Pancreatic Cancer Stem Cell Characteristics Research Articles

Abstract 6030: Role of immature CD44 o-glycosylation in enrichment of cancer stem cell population for aggressive pancreatic cancer

Abstract Pancreatic cancer (PC) is an aggressive malignancy expected to be the second leading cause of cancer death by 2030. Accumulating reports demonstrate that truncated o-glycans are largely observed in PC and correlates with poor prognosis and reduced patient survival. We have previously shown that loss of C1GALT1 (Core 1 n-acetylgalactose transferase) expression in PC results in the upregulation of genes associated with aggressive PC and metastasis. To study the involvement of immature O-glycophenotype in pancreatic cancer stem cells (CSCs), we selected the single-transmembrane CD44 glycoprotein based on our unbiased approach using proteomics and RNA-sequence analysis.This highly o-glycosylated protein with diverse cellular activity is essential in cell-cell recognition and is a widely occurring CSC marker identified in several cancers. However, the functional properties of CD44 o-glycan variation in pancreatic CSCs are not well understood. Thus, we hypothesize that truncation of o-glycans on CD44 imparts activation of downstream targets responsible for self-renewal and maintenance of CSC in aggressive PC. Using a proteomics approach of enriched truncated o-glycans, we discovered CD44 as one of the top identified proteins in CRISPR/Cas9 C1GALT1 knockout PC cells. We observed enhanced expression of markers responsible for CSC self-renewal and characteristics as measured by FACS-based side-population analysis and tumor sphere formation as a result of a loss in C1GALT1. To determine a potential mechanism by which truncated o-glycans on CD44 enhances CSC properties, we deleted C1GALT1 and CD44 in PC cells. A loss of CD44 o-glycan truncation significantly reduced expression of the self-renewal CSC marker Nanog and phosphorylated p65, a major component in the NF-κB signaling pathway. Furthermore, pancreatic tumorigenesis was significantly reduced in athymic mice injected with PC cells as a result of C1GALT1 and CD44 deletion. These results provide a novel mechanism by which truncated o-glycans on CD44 is responsible for activating downstream targets for self-renewal and maintenance of CSCs. Our findings begin to contextualize the contributions of aberrant glycosylation on glycoproteins to accelerate metastatic properties by enhanced expression and characteristics of pancreatic CSC. Citation Format: Frank Leon, Rama K. Nimmakayala, Seema Chugh, Rohitesh Gupta, Satyanarayana Rachagani, Surinder K. Batra, Moorthy P. Ponnusamy. Role of immature CD44 o-glycosylation in enrichment of cancer stem cell population for aggressive pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6030.

Inhibition of pancreatic cancer stem cell characteristics by α-Mangostin: Molecular mechanisms involving Sonic hedgehog and Nanog.

The current investigation was intended to elucidate the molecular mechanism of α‐Mangostin in the regulation of pancreatic cancer stem cell (CSC) characteristics. Here, we demonstrate that α‐Mangostin inhibited cell proliferation in pancreatic CSCs and cancer cell lines while it showed no effect on human pancreatic normal ductal epithelial cells. Also, α‐Mangostin inhibited colony formation and induced apoptosis in these cells. Further, α‐Mangostin inhibited the self‐renewal capacity of CSCs isolated from human primary tumours and KrasG12D mice. Furthermore, α‐Mangostin inhibited the invasive and metastatic ability of pancreatic CSCs by suppressing the epithelial‐to‐mesenchymal transition (EMT) via up‐regulation of E‐cadherin and down‐regulation of mesenchymal phenotype by inhibiting N‐cadherin, Snail and Slug expression. Interestingly, the pluripotency maintaining factors and CSC markers were inhibited by α‐Mangostin thus suggesting that α‐Mangostin can target CSCs to inhibit pancreatic cancer effectively. Gli signalling plays a crucial role in the self‐renewal and pluripotency of CSCs. α‐Mangostin inhibited the Gli transcription and the expression of Gli target genes (Nanog, Oct4, c‐Myc, Sox‐2 and KLF4) in CSCs. Using ChIP assay, we demonstrated that Nanog could directly bind to promoters of Cdk2, Cdk6, FGF4, c‐Myc and α‐Mangostin inhibited Nanog binding to these promoters. Conversely, the inhibitory effects of the α‐Mangostin on CSC proliferation and Gli or Nanog transcription and their targets were abrogated by either enforced activation of sonic hedgehog (Shh) or by the overexpression of Nanog. Taken together, our studies suggest that α‐Mangostin may act as Gli inhibitor and establishes the pre‐clinical significance of α‐Mangostin for the prevention and treatment of pancreatic cancer.

Pancreatic cancer stem cells: A state or an entity?

Pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer, has a median overall survival of 6-12 months and a 5-year survival of less than 7%. While PDAC currently represents the 4th most frequent cause of death due to cancer worldwide, it is expected to become the second leading cause of cancer-related death by 2030. These alarming statistics are primarily due to both the inherent chemoresistant and metastatic nature of this tumor, and the existence of a subpopulation of highly plastic "stem"-like cells within the tumor, known as cancer stem cells (CSCs). Since their discovery in PDAC in 2007, we have come to realize that pancreatic CSCs have unique metabolic, autophagic, invasive, and chemoresistance properties that allow them to continuously self-renew and escape chemo-therapeutic elimination. More importantly, the concept of the CSC as a fixed entity within the tumor has also evolved, and current data suggest that CSCs are states rather than defined entities. Consequently, current treatments for the majority of PDAC patients are not effective, and do not significantly impact overall patient survival, as they do not adequately target the plastic CSC sub-population nor the transient/hybrid cells that can replenish the CSC pool. Thus, it is necessary that we improve our understanding of the characteristics and signals that maintain and drive the pancreatic CSC population in order to develop new therapies to target these cells. Herein, we will provide the latest updates and knowledge on the inherent characteristics of pancreatic CSCs and the CSC niche, specifically the cross-talk that exists between CSCs and niche resident cells. Lastly, we will address the question of whether a CSC is a state or an entity and discuss how the answer to this question can impact treatment approaches.

Abstract 3728: α-Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic carcinogenesis by targeting cancer stem cells in KC and KPC mice

Abstract Activation of sonic Hedgehog (Shh) in cancer stem cell (CSC) has been demonstrated with aggressiveness of pancreatic cancer. In order to enhance the biologic activity of α-mangostin, we formulated mangostin-encapsulated PLGA nanoparticles (Mang-NPs) and examined the molecular mechanisms by which they inhibit human and KC mice (PdxCre;LSL-KrasG12D) pancreatic CSC characteristics in vitro, and pancreatic carcinogenesis in KPC (PdxCre;LSLKrasG12D;LSL-Trp53R172H) mice. Mang-NPs inhibited human and KrasG12D mice pancreatic CSC characteristics in vitro. Mang-NPs also inhibited EMT by upregulating E-cadherin and inhibiting N-cadherin and transcription factors Slug, and pluripotency maintaining factors Nanog, c-Myc, and Oct4. Furthermore, Mang-NPs inhibited the components of Shh pathway and Gli targets. In vivo, Mang-NPs inhibited the progression of pancreatic intraneoplasia to pancreatic ductal adenocarcinoma and liver metastasis in KPC mice. The inhibitory effects of Mang-NPs on carcinogenesis in KPC mice were associated with downregulation of pluripotency-maintaining factors (c-Myc, Nanog and Oct4), stem cell markers (CD24 and CD133), components of Shh pathway (Gli1, Gli2, Patched1/2, and Smoothened), Gli targets (Bcl-2, XIAP and Cyclin D1), and EMT markers and transcription factors (N-cadherin, Slug, Snail and Zeb1), and upregulation of E-cadherin. Overall, our data suggest that Mang-NPs can inhibit pancreatic cancer growth, development and metastasis by targeting Shh pathway. Citation Format: Raj K. Verma, Wei Yu, Anju Shrivastava, Rakesh K. Srivastava, Sharmila Shankar. α-Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic carcinogenesis by targeting cancer stem cells in KC and KPC mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3728.

Sanguinarine inhibits pancreatic cancer stem cell characteristics by inducing oxidative stress and suppressing sonic hedgehog-Gli-Nanog pathway.

Sonic hedgehog pathway is highly activated in pancreatic cancer stem cells (CSC) which play crucial roles in cancer initiation, progression and metastasis. However, the molecular mechanisms by which sanguinarine regulates pancreatic CSC characteristics is not well understood. The objectives of this study were to examine the molecular mechanisms by which sanguinarine regulates pancreatic CSC characteristics. Sanguinarine inhibited cell proliferation and colony formation and induced apoptosis through oxidative damage. Sanguinarine inhibited self-renewal capacity of pancreatic CSCs isolated from human and KrasG12D mice. Furthermore, sanguinarine suppressed epithelial-mesenchymal transition (EMT) by up-regulating E-cadherin and inhibiting N-cadherin. Significant decrease in expression level of Snail, Slug and Zeb1 corroborated the suppression of EMT in sanguinarine treated pancreatic CSCS. The ability of sanguinarine to inhibit pluripotency maintaining factors and CSC markers suggest that sanguinarine can be an effective agent for inhibiting pancreatic cancer growth and development by targeting CSCs. Furthermore, sanguinarine inhibited Shh-Gli pathway leading to modulation of Gli target genes in pancreatic CSCs. Chromatin immunoprecipitation assay demonstrated that Nanog directly binds to promoters of Cdk2, Cdk6, FGF4, c-Myc and Oct4, and sanguinarine inhibits the binding of Nanog with these genes, suggesting the direct involvement of Nanog in cell cycle, pluripotency and self-renewal. To further investigate the role of Shh-Gli-Nanog pathway, we regulated Shh signaling either by Shh protein or Nanog overexpression. Enforced activation of Shh or overexpression of Nanog counteracted the inhibitory effects of sanguinarine on pancreatic CSC proliferation, suggesting the actions of sanguinarine are mediated, at least in part, through Shh-Gli-Nanog pathway. Our studies suggest that sanguinarine can be used for the treatment and/or prevention of pancreatic cancer by targeting CSCs.

Targeting Cancer Stem Cells and Their Niche: Current Therapeutic Implications and Challenges in Pancreatic Cancer.

Cancer stem cells (CSCs) have been identified as a subpopulation of stem-like cancer cells with the ability of self-renewal and differentiation in hematological malignancies and solid tumors. Pancreatic cancer is one of the most lethal cancers worldwide. CSCs are thought to be responsible for cancer initiation, progression, metastasis, chemoresistance, and recurrence in pancreatic cancer. In this review, we summarize the characteristics of pancreatic CSCs and discuss the mechanisms involved in resistance to chemotherapy, the interactions with the niche, and the potential role in cancer immunoediting. We propose that immunotherapy targeting pancreatic CSCs, in combination with targeting the niche components, may provide a novel treatment strategy to eradicate pancreatic CSCs and hence improve outcomes in pancreatic cancer.

Proteomic analysis of pancreatic cancer stem cells: Functional role of fatty acid synthesis and mevalonate pathways

Recently, we have shown that the secretome of pancreatic cancer stem cells (CSCs) is characterized by proteins that participate in cancer differentiation, invasion, and metastasis. However, the differentially expressed intracellular proteins that lead to the specific characteristics of pancreatic CSCs have not yet been identified, and as a consequence the deranged metabolic pathways are yet to be elucidated.To identify the modulated proteins of pancreatic CSCs, iTRAQ-based proteomic analysis was performed to compare the proteome of Panc1 CSCs and Panc1 parental cells, identifying 230 modulated proteins. Pathway analysis revealed activation of glycolysis, the pentose phosphate pathway, the pyruvate-malate cycle, and lipid metabolism as well as downregulation of the Krebs cycle, the splicesome and non-homologous end joining. These findings were supported by metabolomics and immunoblotting analysis. It was also found that inhibition of fatty acid synthase by cerulenin and of mevalonate pathways by atorvastatin have a greater anti-proliferative effect on cancer stem cells than parental cells.Taken together, these results clarify some important aspects of the metabolic network signature of pancreatic cancer stem cells, shedding light on key and novel therapeutic targets and suggesting that fatty acid synthesis and mevalonate pathways play a key role in ensuring their viability. Biological significanceTo better understand the altered metabolic pathways of pancreatic cancer stem cells (CSCs), a comprehensive proteomic analysis and metabolite profiling investigation of Panc1 and Panc1 CSCs were carried out. The findings obtained indicate that Panc1 CSCs are characterized by upregulation of glycolysis, pentose phosphate pathway, pyruvate-malate cycle, and lipid metabolism and by downregulation of Krebs cycle, spliceosome and non-homologous end joining. Moreover, fatty acid synthesis and mevalonate pathways are shown to play a critical contribution to the survival of pancreatic cancer stem cells. This study is helpful for broadening the knowledge of pancreatic cancer stem cells and could accelerate the development of novel therapeutic strategies.

α-Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic carcinogenesis by targeting cancer stem cells in human, and transgenic (Kras(G12D), and Kras(G12D)/tp53R270H) mice.

Activation of sonic hedgehog (Shh) in cancer stem cell (CSC) has been demonstrated with aggressiveness of pancreatic cancer. In order to enhance the biological activity of α-mangostin, we formulated mangostin-encapsulated PLGA nanoparticles (Mang-NPs) and examined the molecular mechanisms by which they inhibit human and KC mice (PdxCre;LSL-KrasG12D) pancreatic CSC characteristics in vitro, and pancreatic carcinogenesis in KPC (PdxCre;LSLKrasG12D;LSL-Trp53R172H) mice. Mang-NPs inhibited human and KrasG12D mice pancreatic CSC characteristics in vitro. Mang-NPs also inhibited EMT by up-regulating E-cadherin and inhibiting N-cadherin and transcription factors Slug, and pluripotency maintaining factors Nanog, c-Myc, and Oct4. Furthermore, Mang-NPs inhibited the components of Shh pathway and Gli targets. In vivo, Mang-NPs inhibited the progression of pancreatic intraneoplasia to pancreatic ductal adenocarcinoma and liver metastasis in KPC mice. The inhibitory effects of Mang-NPs on carcinogenesis in KPC mice were associated with downregulation of pluripotency maintaining factors (c-Myc, Nanog and Oct4), stem cell markers (CD24 and CD133), components of Shh pathway (Gli1, Gli2, Patched1/2, and Smoothened), Gli targets (Bcl-2, XIAP and Cyclin D1), and EMT markers and transcription factors (N-cadherin, Slug, Snail and Zeb1), and upregulation of E-cadherin. Overall, our data suggest that Mang-NPs can inhibit pancreatic cancer growth, development and metastasis by targeting Shh pathway.

Abstract 2074: Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic cancer stem cell characteristics by suppressing sonic hedgehog pathway

Abstract Pancreatic cancer is the fourth leading cause of cancer-related deaths in US, with a 5-year survival rate of approximately 6%. Patients develop resistance to currently used anticancer drugs which are mainly toxic and also demonstrate severe side effects. Cancer stem cells (CSCs) / tumor initiating cells (TICs) have been proposed for tumor initiation, promotion, metastasis, and chemotherapy failure. Thus, there is an urgent need to develop novel strategies for the management of pancreatic cancer. Sonic hedgehog (Shh) pathway has been implicated in pancreatic carcinogenesis and is constitutively active in CSCs. The α-Mangostin is derived from the plant mangosteen (Garcinia mangostana) which is a tropical evergreen tree. Mangosteen contains Mangnoids, such as α-Mangostin, and other phytochemicals. The main objective of the study was to demonstrate the biological activities of α-Mangostin-encapsulated PLGA nanoparticles (Mang-NPs) in human pancreatic CSCs and cells lines. We have also performed docking of α-Mangostin on to the DNA binding sites of Gli to assess whether α-Mangostin disrupts Gli-DNA binding and acts as a Gli inhibitor. Mang-NPs inhibited cell proliferation, colony formation, and induced apoptosis in pancreatic cancer AsPC-1, PANC-1 and Mia-Paca-2 cell lines, and CSCs. However, Mang-NPs had no effect on human pancreatic normal ductal epithelial cells. In addition, Mang-NPs inhibited epithelial to mesenchymal transition. α-Mangostin disrupted Gli-DNA binding activity. Western blot analysis has demonstrated that Mang-NPs inhibited pluripotency maintaining factors Nanog and c-Myc. Furthermore, Mang-NPs inhibited the expression of Gli1, Gli2, Patched1 and Patched2, and Gli transcriptional activity in pancreatic CSCs. Our data demonstrate that Mang-NPs can inhibit pancreatic CSC characteristics by disrupting Gli-DNA binding activity and may act as a Gli transcription inhibitor. Citation Format: Wei Yu, Yiming Ma, Sharmila Shankar, Rakesh K. Srivastava. Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic cancer stem cell characteristics by suppressing sonic hedgehog pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2074.

PI3K/AKT/mTOR and sonic hedgehog pathways cooperate together to inhibit human pancreatic cancer stem cell characteristics and tumor growth.

Cancer stem cells (CSCs) play major roles in cancer initiation, progression, and metastasis. It is evident from growing reports that PI3K/Akt/mTOR and Sonic Hedgehog (Shh) signaling pathways are aberrantly reactivated in pancreatic CSCs. Here, we examined the efficacy of combining NVP-LDE-225 (PI3K/mTOR inhibitor) and NVP-BEZ-235 (Smoothened inhibitor) on pancreatic CSCs characteristics, microRNA regulatory network, and tumor growth. NVP-LDE-225 co-operated with NVP-BEZ-235 in inhibiting pancreatic CSC's characteristics and tumor growth in mice by acting at the level of Gli. Combination of NVP-LDE-225 and NVP-BEZ-235 inhibited self-renewal capacity of CSCs by suppressing the expression of pluripotency maintaining factors Nanog, Oct-4, Sox-2 and c-Myc, and transcription of Gli. NVP-LDE-225 co-operated with NVP-BEZ-235 to inhibit Lin28/Let7a/Kras axis in pancreatic CSCs. Furthermore, a superior interaction of these drugs was observed on spheroid formation by pancreatic CSCs isolated from Pankras/p53 mice. The combination of these drugs also showed superior effects on the expression of proteins involved in cell proliferation, survival and apoptosis. In addition, NVP-LDE-225 co-operated with NVP-BEZ-235 in inhibiting EMT through modulation of cadherin, vimentin and transcription factors Snail, Slug and Zeb1. In conclusion, these data suggest that the combined inhibition of PI3K/Akt/mTOR and Shh pathways may be beneficial for the treatment of pancreatic cancer.

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

The impact of octamer-binding transcription factor 4 and Nanog gene on pancreatic cancer stem cells in vivo

Objective To studythe impact of octamer- binding transcription factor 4(Oct- 4) and Nanog gene on the biological characteristics of pancreatic cancer stem cells(PCSCs) in vivo. Methods The CD44+ CD24+ epithelial specific antigen(ESA)+ pancreatic cancer stem cells were sorted out via flow cytometry.The expression of Oct4 and Nanog genes in PCSCs were silenced by specific short hairpin RNA(shRNA)lentiviral vector.The interfering efficiency was detected by western blotting assay.The ectopic xenograft model was established via subcutaneous and intraperitoneal injection of Oct- 4 and Nanog silenced PCSCs, normal PCSCs and PANC- 1 cells into BALB/c nude mice, respectively. The influence of Oct4 and Nanog silencing on tumorigenicity, drug resistance and invasiveness were observed in vivo. Results The PCSCs accounted for 0. 1%-0. 8% in PANC- 1 cells.The efficiency of sh RNA silencing Oct4 and Nanog were(46. 00±0. 08)% and(78. 00±0. 12)% respectively.Silencing of Oct4 and Nanog significantly inhibited the tumorigenicity and invasiveness of PCSCs in nude mice and reversed the resistance to gemcitabine. Conclusion The lentiviral vector- mediated silencing of Oct4 and Nanog could inhibit the stem cell- like biological characteristics of PCSCs in vivo. Key words: Pancreatic cancer; Cancer stem cells; Octamer-binding transcription factor 4; Nanog; Gene silencing

Abstract 4123: Exploiting sonic hedgehog pathway in pancreatic carcinogenesis as a potential target for chemoprevention

Abstract Dysregulation of the sonic hedgehog (Shh) signaling pathway has been associated with cancer stem cells (CSC) and implicated in the initiation of pancreatic cancer. The lethal nature of pancreatic cancer stems from its propensity to rapidly disseminate accompanied by resistance to current therapies contributes to the high mortality in patients with pancreatic cancer. Thus, the objective of this study was to investigate the role of Shh pathway in pancreatic cancer and to examine the molecular mechanisms by which sulforaphane (SFN), an active compound in cruciferous vegetables, inhibits self-renewal capacity of human pancreatic CSCs. Interestingly, we demonstrate here that Shh pathway is highly activated in pancreatic CSCs and plays important role in maintaining stemness by regulating the expression of stemness genes. Given the requirement for Hedgehog in pancreatic cancer, we investigated whether hedgehog blockade by SFN could target the stem cell population in pancreatic cancer. In an in vitro model, human pancreatic CSCs derived spheres were significantly inhibited on treatment with SFN, suggesting the clonogenic depletion of the CSCs. Interestingly, SFN inhibited the components of Shh pathway and Gli transcriptional activity. Interference of Shh-Gli signaling significantly blocked SFN-induced inhibitory effects demonstrating the requirement of an active pathway for the growth of pancreatic CSCs. We also show here for the first time, that sulforaphane treatment resulted in a significant reduction in the tumor growth, of orthotopically implanted primary pancreatic CSCs isolated from human pancreatic tumors into the pancreas of NOD/SCID/IL2Rgamma mice, which is mediated through the modulation of Sonic hedgehog-GLI signaling. Hedgehog pathway blockade by SFN at a dose of 20 mg/kg resulted in a 45% reduction in growth of pancreatic cancer tumors. SFN also inhibited downstream targets of Gli transcription by suppressing the expression of pluripotency maintaining factors (Nanog and Oct-4) as well as PDGFRα and Cyclin D1. Furthermore, SFN treatment resulted in a significant reduction in EMT markers Zeb-1, which correlated with increase in E-Cadherin expression suggesting the blockade of signaling involved in early metastasis. Interestingly, SFN downregulated the expression of Bcl-2 and XIAP to induce apoptosis. Our data reveal the essential role of Shh-Gli signaling in controlling the characteristics of pancreatic CSCs. Such information will not only allow rational design of SFN-based strategies for prevention and/ or treatment of pancreatic cancer but could also facilitate development of mechanism-driven protocols for optimal clinical effects. Thus Sulforaphane potentially represents an inexpensive, safe and effective alternative for the management of pancreatic cancer. Citation Format: Rakesh Srivastava, Sharmila Shankar. Exploiting sonic hedgehog pathway in pancreatic carcinogenesis as a potential target for chemoprevention. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4123. doi:10.1158/1538-7445.AM2014-4123

GLRX3, a novel secretory biomarker of pancreatic cancer based on pancreatic cancer stem cell characteristics

GLRX3, a novel secretory biomarker of pancreatic cancer based on pancreatic cancer stem cell characteristics

Abstract 200: EGCG inhibits pancreatic cancer stem cell characteristics in human primary tumors, and carcinogenesis in KrasG12D transgenic mice.

Abstract EGCG, a component of green tea, exerts antiproliferative and proapoptotic actions on a number of different cancer cell lines through diverse mechanisms. However, the mechanisms by which EGCG inhibits cell proliferation and induces apoptosis are poorly understood. The objectives of this study were to examine the molecular mechanisms by which EGCG inhibits stem cell characteristics of pancreatic cancer stem cells (CSCs) derived from human primary tumors and carcinogenesis in KrasG12D transgenic mice. Human pancreatic CSCs (CD133+CD44+CD24+ESA+) are highly tumorigenic and form subcutaneous tumors in NOD/SCID IL2R gamma mice. Human pancreatic CSCs expressing high levels of CD133, CD24, CD44, ESA, and aldehyde dehydrogenase also express significantly more Nanog, Oct-4, Notch1, MDR1 and ABCG2 than normal pancreatic tissues and primary pancreatic cancer cells. Similarly, CSCs from KrasG12D mice express significantly high levels of pluripotency maintaining factors than pancreatic tissues from Pdx-Cre mice. EGCG inhibits the growth (size and weight) and development (PanIN lesions) of pancreatic cancer in KrasG12D mice by suppressing the activation of Kras, ERK, AKT and FoxO. EGCG inhibits the self-renewal capacity of pancreatic CSCs derived from human primary tumors and KrasG12D mice. EGCG inhibits pluripotency maintaining factors (Nanog, Sox-2, c-Myc and Oct-4) and drug resistance gene ABCG2 in CSCs. Inhibition of Nanog by shRNA enhances the inhibitory effects of EGCG on self-renewal capacity of CSCs. Finally, EGCG inhibits CSC's migration and invasion and markers of epithelial-mesenchymal transition (Zeb-1, Slug and Snail). These data suggest that EGCG can inhibit pancreatic cancer development in human and KrasG12D transgenic mice by inhibiting stem cell characteristics. In conclusion, EGCG can be used for the management of pancreatic cancer. Citation Format: Luke Marsh, Su-Ni Tang, Dara Nall, Chris Jackman, Sharmila Shankar, Rakesh K. Srivastava. EGCG inhibits pancreatic cancer stem cell characteristics in human primary tumors, and carcinogenesis in KrasG12D transgenic mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 200. doi:10.1158/1538-7445.AM2013-200

Abstract 3685: Targeting of pancreatic cancer stem cell self-renewal by Sulforaphane involves modulation of Sonic hedgehog signaling.

Abstract Pancreatic CSCs are rare tumor cells characterized by their ability to self-renew, are responsible for tumor recurrence accompanied by resistance to current therapies. The lethality of these incurable, aggressive and invasive pancreatic tumors remains a daunting clinical challenge. Dysregulation of the sonic hedgehog (Shh) signaling pathway has been associated with cancer stem cells (CSC) and implicated in the initiation of pancreatic cancer. Thus, the objective of this study was to investigate the role of Shh pathway in pancreatic cancer and to examine the molecular mechanisms by which sulforaphane (SFN), an active compound in cruciferous vegetables, inhibits self-renewal capacity of human pancreatic CSCs. Interestingly, we demonstrate here that Shh pathway is highly activated in pancreatic CSCs and plays important role in maintaining stemness by regulating the expression of stemness genes. Given the requirement for Hedgehog in pancreatic cancer, we investigated whether hedgehog blockade by SFN could target the stem cell population in pancreatic cancer. In an in vitro model, human pancreatic CSCs derived spheres were significantly inhibited on treatment with SFN, suggesting the clonogenic depletion of the CSCs. Interestingly, SFN inhibited the components of Shh pathway and Gli transcriptional activity. Interference of Shh-Gli signaling significantly blocked SFN-induced inhibitory effects demonstrating the requirement of an active pathway for the growth of pancreatic CSCs. We also show here for the first time, that sulforaphane treatment resulted in a significant reduction in the tumor growth, of orthotopically implanted primary pancreatic CSCs isolated from human pancreatic tumors into the pancreas of NOD/SCID/IL2Rgamma mice, which is mediated through the modulation of Sonic hedgehog-GLI signaling. Hedgehog pathway blockade by SFN at a dose of 20 mg/kg resulted in a 45% reduction in growth of pancreatic cancer tumors. SFN also inhibited downstream targets of Gli transcription by suppressing the expression of pluripotency maintaining factors (Nanog and Oct-4) as well as PDGFRα and Cyclin D1. Furthermore, SFN treatment resulted in a significant reduction in EMT markers Zeb-1, which correlated with increase in E-Cadherin expression suggesting the blockade of signaling involved in early metastasis. Interestingly, SFN down regulated the expression of Bcl-2 and XIAP to induce apoptosis. Our data reveal the essential role of Shh-Gli signaling in controlling the characteristics of pancreatic CSCs. Such information will not only allow rational design of SFN-based strategies for prevention and/ or treatment of pancreatic cancer but could also facilitate development of mechanism-driven protocols for optimal clinical effects. Thus Sulforaphane potentially represents an inexpensive, safe and effective alternative for the management of pancreatic cancer. Citation Format: Rakesh Srivastava, Sharmila Shankar. Targeting of pancreatic cancer stem cell self-renewal by Sulforaphane involves modulation of Sonic hedgehog signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3685. doi:10.1158/1538-7445.AM2013-3685

Inhibition of pancreatic cancer stem cell characteristics and tumor growth by Gli inhibitor GANT61

s / Pancreatology 13 (2013) e1–e94 e11 mTOR signaling in PaSC. Further, conditioned medium from PanIN cells isolated from KRAS mice enhanced fibrogenic responses in PaSC. Histological analysis of tissue microarrays of patient-matched normal human pancreas parenchyma and PanIN lesions showed profuse active PaSC surrounding both low-grade and advanced PanINs, and acini in adjacent normal parenchymal sections. Conclusions: Our data support a key role for Akt/mTOR signaling in PaSC fibrogenic responses, and indicate that interplay between PanINs and PaSC provide the mechanistic underpinnings of environmental risk factor promotion of tumor progression.

Sonic hedgehog signaling inhibition provides opportunities for targeted therapy by sulforaphane in regulating pancreatic cancer stem cell self-renewal.

Dysregulation of the sonic hedgehog (Shh) signaling pathway has been associated with cancer stem cells (CSC) and implicated in the initiation of pancreatic cancer. Pancreatic CSCs are rare tumor cells characterized by their ability to self-renew, and are responsible for tumor recurrence accompanied by resistance to current therapies. The lethality of these incurable, aggressive and invasive pancreatic tumors remains a daunting clinical challenge. Thus, the objective of this study was to investigate the role of Shh pathway in pancreatic cancer and to examine the molecular mechanisms by which sulforaphane (SFN), an active compound in cruciferous vegetables, inhibits self-renewal capacity of human pancreatic CSCs. Interestingly, we demonstrate here that Shh pathway is highly activated in pancreatic CSCs and plays important role in maintaining stemness by regulating the expression of stemness genes. Given the requirement for Hedgehog in pancreatic cancer, we investigated whether hedgehog blockade by SFN could target the stem cell population in pancreatic cancer. In an in vitro model, human pancreatic CSCs derived spheres were significantly inhibited on treatment with SFN, suggesting the clonogenic depletion of the CSCs. Interestingly, SFN inhibited the components of Shh pathway and Gli transcriptional activity. Interference of Shh-Gli signaling significantly blocked SFN-induced inhibitory effects demonstrating the requirement of an active pathway for the growth of pancreatic CSCs. SFN also inhibited downstream targets of Gli transcription by suppressing the expression of pluripotency maintaining factors (Nanog and Oct-4) as well as PDGFRα and Cyclin D1. Furthermore, SFN induced apoptosis by inhibition of BCL-2 and activation of caspases. Our data reveal the essential role of Shh-Gli signaling in controlling the characteristics of pancreatic CSCs. We propose that pancreatic cancer preventative effects of SFN may result from inhibition of the Shh pathway. Thus Sulforaphane potentially represents an inexpensive, safe and effective alternative for the management of pancreatic cancer.

Abstract 713: GLRX3, a secretory biomarker of pancreatic cancer based on pancreatic cancer stem cell characteristics

Abstract A. BACKGROUND/AIMS Pancreatic cancer is one of the most lethal diseases, which is difficult to diagnose and resistant to conventional treatment such as chemotherapy and radiotherapy. Cancer stem cells are involved in carcinogenesis, cancer progression and recurrence. We tried to find secretory biomarkers associated with pancreatic cancer stem cells using proteomic analysis. B. METHODS Pancreatic cancer stem like cells were enriched using sphere culture method. Proteomic analysis was done with secreted protein in culture medium of sphere and adherent cells. Identified proteins were confirmed with Western blot and immunohistochemical staining. ELISA was done to detect blood levels of validated proteins in pancreatic cancer patients. shRNA for GLRX3 was used to study the function of GLRX3 in pancreatic cancer. C. RESULTS Proteomic analysis showed that total of 55 protein spots from sphere cells were increased compared to adherent cells. Among them, 41 spots were identified using MALDI-TOF. Proteins known to be associated with cancer stem cells such as HSP90AB1, ALDH, and vimentin were expressed in sphere cells. The overexpression of 5 proteins including GLRX3 was confirmed by western blot in sphere cells and patient serum. Immunohistochemical staining in TMA of human pancreatic cancer tissue revealed that GLRX3 was detected in 15 (57.7%) out of 25 pancreatic cancer tissues regardless of cancer stages. To examine GLRX3 involvement in pancreatic carcinogenesis, we used shRNA to generate GLRX3-knockdown cells. Compared to the mock, knockdown of GLRX3 in human pancreatic cancer cell lines decreased in vitro proliferation, clonogenecity, and sphere formation. GLRX3 knockdown reverses epithelial-mesenchymal transition in pancreatic cancer cells. ELISA showed patients with unresectable pancreatic cancer and high blood GLRX3 levels had poor survival compared to those with low blood GLRX3 levels (155 days vs 463 days, p-value <0.000). D. CONCLUSION This study shows that secretory protein GLRX3 may be a useful prognostic marker in pancreatic cancer. More researches are needed to find their roles in cancer stem cells and clinical implication. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 713. doi:1538-7445.AM2012-713