A pilot study to develop a diagnostic test for pancreatic ductal adenocarcinoma based on differential expression of select miRNA in plasma and bile.

OBJECTIVES: Accurate peripheral markers for the diagnosis of pancreatic ductal adenocarcinoma (PDAC) are lacking. We measured the differential expression of select microRNAs (miRNAs) in plasma and bile among patients with PDAC, chronic pancreatitis (CP), and controls. METHODS: We identified patients (n=215) with treatment-naive PDAC (n=77), CP with bile/pancreatic duct pathology (n=67), and controls (n=71) who had been prospectively enrolled in a Pancreatobiliary Biorepository at the time of endoscopic retrograde cholangiopancreatography or endoscopic ultrasound. Controls were patients with choledocholithiasis but normal pancreata. The sample was separated into training (n=95) and validation (n=120) cohorts to establish and then test the performance of PDAC Signature Panels in diagnosing PDAC. The training cohort (n=95) included age-matched patients with PDAC, CP, and controls. Panels were derived from the differential expression of 10 candidate miRNAs in plasma or bile. We selected miRNAs having excellent accuracy for inclusion in regression models. RESULTS: Using the training cohort, we confirmed the differential expression of 9/10 miRNAs in plasma (miR-10b, -30c, -106b, -132, -155, -181a, -181b, -196a, and -212) and 7/10 in bile (excluding miR-21, -132, and -181b). Of these, five (miR-10b, -155, -106b, -30c, and -212) had excellent accuracy for distinguishing PDAC. In the training and validation cohorts, the sensitivity/specificity for a PDAC Panel derived from plasma was 95/100% and 100/100%, respectively; in bile, these were 96/100% and 100/100%. CONCLUSIONS: Increased expression of miRNA-10b, -155, and -106b in plasma appears highly accurate in diagnosing PDAC. Additional studies are needed to confirm this Panel and explore its value as a prognostic test.

DUSP1 is a novel target for enhancing pancreatic cancer cell sensitivity to gemcitabine.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer with a poor prognosis that is characterized by excessive mitogenic pathway activation and marked chemoresistance to a broad spectrum of chemotherapeutic drugs. Dual specificity protein phosphatase 1 (DUSP1) is a key negative regulator of mitogen activated protein kinases (MAPKs). Yet, DUSP1 is overexpressed in pancreatic cancer cells (PCCs) in PDAC where it paradoxically enhances colony formation in soft agar and promotes in vivo tumorigenicity. However, it is not known whether DUSP1 overexpression contributes to PDAC chemoresistance. Using BxPC3 and COLO-357 human PCCs, we show that gemcitabine activates c-JUN N-terminal kinase (JNK) and p38 mitogen activated protein kinase (p38 MAPK), key kinases in two major stress-activated signaling pathways. Gemcitabine-induced JNK and p38 MAPK activation mediates increased apoptosis, but also transcriptionally upregulates DUSP1, as evidenced by increased DUSP1 mRNA levels and RNA polymerase II loading at DUSP1 gene body. Conversely, shRNA-mediated inhibition of DUSP1 enhances JNK and p38 MAPK activation and gemcitabine chemosensitivity. Using doxycycline-inducible knockdown of DUSP1 in established orthotopic pancreatic tumors, we found that combining gemcitabine with DUSP1 inhibition improves animal survival, attenuates angiogenesis, and enhances apoptotic cell death, as compared with gemcitabine alone. Taken together, these results suggest that gemcitabine-mediated upregulation of DUSP1 contributes to a negative feedback loop that attenuates its beneficial actions on stress pathways and apoptosis, raising the possibility that targeting DUSP1 in PDAC may have the advantage of enhancing gemcitabine chemosensitivity while suppressing angiogenesis.

Pancreatic cancer-associated retinoblastoma 1 dysfunction enables TGF-ß to promote proliferation.

Pancreatic ductal adenocarcinoma (PDAC) is often associated with overexpression of TGF-ß. Given its tumor suppressor functions, it is unclear whether TGF-ß is a valid therapeutic target for PDAC. Here, we found that proliferating pancreatic cancer cells (PCCs) from human PDAC patients and multiple murine models of PDAC (mPDAC) often exhibit abundant levels of phosphorylated retinoblastoma 1 (RB) and Smad2. TGF-ß1 treatment enhanced proliferation of PCCs isolated from KrasG12D-driven mPDAC that lacked RB (KRC cells). This mitogenic effect was abrogated by pharmacological inhibition of type I TGF-ß receptor kinase, combined inhibition of MEK/Src or MEK/PI3K, and restoration of RB expression. TGF-ß1 promoted epithelial-to-mesenchymal transition (EMT), invasion, Smad2/3 phosphorylation, Src activation, Wnt reporter activity, and Smad-dependent upregulation of Wnt7b in KRC cells. Importantly, TGF-ß1-induced mitogenesis was markedly attenuated by inhibition of Wnt secretion. In an in vivo syngeneic orthotopic model, inhibition of TGF-ß signaling suppressed KRC cell proliferation, tumor growth, stroma formation, EMT, metastasis, ascites formation, and Wnt7b expression, and markedly prolonged survival. Together, these data indicate that RB dysfunction converts TGF-ß to a mitogen that activates known oncogenic signaling pathways and upregulates Wnt7b, which synergize to promote PCC invasion, survival, and mitogenesis. Furthermore, this study suggests that concomitantly targeting TGF-ß and Wnt7b signaling in PDAC may disrupt these aberrant pathways, which warrants further evaluation in preclinical models.

Deletion of Rb accelerates pancreatic carcinogenesis by oncogenic Kras and impairs senescence in premalignant lesions.

BACKGROUND & AIMS: Rb1 encodes a cell-cycle regulator that is functionally disrupted in most human cancers. Pancreatic ductal adenocarcinomas (PDACs) have a high frequency of mutations in KRAS and INK4A/CDKN2A that might allow cells to bypass the regulatory actions of retinoblastoma (RB). To determine the role of loss of RB function in PDAC progression, we investigated the effects of Rb disruption during pancreatic malignant transformation initiated by oncogenic Kras. METHODS: We generated mice with pancreas-specific disruption of Rb, in the absence or presence of oncogenic Kras, to examine the role of RB in pancreatic carcinogenesis. RESULTS: In the presence of oncogenic Kras, loss of Rb from the pancreatic epithelium accelerated formation of pancreatic intraepithelial neoplasia (PanIN), increased the frequency of cystic neoplasms, and promoted rapid progression toward PDAC. Early stage cancers were characterized by acute pancreatic inflammation, associated with up-regulation of proinflammatory cytokines within the pancreas. Despite the presence of markers associated with oncogene-induced senescence, low-grade PanIN were highly proliferative and expressed high levels of p53. Pancreatic cancer cell lines derived from these mice expressed high levels of cytokines, and transcriptional activity of p53 was impaired. CONCLUSIONS: Rb encodes a tumor suppressor that attenuates progression of oncogenic Kras-induced carcinogenesis in the pancreas by mediating the senescence response and promoting activity of the tumor suppressor p53.

Expression and regulation of the beta-subunit of ovine follicle-stimulating hormone relies heavily on a promoter sequence likely to bind Smad-associated proteins.

FSH is essential for normal gonadal function in mammals. Expression of its beta-subunit (FSHB) controls overall production/secretion of FSH and is induced by activin. Studies with ovine FSHB promoter/reporter constructs in L beta T2 gonadotropes show that induction by activin requires a putative Smad binding element in the ovine FSHB promoter (-162AGAC-159). Similar studies reported here show that another site, juxtaposed to the Smad binding element, was also required for 81% activin induction in L beta T2 cells. This site was similar to several that bind proteins known to partner with Smads. When this site (-171ACTgcgtTT-163) was mutated by changing the nucleotides shown in lowercase letters, the resulting ovine-derived construct (mut-oFSHBLuc) was expressed poorly as a transgene in primary mouse gonadotropes (<0.001 times compared with ovine wild-type transgenes). This decrease in expression demonstrated the importance of this site for activin induction and, perhaps, basal expression, although studies with L beta T2 cells did not suggest this latter possibility. Expression of mut-oFSHBLuc in male mouse gonadotropes in vivo was at least 644 times greater than expression in all but one nongonadotrope tissue tested, indicating that mut-oFSHBLuc retained significant gonadotrope-specific expression. An increase in FSHB expression occurs during estrus in mice and is faithfully reproduced with wild-type ovine FSHBLuc transgenes, but not with mut-oFSHBLuc, indicating that the mutated site is needed for this secondary FSH surge. These data suggest that activin gathers Smads and Smad-associated proteins at the -171/-159 promoter region to regulate expression of the ovine FSHB and overall FSH production.

Differential regulation of follicle stimulating hormone by activin A and TGFB1 in murine gonadotropes.

BACKGROUND: Activins stimulate the synthesis of follicle stimulating hormone (FSH) in pituitary gonadotropes, at least in part, by inducing transcription of its beta subunit (Fshb). Evidence from several laboratories studying transformed murine LbetaT2 gonadotropes indicates that activins signal through Smad-dependent and/or Smad-independent pathways, similar to those used by transforming growth factor beta-1 (TGFB1) in other cell types. Therefore, given common intracellular signaling mechanisms of these two ligands, we examined whether TGFBs can also induce transcription of Fshb in LbetaT2 cells as well as in purified primary murine gonadotropes. METHODS: Murine Fshb promoter-reporter (-1990/+1 mFshb-luc) activity was measured in LbetaT2 cells treated with activin A or TGFB1, and in cells transfected with either activin or TGFB receptors. The ability of the ligands to stimulate phosphorylation of Smads 2 and 3 in LbetaT2 cells was measured by western blot analysis, and expression of TGFB type I and II receptors was assessed by reverse transcriptase polymerase chain reaction in both LbetaT2 cells and primary gonadotropes purified from male mice of different ages. Finally, regulation of endogenous murine Fshb mRNA levels by activin A and TGFB1 in purified gonadotropes and whole pituitary cultures was measured using quantitative RT-PCR. RESULTS: Activin A dose-dependently stimulated -1990/+1 mFshb-luc activity in LbetaT2 cells, but TGFB1 had no effect at doses up to 5 nM. Similarly, activin A, but not TGFB1, stimulated Smad 2 and 3 phosphorylation in these cells. Constitutively active forms of the activin (Acvr1b-T206D) and TGFB (TGFBR1-T204D) type I receptors strongly stimulated -1990/+1 mFshb-luc activity, showing that mechanisms down stream of Tgfbr1 seem to be intact in LbetaT2 cells. RT-PCR analysis of LbetaT2 cells and whole adult murine pituitaries indicated that both expressed Tgfbr1 mRNA, but that Tgfbr2 was not detected in LbetaT2 cells. When cells were transfected with a human TGFBR2 expression construct, TGFB1 acquired the ability to significantly stimulate -1990/+1 mFshb-luc activity. In contrast to LbetaT2 cells, primary murine gonadotropes from young mice (8-10 weeks) contained low, but detectable levels of Tgfbr2 mRNA and these levels increased in older mice (1 yr). A second surprise was the finding that treatment of purified primary gonadotropes with TGFB1 decreased murine Fshb mRNA expression by 95% whereas activin A stimulated expression by 31-fold. CONCLUSION: These data indicate that TGFB1-insensitivity in LbetaT2 cells results from a deficiency in Tgfbr2 expression. In primary gonadotropes, however, expression of Tgfbr2 does occur, and its presence permits TGFB1 to inhibit Fshb transcription, whereas activin A stimulates it. These divergent actions of activin A and TGFB1 were unexpected and show that the two ligands may act through distinct pathways to cause opposing biological effects in primary murine gonadotropes.

Transforming growth factor beta-activated kinase 1 is a key mediator of ovine follicle-stimulating hormone beta-subunit expression.

FSH, a key regulator of gonadal function, contains a beta-subunit (FSHbeta) that is transcriptionally induced by activin, a member of the TGFbeta-superfamily. This study used 4.7 kb of the ovine FSHbeta-promoter linked to luciferase (oFSHbetaLuc) plus a well-characterized activin-responsive construct, p3TPLuc, to investigate the hypothesis that Smad3, TGFbeta-activated kinase 1 (TAK1), or both cause activin-mediated induction of FSH. Overexpression of either Smad3 or TAK1 induced oFSHbetaLuc in gonadotrope-derived LbetaT2 cells as much as activin itself. Induction of p3TPLuc by activin is known to require Smad3 activation in many cell types, and this was true in LbetaT2 cells, where 10-fold induction by activin (2-8 h after activin treatment) was blocked more than 90% by two dominant negative (DN) inhibitors of Smad3 [DN-Smad3 (3SA) and DN-Smad3 (D407E)]. By contrast, 6.5-fold induction of oFSHbetaLuc by activin (10-24 h after activin treatment) was not blocked by either DN-Smad inhibitor, suggesting that activation of Smad3 did not trigger induction of oFSHbetaLuc. By contrast, inhibition of TAK1 by a DN-TAK1 construct led to a 50% decrease in activin-mediated induction of oFSHbetaLuc, and a specific inhibitor of TAK1 (5Z-7-Oxozeanol) blocked induction by 100%, indicating that TAK1 is necessary for activin induction of oFSHbetaLuc. Finally, inhibiting p38-MAPK (often activated by TAK1) blocked induction of oFSHbetaLuc by 60%. In conclusion, the data presented here indicate that activation of TAK1 (and probably p38-MAPK), but not Smad3, is necessary for triggering induction of oFSHbeta by activin.

Conditional induction of ovulation in mice.

Follicle-stimulating hormone controls the maturation of mammalian ovarian follicles. In excess, it can increase ovulation (egg production). Reported here is a transgenic doxycycline-activated switch, tested in mice, that produced more FSHB subunit (therefore more FSH) and increased ovulation by the simple feeding of doxycycline (Dox). The transgenic switch was expressed selectively in pituitary gonadotropes and was designed to enhance normal expression of FSH when exposed to Dox, but to be regulated by all the hormones that normally control FSH production in vivo. Feeding maximally effective levels of Dox increased overall mRNA for FSHB and serum FSH by over half in males, and Dox treatment more than doubled the normal ovulation rate of female mice for up to 10 reproductive cycles. Lower levels of Dox increased the number of developing embryos by 30%. Ovarian structure and function appeared normal. In summary, gene switch technology and normal FSH regulation were combined to effectively enhance ovulation in mice. Theoretically, the same strategy can be used with any genetic switch to increase ovulation (or any highly conserved physiology) in any mammal.