neoplasm progression of CRC. Thus, the expression and function needs further investigation. of HOXB7 might be tissue specific or individual specific. There were 2 opposite functions of HOXB7 that were Although HOXB7 has been linked to regulation of documented in different cellular contexts. Majority studies proliferation (and thus transformation), the molecular supported that HOXB7 might play a role in promotion of mechanisms remain poorly identified. Majority multistep process of tumor formation and progression, researchesonlyreportedthatbFGF,oneofdirecttargets including transformation, proliferation, angiogenesis, of HOXB7, contributed to HOXB7-induced cellular pro- invasion, and metastasis (17, 18, 20–25). On the other liferation and transformation (15, 16, 18, 25). Here, more hand, some researchers observed a promoting role of specifically, we showed the molecular mechanisms might HOXB7 in differentiation in hematopoietic stem cells be the acceleration of G1–S transition, upregulation of

3576 Clin Cancer Res; 17(11) June 1, 2011 Clinical Cancer Research

HOXB7 Accelerates Progression of Colorectal Cancer

cyclin D1 and downregulation of p27Kip1 under Similarly, cyclin D1 was regulated at both transcript and enforced expression of HOXB7. Previous studies revealed protein levels by HOXB7 in this study. Thus, the effect of that PI3K/AKT and MAPK signal transduction cascades, HOXB7 on cyclin D1 in CRC cells may also be regulated required for cell cycle progression through G1 phase, were mainly at transcriptional level. However, whether HOXB7 frequently involved in proliferation (31). In addition, could directly promote transcription of cyclin D1 need activation of RAS and PI3K/AKT decreased the cellular further investigation. levels of p27Kip1 and induced cyclin D1 mRNA and In conclusion, our findings suggest that upregulation of protein, thereby promoting cell proliferation (32). More- HOXB7 might be a valuable prognostic marker of CRC over, HOXB7 was shown to activate the Ras-RAF-MAPK progression. Altered expression of HOXB7 genes could be pathway in breast cancer cell lines (17). Here, we showed important for tumorigenesis and progression of CRC. that PI3K/AKT and MAPK pathways were activated Modulation of the tumor proliferation effect through inhi- because p-ERK, p-AKT and p-GSK3-b were upregulated biting PI3K/AKT or MAPK activation mediated by HOXB7 by HOXB7. Therefore, the regulation of p27Kip1 and overexpression might be used as a potential target for CRC cyclin D1 by HOXB7 probably resulted from enhanced prevention and therapy. PI3K/AKT and MAPK pathway activity. This thus explains the accelerating G1–S transition induced by HOXB7. Disclosure of Potential Conflicts of Interest Taken together, our observations link HOXB7 to the basic cell cycle regulation, which helps provide evidence for No potential conflicts of interest were disclosed. diverse molecular mechanisms by which HOXB7 promote cell growth and proliferation. Grant Support Both MAPK and PI3K/Akt pathways directly regulate p27Kip1 and cyclin D1 (31). The regulation occurs not This work was supported by the National Natural Science Foundation of China (No. 30901791, 30670967, 30770977, 81071735, 81090422/H1606), the only at transcriptional level but also at posttranslational Research Fund for the Doctoral Program of Higher Education of China (No. level via ubiquitin-proteasome proteolysis (33–35). 2009443312009, 20094433110001), National Basic Research Program of China p27Kip1 promoter activity could be regulated by FOXO (973 Program, No. 2010CB529403) and Innovative Research Team Foundation in University (No. IRT0731), Universities in Guangdong Province 211 key con- proteins (FOXO4, FOXO3a, and FOXO1) which, in turn, struction projects, Guangdong Provincial Key Science and Technology Innova- could be modulated by PI3K/AKT and MAPK pathways (32, tion Fund for Higher Education (No. GXZD1016), Guangdong Natural Science 36). In this study we showed that HOXB7 overexpression Foundation of China (2010B031500012). The costs of publication of this article were defrayed in part by the could downregulate p27Kip1 expression both at mRNA payment of page charges. This article must therefore be hereby marked and protein levels, with little difference between the 2 levels advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate in the change fold of p27Kip1. Therefore, the effect of this fact. HOXB7 on p27Kip1 in CRC cells may be mediated via Received September 21, 2010; revised March 26, 2011; accepted March PI3K/AKT and MAPK pathways at transcriptional level. 28, 2011; published OnlineFirst April 7, 2011.

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3578 Clin Cancer Res; 17(11) June 1, 2011 Clinical Cancer Research

HOXB7 as a Prognostic Factor and Mediator of Colorectal Cancer Progression

Wen-Ting Liao, Dan Jiang, Jian Yuan, et al.

Clin Cancer Res 2011;17:3569-3578. Published OnlineFirst April 7, 2011.

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