The fluorinated analog of deoxycytidine, Gemcitabine (Gemzar), is the main chemotherapeutic drug in pancreatic cancer, but survival remains weak mainly because of the high resistance of tumors to the drug. Recent works have shown that the mucin MUC4 may confer an advantage to pancreatic tumor cells by modifying their susceptibility to drugs. However, the cellular mechanism(s) responsible for this MUC4-mediated resistance is unknown. The aim of this work was to identify the cellular mechanisms responsible for gemcitabine resistance linked to MUC4 expression. CAPAN-2 and CAPAN-1 adenocarcinomatous pancreatic cancer (PC) cell lines were used to establish stable MUC4-deficient clones (MUC4-KD) by shRNA interference. Measurement of the IC 50 index using tetrazolium salt test indicated that MUC4-deficient cells were more sensitive to gemcitabine. This was correlated with increased Bax/Bcl XL ratio and apoptotic cell number. Expression of Equilibrative/Concentrative Nucleoside Transporter (hENT1, hCNT1/3), deoxycytidine kinase (dCK), ribonucleotide reductase (RRM1/2) and Multidrug-Resistance Protein (MRP3/4/5) was evaluated by quantitative RT-PCR (qRT-PCR) and western blotting. Alteration of MRP3, MRP4, hCNT1 and hCNT3 expression was observed in MUC4-KD cells, but only hCNT1 alteration was correlated to MUC4 expression and sensitivity to gemcitabine. Decreased activation of MAPK, JNK and NF-kB pathways was observed in MUC4-deficient cells, in which the NF-kB pathway was found to have an important role in both sensitivity to gemcitabine and hCNT1 regulation. Finally, and in accordance with our in vitro data, we found that MUC4 expression was conversely correlated to that of hCNT1 in tissues from patients with pancreatic adenocarcinoma. This work describes a new mechanism of PC cell resistance to gemcitabine, in which the MUC4 mucin negatively regulates the hCNT1 transporter expression via the NF-kB pathway. Altogether, these data point out to MUC4 and hCNT1 as potential targets to ameliorate the response of pancreatic tumors to gemcitabine treatment.

To determine whether glucocorticoids are involved in pancreas development, glucocorticoid treatment of rat pancreatic buds in vitro was combined with the analysis of transgenic mice lacking the glucocorticoid receptor (GR) in specific pancreatic cells. In vitro treatment of embryonic pancreata with dexamethasone, a glucocorticoid agonist, induced a decrease of insulin-expressing cell numbers and a doubling of acinar cell area, indicating that glucocorticoids favored acinar differentiation; in line with this, expression of Pdx-1, Pax-6, and Nkx6.1 was downregulated, whereas the mRNA levels of Ptf1-p48 and Hes-1 were increased. The selective inactivation of the GR gene in insulin-expressing ␤-cells in mice (using a RIP-Cre transgene) had no measurable consequences on ␤-or ␣-cell mass, whereas the absence of GR in the expression domain of Pdx-1 (Pdx-Cre transgene) led to a twofold increased ␤-cell mass, with increased islet numbers and size but normal ␣-cell mass in adults. These results demonstrate that glucocorticoids play an important role in pancreatic ␤-cell lineage, acting before hormone gene expression onset and possibly also modulating the balance between endocrine and exocrine cell differentiation. Diabetes 53:2322-2329, 2004 I ncreasing evidence from epidemiological studies led to the concept of the early-life origins of adult diseases, suggesting that late-onset disorders such as type 2 diabetes, glucose intolerance, or hypertension may be programmed by nutritional inadequacy in utero (1-5). Excess glucocorticoids also retard fetal growth, and overexposure to these hormones during intrauterine life has been shown to play a role in fetal programming in both humans (6) and rodents (7). The link between glucocorticoid overexposure in utero and the occurrence of metabolic diseases in adulthood has been well documented in rats. Maternal treatment with dexamethasone (DEX), a glucocorticoid agonist, induces in the offspring growth retardation at birth as well as hyperglycemia and increased systolic blood pressure at adult age (8 -10).Similarly, inhibition of the placental 11␤-hydroxysteroid dehydrogenase type 2, the enzyme that protects the fetus from maternal glucocorticoids, induces intrauterine growth retardation as well as glucose intolerance and hypertension in adults (11).We have previously shown that maternal general food restriction during late pregnancy decreased the ␤-cell mass of newborn rats (12). This reduction was irreversible and persisted in adults despite restoration of normal nutrition from weaning (13), ultimately leading to impaired glucose tolerance associated with aging (14) or pregnancy (15). These results sustain the notion that some of the late alterations observed in humans born with intrauterine growth retardation may result from altered ␤-cell development in utero, as initially suggested by Hales and Barker (2). Additionally, we have recently demonstrated that maternal general food restriction in the rat induced a rise in both maternal and fetal corticosterone levels, which in turn w...