Hypoxia inducible BHLHB2 is a novel and independent prognostic marker in pancreatic ductal adenocarcinoma.
OBJECTIVE
The cyclic adenosine monophosphate-inducible basic helix-loop-helix (bHLH) domain containing class-B2 transcriptional factor BHLHB2 is differentially expressed in a number of human malignancies. In the present study, the expression, regulation, functions and prognostic impact of BHLHB2 in pancreatic cancer were investigated.


METHODS
Expression analyses were carried out in tissues of the normal pancreas (n=10) and pancreatic ductal adenocarcinoma (n=77) as well as in eight pancreatic cancer cell lines using quantitative RT-PCR, semiquantitative immunohistochemistry, and immunoblot analyses. In vitro functional experiments were conducted using siRNA transfection, hypoxia, serum starvation, apoptosis induction with gemcitabine and actinomycin-D, and invasion assays. Survival analysis was performed using the Kaplan-Meier method. Prognostic factors were determined in a multivariable analysis using a Cox proportional hazards model.


RESULTS
BHLHB2 mRNA and protein expressions were strongly induced by hypoxia and by serum starvation in pancreatic cancer cell lines. BHLHB2 silencing with RNAi had no significant effects on growth and invasion but increased apoptosis resistance against gemcitabine by reducing caspace-3 cleavage. In BHLHB2 silenced cells the ED50 of gemcitabine increased from 13.95 ± 1.353 to 38.70 ± 5.262 nM (p<0.05). Ex vivo, the weak/absent nuclear staining in normal pancreatic ducts and acinar cells was replaced by moderate to strong nuclear/cytoplasmic staining in PanIN lesions and pancreatic cancer cells. Patients with weak/absent nuclear BHLHB2 staining had significantly worse median survival compared to those with strong staining (13 months vs. 27 months, p=0.03). In a multivariable analysis, BHLHB2 staining was an independent prognostic factor (Hazard-Ratio=2.348, 95% CI=1.250-4.411, p=0.008).


CONCLUSIONS
Hypoxia-inducible BHLHB2 expression is a novel independent prognostic marker in pancreatic cancer patients and indicates increased chemosensitivity towards gemcitabine.
Wang
Weibin
W
Department of Surgery, Technische Universität München, Munich, Germany.
Reiser-Erkan
Carolin
C
Michalski
Christoph W
CW
Raggi
Matthias C
MC
Quan
Liao
L
Yupei
Zhao
Z
Friess
Helmut
H
Erkan
Mert
M
Kleeff
Jörg
J
D000368:Aged; D000964:Antimetabolites, Antineoplastic / Q000627:therapeutic use*; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism*; D021441:Carcinoma, Pancreatic Ductal / Q000188:drug therapy / Q000378:metabolism / Q000401:mortality*; D015687:Cell Hypoxia; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000627:therapeutic use; D019008:Drug Resistance, Neoplasm; D005260:Female; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008297:Male; D010179:Pancreas / Q000378:metabolism / Q000473:pathology; D010190:Pancreatic Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000401:mortality*; D011379:Prognosis; D034622:RNA Interference; D000093542:Gemcitabine
D000964:Antimetabolites, Antineoplastic; D051792:Basic Helix-Loop-Helix Proteins; D014408:Biomarkers, Tumor; D003841:Deoxycytidine; D018398:Homeodomain Proteins; D000093542:Gemcitabine; C107989:BHLHE40 protein, human