Finding and Killing the CRABs of Pancreatic Cancer

Abstract

See “Combined targeted treatment to eliminate tumorigenic cancer stem cells in human pancreatic cancer” by Mueller M–T, Hermann PC, Witthauer J, et al, on page 1102. See “Combined targeted treatment to eliminate tumorigenic cancer stem cells in human pancreatic cancer” by Mueller M–T, Hermann PC, Witthauer J, et al, on page 1102. Pancreatic ductal adenocarcinoma (PDA) is a rapidly lethal disease, largely because it is resistant to therapeutic intervention. Potential explanations for medical failure in PDA include classical pharmacological resistance,1Li D. Xie K. Wolff R. et al.Pancreatic cancer.Lancet. 2004; 363: 1049-1057Abstract Full Text Full Text PDF PubMed Scopus (1750) Google Scholar insufficient drug delivery,2Olive K.P. Jacobetz M.A. Davidson C.J. et al.Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer.Science. 2009; 324: 1457-1461Crossref PubMed Scopus (2448) Google Scholar and innate cellular features such as activated survival pathways.3Bhanot U. Heydrich R. Moller P. et al.Survivin expression in pancreatic intraepithelial neoplasia (PanIN): steady increase along the developmental stages of pancreatic ductal adenocarcinoma.Am J Surg Pathol. 2006; 30: 754-759Crossref PubMed Scopus (46) Google Scholar Recently, several groups reported that a subpopulation of PDA cells possess enhanced resilience to various stresses, and such cells are termed cancer stem cells (CSC), because their tenaciousness could lead logically to the continual regeneration of the neoplasm.4Li C. Heidt D.G. Dalerba P. et al.Identification of pancreatic cancer stem cells.Cancer Res. 2007; 67: 1030-1037Crossref PubMed Scopus (2764) Google Scholar, 5Hermann P.C. Huber S.L. Herrler T. et al.Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer.Cell Stem Cell. 2007; 1: 313-323Abstract Full Text Full Text PDF PubMed Scopus (2284) Google Scholar The CSC premise posits that tumors contain a hierarchical arrangement of cells to ensure tumor propagation, with the core or “stem” population directing a pattern of asymmetric proliferation and differentiation akin to the developmental models proposed for stem cells in normal tissues.6Bonnet D. Dick J.E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.Nat Med. 1997; 3: 730-737Crossref PubMed Scopus (5490) Google Scholar, 7Reya T. Morrison S.J. Clarke M.F. et al.Stem cells, cancer, and cancer stem cells.Nature. 2001; 414: 105-111Crossref PubMed Scopus (7887) Google Scholar The small fraction of CSCs are projected to have a slower proliferation rate than the majority of the bulk tumor, thus minimizing the effects of cytotoxic drugs owing to longer times available for DNA damage repair. In addition, CSCs have been reported to express drug efflux pumps,8Chaudhary P.M. Roninson I.B. Expression and activity of P-glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells.Cell. 1991; 66: 85-94Abstract Full Text PDF PubMed Scopus (904) Google Scholar, 9Schatton T. Frank M.H. Cancer stem cells and human malignant melanoma.Pigment Cell Melanoma Res. 2008; 21: 39-55Crossref PubMed Scopus (168) Google Scholar thereby contributing to the evasion of cytotoxic and targeted drugs. Such characteristics of CSCs offer explanations for both therapeutic resistance and tumor dormancy, central problems in the treatment of most incurable neoplasms. Two prior studies had reported the isolation of CSCs from PDA, characterized by an increased ability of that population to survive orthotopic transplantation in an immunocompromised mouse. CSCs expressed either CD24/44/ESA4Li C. Heidt D.G. Dalerba P. et al.Identification of pancreatic cancer stem cells.Cancer Res. 2007; 67: 1030-1037Crossref PubMed Scopus (2764) Google Scholar or CD1335Hermann P.C. Huber S.L. Herrler T. et al.Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer.Cell Stem Cell. 2007; 1: 313-323Abstract Full Text Full Text PDF PubMed Scopus (2284) Google Scholar and, consistent with the concept that CSCs harbor therapeutic resistance properties, gemcitabine treatment increased the proportion of the tumor cells that express these markers.5Hermann P.C. Huber S.L. Herrler T. et al.Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer.Cell Stem Cell. 2007; 1: 313-323Abstract Full Text Full Text PDF PubMed Scopus (2284) Google Scholar Given the resistance of these CSC to therapeutic intervention, Mueller et al10Mueller M–T. Hermann P.C. Witthauer J. et al.Combined targeted treatment to eliminate tumorigenic cancer stem cells in human pancreatic cancer.Gastroenterology. 2009; 137: 1102-1113Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar set out to design rational therapeutic strategies based upon the known or suspected biology of CSCs, as reported in this issue of Gastroenterology. Because CSCs were previously reported to express elevated Hedgehog (Hh) ligand expression, CD133+ CSC were treated with several Hh pathway inhibitors alone and in combination with gemcitabine and the Raptor/TOR inhibitor rapamycin. Herein, Mueller et al10Mueller M–T. Hermann P.C. Witthauer J. et al.Combined targeted treatment to eliminate tumorigenic cancer stem cells in human pancreatic cancer.Gastroenterology. 2009; 137: 1102-1113Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar report that combined treatment with all 3 agents led to a profound depletion of the CSC compartment, shrinkage of established tumors, reduction in tumor metastasis, and a remarkably prolonged survival benefit. These exciting results prompt several questions regarding the general properties of CSCs and their relationship to pancreatic cancer, and consideration of such might assist in the optimal translation of these findings to the clinic. First, do all PDA cells have the potential of being a CSC? Recently, Morrison et al reported that unsorted melanoma cells are highly tumorigenic when implanted with matrigel in an extremely immunocompromised mouse, such that one third of cells can develop into a xenograft.11Quintana E. Shackleton E. Sabel M.S. et al.Efficient tumor formation by single human melanoma cells.Nature. 2008; 456: 593-598Crossref PubMed Scopus (1487) Google Scholar If applicable to epithelial tumors such as PDA, then this level of CSC content makes the term superfluous. One possible explanation for having such a high degree of “stemness” is that neoplastic cells are never fixed in a stem cell state, but rather exist in a dynamic equilibrium with non–stem cells owing to stochastic epigenetic alterations and the microenvironment. Indeed, it was reported recently that immortalized mammary epithelial cells acquired stem-like properties upon the induction of epithelial-mesenchymal transition by transforming growth factor-β exposure or the ectopic expression of Snail or Slug.12Mani S.A. Guo W. Liao M.J. et al.The epithelial-mesenchymal transition generates cells with properties of stem cells.Cell. 2008; 133: 704-715Abstract Full Text Full Text PDF PubMed Scopus (6787) Google Scholar An alternative explanation is that a stem cell phenotype is inconsequential for the proliferation of neoplastic cells in vitro and propagation in vivo because uncontrolled cellular proliferation is a cardinal feature of most cancer cells. Rather, stemness per se becomes relevant when environmental stress is present. Because the assays that are used traditionally to measure stemness in neoplasms are reflective directly of the survival capabilities of the cancer cells when insulted with chemotherapy, radiation, or transplantation, the CSCs in solid tumors might be more aptly described as chemoradiotherapy resistant anoikis blunted cells (CRABs). CRABs would then denote the subpopulation of cells harboring increased stress survival properties at any given moment, and all cancer cells possess the potential of becoming CRABs (Figure 1). This would distinguish CRABs thematically more clearly from normal tissue stem cells. Second, is the hedgehog pathway a cell-autonomous target in neoplastic PDA cells? Because cyclopamine and other smoothened receptor inhibitors work at the level of ligand-dependent Hh signaling, it is important to determine whether CSCs/CRABs requires intact smoothened function to mediate their effects. Initial mouse models and cell culture work supported the concept that the Hh pathway is operant in a cell-autonomous fashion in the development of pancreatic metaplasia13Thayer S.P. di Magliano M.P. Heiser P.W. et al.Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis.Nature. 2003; 425: 851-856Crossref PubMed Scopus (1317) Google Scholar and pancreatic intraepithelial neoplasms (PanIN) and PDA.14Argani P. Rosty C. Reiter R.E. et al.Discovery of new markers of cancer through serial analysis of gene expression: prostate stem cell antigen is overexpressed in pancreatic adenocarcinoma.Cancer Res. 2001; 61: 4320-4324PubMed Google Scholar Additionally, ectopic Gli-1,15Prasad N.B. Biankin A.V. Fukushima N. et al.Gene expression profiles in pancreatic intraepithelial neoplasia reflect the effects of Hedgehog signaling on pancreatic ductal epithelial cells.Cancer Res. 2005; 65: 1619-1626Crossref PubMed Scopus (199) Google Scholar, 16Pasca di Magliano M. Biankin A.V. Heiser P.W. et al.Common activation of canonical Wnt signaling in pancreatic adenocarcinoma.PLoS One. 2007; 2: e1155Crossref PubMed Scopus (187) Google Scholar Gli-2,17Pasca di Magliano M. Sekine S. Ermilov A. et al.Hedgehog/Ras interactions regulate early stages of pancreatic cancer.Genes Dev. 2006; 20: 3161-3173Crossref PubMed Scopus (260) Google Scholar and Shh18Morton J.P. Mongeau M.E. Klimstra D.A. et al.Sonic hedgehog acts at multiple stages during pancreatic tumorigenesis.Proc Natl Acad Sci U S A. 2007; 104: 5103-5108Crossref PubMed Scopus (190) Google Scholar increased the transformation of human and murine ductal epithelial cells in vitro and in vivo; whereas conversely smoothened inhibitors decreased proliferation and Gli target gene expression in PDA cells.13Thayer S.P. di Magliano M.P. Heiser P.W. et al.Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis.Nature. 2003; 425: 851-856Crossref PubMed Scopus (1317) Google Scholar, 19Feldmann G. Fendrich V. McGovern K. et al.An orally bioavailable small-molecule inhibitor of Hedgehog signaling inhibits tumor initiation and metastasis in pancreatic cancer.Mol Cancer Ther. 2008; 7: 2725-2735Crossref PubMed Scopus (234) Google Scholar Furthermore, Shh ligand expression is very high in CD24/CD44/ESA+ PDA cells4Li C. Heidt D.G. Dalerba P. et al.Identification of pancreatic cancer stem cells.Cancer Res. 2007; 67: 1030-1037Crossref PubMed Scopus (2764) Google Scholar and another CSC-like compartment defined by high ALDH expression,20Feldmann G. Dhara S. Fendrich V. et al.Blockade of hedgehog signaling inhibits pancreatic cancer invasion and metastases: a new paradigm for combination therapy in solid cancers.Cancer Res. 2007; 67: 2187-2196Crossref PubMed Scopus (613) Google Scholar reinforcing the potential importance of this pathway and collectively suggesting that the Hh pathway functions in an autocrine fashion in PDA. Indeed, Mueller et al10Mueller M–T. Hermann P.C. Witthauer J. et al.Combined targeted treatment to eliminate tumorigenic cancer stem cells in human pancreatic cancer.Gastroenterology. 2009; 137: 1102-1113Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar show a clear reduction in metastatic potential when CD133+ CSCs are pretreated in culture with cyclopamine based regimens. However, recent findings have revealed that the Hh pathway functions also in a paracrine manner in PDA. First, smoothened inhibitors were shown to function by targeting stromal cells and not epithelial cells in xenografts21Yauch R.L. Gould S.E. Scales S.J. et al.A paracrine requirement for hedgehog signalling in cancer.Nature. 2008; 455: 406-410Crossref PubMed Scopus (822) Google Scholar with Gli activity only detectable in the neighboring fibroblasts.22Tian H. Callahan C.A. DuPree K.J. et al.Hedgehog signaling is restricted to the stromal compartment during pancreatic carcinogenesis.Proc Natl Acad Sci U S A. 2009; 106: 4254-4259Crossref PubMed Scopus (344) Google Scholar Second, the ectopic expression of Shh-1 was shown to induce a proliferative desmoplastic stroma in orthotopic tumors.23Bailey J.M. Swanson B.J. Hamada T. et al.Sonic hedgehog promotes desmoplasia in pancreatic cancer.Clin Cancer Res. 2008; 14: 5995-6004Crossref PubMed Scopus (417) Google Scholar Third, primary cilia, the cellular structure required to assemble the Smoothened-Patched receptors to receive extracellular stimulation, are absent on PanIN and PDA cells.24Seeley E.S. Carriere C. Goetze T. et al.Pancreatic cancer and precursor pancreatic intraepithelial neoplasia lesions are devoid of primary cilia.Cancer Res. 2009; 69: 422-430Crossref PubMed Scopus (193) Google Scholar Fourth, the genetic ablation of smoothened in mouse models in pancreatic progenitor cells did not delay the development of PanIN or PDA, demonstrating that the ligand-dependent stimulation by Hh was unnecessary for tumor formation. Finally, we reported recently that although smoothened inhibitors did not diminish the proliferation of PDA cells, they had a profound impact on the proliferation and intratumoral content of stromal fibroblasts.2Olive K.P. Jacobetz M.A. Davidson C.J. et al.Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer.Science. 2009; 324: 1457-1461Crossref PubMed Scopus (2448) Google Scholar Although these studies do not refute the importance of ligand-independent Gli signaling in PanIN or PDAC cells,25Nolan-Stevaux O. Lau J. Truitt M.L. et al.GLI1 is regulated through Smoothened-independent mechanisms in neoplastic pancreatic ducts and mediates PDAC cell survival and transformation.Genes Dev. 2009; 23: 24-36Crossref PubMed Scopus (330) Google Scholar they serve to redirect our focus regarding Hh inhibitors to the tumor microenvironment interface. Genetic interference of Smoothened in human PDA cells prior to the transplantation assays could clarify quickly whether this point is directly relevant for CSCs/CRABs. An intriguing possibility is that CSCs/CRABs may be the most capable of recruiting stromal myofibroblasts through the elevated secretion of Shh, and this may be one important survival characteristic of such cells. Third, should more attention be directed to the development of PDA to understand the origins of CSCs/CRABs? Cancer initiation is a fundamentally different process than cancer progression as the former requires a single genetically altered cell to overcome intrinsic tumor suppressive mechanisms, outcompete adjacent wild-type cells, and yet still evade immune surveillance, stromal inhibition, and epithelial neighbor suppression. Thus, the question remains, what is the CD133 positive counterpart in the wild-type pancreas and the cell of origin in PDA? Possible candidates for the CD133-positive counterpart in the pancreas include the islets of Langerhans, the acinar cells, and the ductal cells; however, there are also several progenitor cell types within the pancreas, specifically those labeled by Bmi1, Hes1, and Nestin. The advent of genetically engineered mouse models has enabled in vivo evaluation of these cell types. Islet β-cells, despite their in vitro plasticity, are an unlikely source for the origins of PDA. Ectopic expression of mutant Hras in β-cells results in diabetes26Efrat S. Fleischer N. Hanahan D. Diabetes induced in male transgenic mice by expression of human H-ras oncoprotein in pancreatic beta cells.Mol Cell Biol. 1990; 10: 1779-1783PubMed Google Scholar and more recent lineage tracing experiments have demonstrated that β-cells contribute to neither acinar nor ductal regeneration in vivo.27Strobel O. Dor Y. Stirman A. et al.Beta cell transdifferentiation does not contribute to preneoplastic/metaplastic ductal lesions of the pancreas by genetic lineage tracing in vivo.Proc Natl Acad Sci U S A. 2007; 104: 4419-4424Crossref PubMed Scopus (48) Google Scholar The acinar compartment has been a favored cell type of origin for PDA and its attendant preneoplasm, PanIN. Guerra et al28Guerra C. Schumacher A.J. Canamero M. et al.Chronic pancreatitis is essential for induction of pancreatic ductal adenocarcinoma by K-Ras oncogenes in adult mice.Cancer Cell. 2007; 11: 291-302Abstract Full Text Full Text PDF PubMed Scopus (877) Google Scholar found that the differentiated acinar compartment could only be induced to form PanIN and PDA in the context of chronic pancreatitis, highlighting the importance of non–cell-autonomous factors in cancer initiation. More recently, using a tamoxifen-activatable Cre allele driven from the acinar-specific Elastase promoter, 2 studies29De La O.J.P. Emerson L.L. Goodman J.L. et al.Notch and Kras reprogram pancreatic acinar cells to ductal intraepithelial neoplasia.Proc Natl Acad Sci U S A. 2008; 105: 18907-18912Crossref PubMed Scopus (314) Google Scholar, 30Habbe N. Shi G. Mequid R.A. et al.Spontaneous induction of murine pancreatic intraepithelial neoplasia (mPanIN) by acinar cell targeting of oncogenic Kras in adult mice.Proc Natl Acad Sci U S A. 2008; 105: 18913-18918Crossref PubMed Scopus (302) Google Scholar have revealed that oncogenic Kras can initiate PanIN in the absence of pancreatitis, further supporting the claim that an adult acinar cell can be the cell of origin for PDA. Interestingly, Sangiorgi and Capecchi31Sangiorgi E. Capecchi M.R. Bmi1 lineage tracing identifies a self-renewing pancreatic acinar cell subpopulation capable of maintaining pancreatic organ homeostasis.Proc Natl Acad Sci U S A. 2009; 106: 7101-7106Crossref PubMed Scopus (77) Google Scholar found that, within the adult acinar compartment, a subpopulation of Bmi1-positive cells are able to maintain long-term acinar cell homeostasis.31Sangiorgi E. Capecchi M.R. Bmi1 lineage tracing identifies a self-renewing pancreatic acinar cell subpopulation capable of maintaining pancreatic organ homeostasis.Proc Natl Acad Sci U S A. 2009; 106: 7101-7106Crossref PubMed Scopus (77) Google Scholar Thus, an Elastase-positive acinar cell that was also Bmi1 positive (and therefore progenitor-like) may have been the cell of origin in the previous 2 studies, confounding the conclusion that fully differentiated acinar cells give rise to PanIN and PDA. Similarly, Nestin- and Hes1-positive cells have both been implicated in PanIN initiation,32Carriere C. Seeley E.S. Goetze T. et al.The Nestin progenitor lineage is the compartment of origin for pancreatic intraepithelial neoplasia.Proc Natl Acad Sci U S A. 2007; 104: 4437-4442Crossref PubMed Scopus (106) Google Scholar, 33Stanger B.Z. Stiles B. Lauwers G.Y. et al.Pten constrains centroacinar cell expansion and malignant transformation in the pancreas.Cancer Cell. 2005; 8: 185-195Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar yet Nestin-positive exocrine progenitors are mutually exclusive of Hes1-positive centroacinar cells, highlighting the potential inherent plasticity of the pancreas. Although pancreatic ductal cells remain an attractive candidate for the cell of origin of PanIN and PDA given the ductal characteristics of the disease, unfortunate limitations in mouse modeling have prevented thorough evaluation of this cell type. Oncogenic Kras ectopically expressed by the pancreatic ductal specific promoter cytokeratin 19 does not result in PanIN or PDA.34Brembeck F.H. Schreiber F.S. Deramaudt T.B. et al.The mutant K-ras oncogene causes pancreatic periductal lymphocytic infiltration and gastric mucous neck cell hyperplasia in transgenic mice.Cancer Res. 2003; 63: 2005-2009PubMed Google Scholar Nonetheless, it is difficult to exclude ductal cells as endogenous expression of KrasG12D has not been evaluated. Current efforts to characterize the origins of PDA should be guided by information gleaned from human studies. Mutational analysis on human PDA samples has shown that Kras mutations can be found in PanIN and their associated metaplastic regions, but never in the adjacent acinar tissue.35Shi C. Hong S.M. Lim P. et al.KRAS2 mutations in human pancreatic acinar-ductal metaplastic lesions are limited to those with PanIN: implications for the human pancreatic cancer cell of origin.Mol Cancer Res. 2009; 7: 230-236Crossref PubMed Scopus (89) Google Scholar Therefore, the cellular origins of PanIN and PDA remain the topic of much debate. Regardless of the cell type in which the initiating Kras mutation occurs, what effect will the mutation have on the behavior of the cell? Cancer initiation and progression depend on mutations conferring fitness advantages relative to their neighbors. Cells that proliferate more, live longer, or resist stress better than their neighbors will over time outcompete for space and resources within the organ, and yet, the current theory regarding pancreatic cancer initiation relies on oncogenic mutations conferring a fitness disadvantage, in the form of oncogene-induced senescence,36Collado M. Gil J. Efevan A. et al.Tumor biology: senescence in premalignant tumors.Nature. 2005; 436: 642Crossref PubMed Scopus (1180) Google Scholar relative to neighboring cells. By contrast, the CSC hypothesis allows for either a stem cell to gain oncogenic properties or for a differentiated cell to acquire ‘stemness’ or long-term regenerative potential. A recent study has demonstrated the impact of these possibilities in vivo. APC deletion in Lgr5+ intestinal crypt progenitor cells leads to rapidly expanding macroadenomas, however, with the same kinetics, deletion of APC in the transiently amplifying population led to smaller, less proliferative, yet not senescent microadenomas.37Barker N. Ridgway R.A. van Es J.H. et al.Crypt stem cells as the cells-of-origin of intestinal cancer.Nature. 2009; 457: 608-611Crossref PubMed Scopus (1639) Google Scholar In this case, 2 cellular compartments within the same organ are competent to form adenomas, but with differing characteristics. In the pancreas, determining immediate cellular behavior in response to acute KrasG12D expression could parse out the contenders for the CD133-positive wild-type counterpart with the potential that >1 lineage may acquire appropriate CSC/CRAB characteristics, and thus, lay claim to the cell of origin of PDA. Combined Targeted Treatment to Eliminate Tumorigenic Cancer Stem Cells in Human Pancreatic CancerGastroenterologyVol. 137Issue 3PreviewPancreatic cancers contain exclusively tumorigenic cancer stem cells (CSCs), which are highly resistant to chemotherapy, resulting in a relative increase in CSC numbers during gemcitabine treatment. Signaling through sonic hedgehog and mammalian target of rapamycin (mTOR), respectively, may be essential for CSC self-renewal and could represent putative targets for novel treatment modalities. Full-Text PDF