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Chemoresistance is a major therapeutic challenge that plays a role in the poor statistical outcomes in pancreatic cancer. Unfolded protein response (UPR) is one of the homeostasis mechanisms in cancer cells that have been correlated with chemoresistance in a number of cancers including pancreatic cancer. In this study, we show that modulating glucose regulatory protein 78 (GRP78), the master regulator of the UPR, can have a profound effect on multiple pathways that mediate chemoresistance. Our study showed for the first time that silencing GRP78 can diminish efflux activity of ATP‐binding cassette (ABC) transporters, and it can decrease the antioxidant response resulting in an accumulation of reactive oxygen species (ROS). We also show that these effects can be mediated by the activity of specificity protein 1 (SP1), a transcription factor overexpressed in pancreatic cancer. Thus, inhibition of SP1 negatively affects the UPR, deregulates the antioxidant response of NRF2, as well as ABC transporter activity by inhibiting GRP78‐mediated ER homeostasis. Sp1 and NRF2 have been classified as nononcogene addiction genes and thus are imperative to understanding the molecular mechanism of resistance. These finding have huge clinical relevance as both Sp1 and GRP78 are overexpressed in pancreatic cancer patients and increased expression of these proteins is indicative of poor prognosis. Understanding how these proteins may regulate chemoresistance phenotype of this aggressive cancer may pave the way for development of efficacious therapy for this devastating disease. Inhibition of the transcription factor, SP1, prevents it from binding to ERSE, resulting in a decrease in GRP78 transcription as well as decreased drug efflux through ABC transporters. Less efflux activity results in chronic UPR activation, which decreases the NRF2‐ARE detoxification mechanism, leading to less chemoresistance in PDAC cells.

Pancreatic tumors are renowned for their extremely hypoxic centers, resulting in upregulation of a number of hypoxia mediated signaling pathways including cell proliferation, metabolism and cell survival. Previous studies from our laboratory have shown that Minnelide, a water-soluble pro-drug of triptolide (anti-cancer compound), decreases viability of cancer cells in vitro as well as in vivo . However, its mechanism of action remain elusive. In the current study we evaluated the effect of Minnelide, on hypoxia mediated oncogenic signaling as well as stemness in pancreatic cancer. Minnelide has just completed Phase 1 trial against GI cancers and is currently awaiting Phase 2 trials. Our results showed that upon treatment with triptolide, HIF-1α protein accumulated in pancreatic cancer cells even though hypoxic response was decreased in them. Our studies showed even though HIF-1α is accumulated in the treated cells, there was no decrease in HIF-1 binding to hypoxia response elements. However, the HIF-1 transcriptional activity was significantly reduced owing to depletion of co-activator p300 upon treatment with triptolide. Further, treatment with triptolide resulted in a decreased activity of Sp1 and NF-kB the two major oncogenic signaling pathway in pancreatic cancer along with a decreased tumor initiating cell (TIC) population in pancreatic tumor.

Endoplasmic reticulum (ER) stress initiates an important mechanism for cell adaptation and survival, named the unfolded protein response (UPR). Severe or chronic/prolonged UPR can breach the threshold for survival and lead to cell death. There is a fundamental gap in knowledge on the molecular mechanism of how chronic ER stress is stimulated and leads to cell death in pancreatic ductal adenocarcinoma (PDAC). Our study shows that downregulating specificity protein 1 (Sp1), a transcription factor that is overexpressed in pancreatic cancer, activates UPR and results in chronic ER stress. In addition, downregulation of Sp1 results in its decreased binding to the ER stress response element present in the promoter region of Grp78, the master regulator of ER stress, thereby preventing homeostasis. We further show that inhibition of Sp1, as well as induction of ER stress, leads to lysosomal membrane permeabilization (LMP), a sustained accumulation of cytosolic calcium, and eventually cell death in pancreatic cancer.

NF-κB has an essential role in the initiation and progression of pancreatic cancer and specifically mediates the induction of epithelial–mesenchymal transition and invasiveness. In this study, we demonstrate the importance of activated NF-κB signaling in EMT induction, lymphovascular metastasis, and neural invasion. Modulation of NF-κB activity was accomplished through the specific NF-κB inhibitor (BAY 11-7085), triptolide, and Minnelide treatment, as well as overexpression of IKBα repressor and IKK activator plasmids. In the classical lymphovascular metastatic cascade, inhibition of NF-κB decreased the expression of several EMT transcription factors (SNAI1, SNAI2, and ZEB1) and mesenchymal markers (VIM and CDH2) and decreased in vitro invasion, which was rescued by IKK activation. This was further demonstrated in vivo via BAY 11-7085 treatment in a orthotopic model of pancreatic cancer. In vivo NF-κB inhibition decreased tumor volume; decreased tumor EMT gene expression, while restoring cell–cell junctions; and decreasing overall metastasis. Furthermore, we demonstrate the importance of active NF-κB signaling in neural invasion. Triptolide treatment inhibits Nerve Growth Factor (NGF) mediated, neural-tumor co-culture in vitro invasion, and dorsal root ganglia (DRG) neural outgrowth through a disruption in tumor-neural cross talk. In vivo, Minnelide treatment decreased neurotrophin expression, nerve density, and sciatic nerve invasion. Taken together, this study demonstrates the importance of NF-κB signaling in the progression of pancreatic cancer through the modulation of EMT induction, lymphovascular invasion, and neural invasion.

Background Pancreatic Ductal Adenocarcinoma (PDAC) is a devastating disease hallmarked by limited patient survival. Resistance to chemotherapy, a major cause of treatment failure in PDAC patients, is often attributed to Cancer Stem Cells (CSCs). Pancreatic CSCs are a small subset of quiescent cells within a tumor represented by surface markers like CD133. These cells are responsible not only for tumor recurrence, but also poor prognosis based on their “stem-like” characteristics. At present, conventional therapy is directed towards rapidly dividing PDAC cells and thus fails to target the CSC population. Methods MIA PaCa-2, S2-013 and AsPC-1 were treated with 12.5 nM triptolide (12 T cells) for 7 days. The surviving cells were recovered briefly in drug-free growth media and then transferred to C ancer S tem cell M edia (CSM). As a control, untreated cells were also transferred to CSM media (CSM). The 12 T and CSM cells were tested for stemness properties using RNA and protein markers. Low numbers of CSM and 12 T cells were implanted subcutaneously in athymic nude mice to study their tumorigenic potential. 12 T and CSM cells were sorted for CD133 expression and assayed for their colony forming ability and sphere forming ability. Invasiveness of 12 T cells, CSM and MIA PaCa-2 were compared using Boyden chamber assays. Results Treated 12 T cells displayed increased expression of the surface marker CD133 and the drug transporter ABCG2 compared to untreated cells (CSM cells). Both 12 T and CSM cells formed subcutaneous tumors in mice confirming their tumor-initiating properties. When tested for invasion, 12 T cells had increased invasiveness compared to CSM cells. CD133 + cells in both CSM and 12 T showed greater colony and sphere forming ability compared to CD133 − cells from each group. Consistent with these data, when injected subcutaneously in mice, CD133 − cells from CSM or 12 T did not form any tumors whereas CD133 + cells from both groups showed tumor formation at a very low cell number. Despite pre-exposure to triptolide in 12 T CD133 + cells, treatment of tumors formed by these cells with Minnelide, a triptolide pro-drug, showed significant tumor regression. Conclusion Our results indicated that triptolide enhanced and enriched the “stemness” in the PDAC cell lines at a low dose of 12.5 nM, but also resulted in the regression of tumors derived from these cells.

A valid preclinical tumor model should recapitulate the tumor microenvironment. Immune and stromal components are absent in immunodeficient models of pancreatic cancer. While these components are present in genetically engineered models such as Kras G12D ; Trp53 R172H ; Pdx-1Cre (KPC), immense variability in development of invasive disease makes them unsuitable for evaluation of novel therapies. We have generated a novel mouse model of pancreatic cancer by implanting tumor fragments from KPC mice into the pancreas of wild type mice. Three-millimeter tumor pieces from KPC mice were implanted into the pancreas of C57BL/6J mice. Four to eight weeks later, tumors were harvested, and stromal and immune components were evaluated. The efficacy of Minnelide, a novel compound which has been shown to be effective against pancreatic cancer in a number of preclinical murine models, was evaluated. In our model, consistent tumor growth and metastases were observed. Tumors demonstrated intense desmoplasia and leukocytic infiltration which was comparable to that in the genetically engineered KPC model and significantly more than that observed in KPC tumor-derived cell line implantation model. Minnelide treatment resulted in a significant decrease in the tumor weight and volume. This novel model demonstrates a consistent growth rate and tumor-associated mortality and recapitulates the tumor microenvironment. This convenient model is a valuable tool to evaluate novel therapies.

Background Rb1 is the most frequently mutated gene in the pediatric cancer retinoblastoma, and its loss causes E2F transcription factors to induce proliferation related genes. However, high E2F levels following pRB loss also induce apoptosis-promoting genes as a safeguard mechanism to suppress emergent tumors. Although p53 accumulation and apoptosis induction is believed to be a primary mechanism to eliminate cells with excess E2F activity, p53 deletion doesn’t suppress RB/E2F induced apoptosis in vivo in the retina. This prompted us to test the PTEN/PI3K/AKT signaling pathway on RB/E2F apoptosis suppression in vivo , to ascertain if the PI3K pathway may provide a potential avenue for retinoblastoma therapy. Methods We developed a mouse model in which Rb1 and Pten were conditionally deleted from retinal progenitor cells using Chx10-Cre , whereas Rbl1 (p107) was constitutively deleted. Pathway components were also tested individually by in vivo electroporation into newborn retinas for an effect on apoptosis and tumor initiation. Mouse retinal tissues were analyzed by immunohistochemistry (IHC) for proliferation, apoptosis, and pathway activation. ShRNAs were used in vitro to assess effects on apoptosis and gene expression. Results Co-deleting Pten with Rb1 and Rbl1 in mouse retinal progenitor cells (RPCs) causes fully penetrant bilateral retinoblastomas by 30 days and strongly suppresses Rb/E2F-induced apoptosis. In vivo electroporation of constitutively active (ca)- Pik3ca , ca- Akt , or dominant-negative (dn)- Foxo1 into apoptosis prone newborn murine retina with deleted Rb/p107 eliminate Rb/E2F induced apoptosis and induce retinoblastoma emergence. Retinal deletion of Pten activates p-AKT and p-FOXO1 signaling in incipient retinoblastoma. An unbiased shRNA screen focusing on Akt phosphorylation targets identified FOXOs as critical mediators of Rb/E2F induced apoptosis and expression of Bim and p73 pro-apoptotic genes. Conclusions These data indicate that we defined a key molecular trigger involving E2F/FOXO functioning to control retinal progenitor cell homeostasis and retinoblastoma tumor initiation. We anticipate that our findings could provide contextual understanding of the proliferation of other progenitor cells, considering the high frequency of co-altered signaling from RB/E2F and PTEN/PI3K/AKT pathways in a wide variety of normal and malignant settings.

Chemoresistance is a major therapeutic challenge that plays a role in the poor statistical outcomes in pancreatic cancer. Unfolded protein response ( UPR ) is one of the homeostasis mechanisms in cancer cells that have been correlated with chemoresistance in a number of cancers including pancreatic cancer. In this study, we show that modulating glucose regulatory protein 78 ( GRP 78), the master regulator of the UPR , can have a profound effect on multiple pathways that mediate chemoresistance. Our study showed for the first time that silencing GRP 78 can diminish efflux activity of ATP ‐binding cassette ( ABC ) transporters, and it can decrease the antioxidant response resulting in an accumulation of reactive oxygen species ( ROS ). We also show that these effects can be mediated by the activity of specificity protein 1 ( SP 1), a transcription factor overexpressed in pancreatic cancer. Thus, inhibition of SP 1 negatively affects the UPR , deregulates the antioxidant response of NRF 2, as well as ABC transporter activity by inhibiting GRP 78‐mediated ER homeostasis. Sp1 and NRF 2 have been classified as nononcogene addiction genes and thus are imperative to understanding the molecular mechanism of resistance. These finding have huge clinical relevance as both Sp1 and GRP 78 are overexpressed in pancreatic cancer patients and increased expression of these proteins is indicative of poor prognosis. Understanding how these proteins may regulate chemoresistance phenotype of this aggressive cancer may pave the way for development of efficacious therapy for this devastating disease.

Pancreatic cancer remains a cancer with the lowest survival rate and shortest median survival. It is predicted to accelerate to the 2nd cause of cancer related death in the next 15 years. Poor survival rates stem from late disease detection, aggressive tumor biology, and unsuccessful treatments. These dismal statistics highlight the urgency for increased knowledge of the biology of pancreatic cancer and the need for effective therapies. Within the past decade, cancer stem cells, a population within the tumor, have been exposed for their role in tumor initiation, progression, chemoresistance, and metastasis. These studies focus to examine the role of CD133 positive cancer stem cells in stemness and metastasis within pancreatic cancer. We demonstrated that CD133 positive pancreatic cancer cells, from human pancreatic cancer cell lines and a spontaneous murine model of pancreatic cancer, are capable of tumor initiation at very low cell numbers in both immunocompromised and immunocompetent mice, respectively. These cells exhibited chemoresistance properties by the upregulation of drug transporters, active drug efflux capabilities, and resistance to cell death upon treatment with conventional chemotherapies, such as gemcitabine and 5-fluorouracil. CD133 positive cancer stem cell population was, however, sensitive to a novel therapy- Minnelide™. In both in vitro and in vivo studies, CD133 positive cells responded to Minnelide™ treatment and underwent apoptosis in addition to the CD133 negative population. These investigations led to questions regarding the function of this surface marker. CD133 played no functional role in conferring the cancer stem cell phenotype to this population. Through overexpression of CD133 in a pancreatic cancer cell line with very low endogenous CD133 expression, we determined that CD133 expression influences both stemness and invasiveness. Cells overexpressing CD133 were capable of initiating tumors at low cell numbers, as compared to control cells, and exhibited an upregulation in pluripotency and developmental signaling gene expression. Additionally, tumors derived from CD133 overexpressing cells demonstrated a marked increase in metastasis to several distant sites. This was shown to occur through the activation of NF-kB signaling and induction of the epithelial-mesenchymal transition; resulting in increased cellular invasiveness. Further, CD133 expressing cells displayed increased expression and secretion of the cytokine, interleukin-1 beta. Inhibition of IL-1 signaling through various methods established a significant role for IL-1 in the induction of the epithelial-mesenchymal transition and cellular invasiveness. Significantly, interleukin-1 beta positively correlates with CD133 gene expression in pancreatic cancer cell lines of varying aggressiveness. In cell lines with high levels of CD133 positive populations, inhibition of IL-1 signaling demonstrated its critical role in epithelial-mesenchymal transition induction and invasiveness. This exhibited that IL-1 signaling functions within CD133 positive populations during the metastasis process. Finally, these studies demonstrated the pivotal role of NF-kB activation in the induction of the epithelial-mesenchymal transition, cellular invasion, and metastasis. Using triptolide and NF-kB signaling inhibitor treatment, as well as, NF-kB signaling pathway modulation via constitutively active or inactive plasmid expression, this signaling pathway was decisively confirmed to mediate invasion and metastasis in pancreatic cancer. Taken together, this work establishes the functional role of the cancer stem cell marker, CD133; IL-1 signaling; and NF-kB activation in pancreatic cancer stemness and metastasis.

Observations of disks with the Atacama Large Millimeter/submillimeter Array (ALMA) allow us to map the chemical makeup of nearby protoplanetary disks with unprecedented spatial resolution and sensitivity. The typical outer Class II disk observed with ALMA is one with an elevated C/O ratio and a lack of oxygen-bearing complex organic molecules, but there are now some interesting exceptions: three transition disks around Herbig Ae stars all show oxygen-rich gas traced via the unique detections of the molecules SO and CH3OH. We present the first results of an ALMA line survey at ≈337–357 GHz of such disks and focus this paper on the first Herbig Ae disk to exhibit this chemical signature—HD 100546. In these data, we detect 19 different molecules including NO, SO2, and CH3OCHO (methyl formate). We also make the first tentative detections of H213CO and 34SO in protoplanetary disks. Multiple molecular species are detected in rings, which are, surprisingly, all peaking just beyond the underlying millimeter continuum ring at ≈200 au. This result demonstrates a clear connection between the large dust distribution and the chemistry in this flat disk. We discuss the physical and/or chemical origin of these substructures in relation to ongoing planet formation in the HD 100546 disk. We also investigate how similar and/or different this molecular makeup of this disk is to other chemically well-characterized Herbig Ae disks. The line-rich data we present motivate the need for more ALMA line surveys to probe the observable chemistry in Herbig Ae systems, which offer unique insight into the composition of disks ices, including complex organic molecules.