HOXB7 promotes invasion and predicts survival in pancreatic adenocarcinoma

Abstract

Background: The homeobox gene HOXB7 is overexpressed across a range of cancers and promotes tumorigenesis through varying effects on proliferation, survival, invasion, and angiogenesis. Although published microarray data suggest HOXB7 is overexpressed in pancreatic ductal adenocarcinoma (PDAC), its function in pancreatic cancer has not been studied.

Methods: HOXB7 message and protein levels were examined in PDAC cell lines and patient samples, as well as in normal pancreas. HOXB7 protein expression in patient tumors was determined by immunohistochemistry and correlated with clinicopathologic factors and survival. The impact of HOXB7 on cell proliferation, growth, and invasion was assessed by knockdown and overexpression in PDAC cell lines. Candidate genes whose expression levels were altered following HOXB7 knockdown were determined by microarray analysis.

Results: HOXB7 message and protein levels were significantly elevated in PDAC cell lines and patient tumor samples relative to normal pancreas. Evaluation of a tissue microarray of 145 resected PDACs found high HOXB7 protein expression was correlated with lymph node metastasis (P = .034) and an independent predictor of worse overall survival in multivariate analysis (hazard ratio = 1.56, 95% confidence interval = 1.02-2.39). HOXB7 knockdown or overexpression in PDAC cell lines resulted in decreased or increased invasion, respectively, without influencing proliferation or cell viability.

Conclusions: HOXB7 is frequently overexpressed in PDAC, specifically promotes invasive phenotype, and is associated with lymph node metastasis and worse survival outcome. HOXB7 and its downstream targets may represent novel clinical biomarkers or targets of therapy for inhibiting the invasive and metastatic capacity of PDAC.

Figure 1

(A) HOXB7 levels in whole sections of human pancreatic ductal adenocarcinoma (PDAC) (N = 43) and normal pancreas (N = 7) patient samples are shown, based on oligonucleotide microarray analysis. (B) HOXB7 levels are shown in a published gene expression data set of laser-capture–microdissected normal pancreatic duct (N = 11) and PDAC (N = 14) patient samples. Levels of HOXB7 message relative to housekeeping gene (ACTB) are shown as determined by quantitative polymerase chain reaction for (C) patient-matched normal pancreas and PDAC tumors and (D) PDAC cell lines versus HPDE cells, an immortalized, nontransformed human pancreatic ductal cell line. (E) Western blots for HOXB7, CCBP2, and ACTB in the same lines.

Figure 2

(A) Representative micrographs show variable nuclear staining for HOXB7 in pancreatic ductal adenocarcinoma (PDAC) tumors. (B) Distribution of semiquantitative histoscores of nuclear HOXB7 expression is shown for all PDAC tumors in the tissue microarray (N = 145). (C) Kaplan-Meier curves for overall survival are shown based on low (histoscore ≤ 110) versus high (histoscore > 110) HOXB7 protein expression.

Figure 3

(A) HOXB7 expression was determined by quantitative PCR in PDAC cell lines following transfection with control small interfering RNA (siRNA), HOXB7 siRNA #1, or HOXB7 siRNA #2, as well as (B) parallel western blots. Corresponding in vitro assays are shown for (C) cell growth by MTT, (D) cell proliferation by bromodeoxyuridine (BrdU) incorporation, (E) invasiveness through Matrigel-coated transwell membrane inserts, or (F) migration through uncoated transwell membrane inserts toward chemoattractant. (G) Migration and matrigel invasion assays and western blots are shown with MiaPaCa-2 cells stably overexpressing GFP or HOXB7. Each sample condition was evaluated in triplicate or quadruplicate, with all assays repeated at least twice. Similar results were obtained using HOXB7 siRNA #3 (data not shown). *P < .05, **P < .005. Abbreviations: mRNA exp., messenger RNA expression; WB, western blot.

Figure 4

(A) Top 10 up-regulated and down-regulated genes on Affymetrix U133 2.0 plus microarray analysis of MiaPaCa-2 cells are shown 48 hours after HOXB7 small interfering RNA (siRNA) transfection. (B) Expression changes are seen with HOXB7 siRNA transfection for genes annotated in the category of cellular movement by Ingenuity Pathway Analysis. (C) CCBP2 levels are shown as measured by quantitative polymerase chain reaction and western blot in separate HOXB7 siRNA transfections of Mia-PaCa-2 and PANC1. (D) Matrigel invasion assay with PANC1 is shown 48 hours after transfection with the indicated combinations of siRNA. *P < .05. Abbreviations: mRNA exp., messenger RNA expression; WB, western blot.