Joseph Dosch

Pancreatic Cancer Stem Cells

Cellular heterogeneity in cancer was observed decades ago by studies in mice which showed that distinct subpopulations of cells within a tumor mass are capable of driving tumorigenesis. Conceptualized from this finding was the stem-cell hypothesis for cancer, which suggests that only a specific subset of cancer cells within each tumor is responsible for tumor initiation and propagation, termed tumor initiating cells or cancer stem cells (CSCs). Recent data has been provided to support the existence of CSCs in human blood cell-derived cancers and solid organ tumors of the breast, brain, prostate, colon, and skin. Study of human pancreatic cancers has also revealed a specific subpopulation of cancer cells that possess the characteristics of CSCs. These pancreatic cancer stem cells express the cell surface markers CD44, CD24, and epithelial-specific antigen, and represent 0.5% to 1.0% of all pancreatic cancer cells. Along with the properties of self-renewal and multilineage differentiation, pancreatic CSCs display upregulation of important developmental genes that maintain self-renewal in normal stem cells, including Sonic hedgehog (SHH) and BMI-1. Signaling cascades that are integral in tumor metastasis are also upregulated in the pancreatic CSC. Understanding the biologic behavior and the molecular pathways that regulate growth, survival, and metastasis of pancreatic CSCs will help to identify novel therapeutic approaches to treat this dismal disease.

c-Met is a marker of pancreatic cancer stem cells and therapeutic target.

BACKGROUND & AIMS Growth of many different tumor types requires a population of self-renewing cancer stem cells (CSCs). c-Met is a marker of normal mouse pancreatic stem and progenitor cells; we investigated whether it is also a marker of human pancreatic CSCs that might be developed as a therapeutic target. METHODS We studied growth of primary human pancreatic adenocarcinoma in NOD SCID mice. The self-renewal capability of pancreatic cancer cells that expressed high levels of c-Met (c-Met(high)) was assessed using in vitro sphere assays and compared with those that were c-Met negative or expressed low levels of c-Met. The tumorigenicity of c-Met(high) pancreatic cancer cells was evaluated in NOD SCID mice. RESULTS c-Met(high) cells readily formed spheres, whereas c-Met-negative cells did not. Use of the c-Met inhibitor XL184 or c-Met knockdown with small hairpin RNAs significantly inhibited tumor sphere formation. c-Met(high) cells had increased tumorigenic potential in mice; those that expressed c-Met and CD44 (0.5%-5% of the pancreatic cancer cells) had the capability for self-renewal and the highest tumorigenic potential of all cell populations studied. In pancreatic tumors established in NOD SCID mice, c-Met inhibitors slowed tumor growth and reduced the population of CSCs when given alone or in combination with gemcitabine. Administration of XL184 for 2 weeks after cardiac injection of cancer cells prevented the development of metastases. CONCLUSIONS c-Met is a new marker for pancreatic CSCs. It is required for growth and metastasis of pancreatic tumors in mice and is a therapeutic target for pancreatic cancer.

Cancer stem cells: a new theory regarding a timeless disease.

Although cancer has been described in early medical texts from antiquity, it remains the second leading cause of death in the United States. Technological improvements in screening modalities have increased detection of smaller tumors, yet current therapies for most types of cancer often fail. Cancer death is usually attributable to the development of metastatic disease. Chemotherapeutic regimens target all proliferating cells based on the principle that tumor cells proliferate at a faster rate than normal cells, resulting in differential cytotoxicity. The increased proliferative capacity of cancer cells is the result of accumulated genetic insults to various cellular pathways. These mutations include those that enhance cellular proliferation or suppress normal growth inhibitory mechanisms and apoptosis. Still other mutations allow tumor cells to evade surveillance and removal by the immune system. Cumulatively, these mutations result in neoplastic tumor growth and subsequent distant metastasis. The tumor microenvironment has also emerged as a critical component in the development of cancer from benign neoplasia, both in secreted factors that modulate tumor cells and in three-dimensional interactions with extracellular matrix proteins. A paradigm shift in cancer biologists’ thinking about solid organ tumors may provide a new understanding of cancer development and progression and has implications for how we think about developing therapies to treat cancer patients. In normal tissues and organs, stem cells reside at the apex of a hierarchical scheme that drives organogenesis. The realization that tumors themselves function like complex organs birthed the theory that cancer cells with the properties of stem cells may be the key drivers of the complex machinery behind tumorigenesis. The cancer stem cell theory is based on the finding that a small number of highly tumorigenic cells within a tumor can produce the heterogeneous populations found within an entire tumor when transplanted into immunocompromised mice. Thus, it is believed that cancer stem cells (CSCs) may be responsible for tumor formation based upon their capacity for selfrenewal and differentiation. These capacities together permit asymmetric division of the stem cell, resulting in maintenance of the parental stem cell population in addition to production of differentiated progeny (Figure 1). The CSC population can be serially transplanted without loss of tumorigenicity due to its ability to undergo self-renewal. Given these findings, many have hypothesized that CSCs arise from genetic mutations that occur in normal stem cells. Normal stem cells possess the longest life span among mammalian cells, and it is thought that these cells are more likely to accumulate mutations over time and ultimately assume a malignant phenotype. Mutation of normal stem cells may create a population of CSCs that cause tumor growth as a result of altered self-renewal mechanisms, although specific mutations have yet to be demonstrated. Recent data supports such a model for colon cancer development. Barker et al. utilized a mouse model where the adenomatous polyposis coli (APC) gene of the Wnt signaling pathway was conditionally inactivated in the intestinal stem cell.1 These stem cells are located at the bottom of the intestinal crypt in both the small intestine and colon and become transformed with APC deletion within days. The transformed stem cells go on to form adenomas, in contrast to APC deletion in the progeny of the stem cells where adenomas rarely form. Alternatively, some studies suggest that CSCs may arise from mutated progenitor cells called transit amplifying cells that develop the capacity for unregulated self-renewal.2,3 Yet in other models, CSCs may arise from differentiated cells that assume a stem-cell phenotype. For example, in genetically engineered mouse models of pancreatic cancer, one study has suggested that the cell of origin of pancreatic cancer is an acinar or centroacinar cell.4 Additional work is being performed in several laboratories to verify this finding. Gene profiling experiments comparing CSCs and nonCSCs have revealed upregulation of other signaling pathways that are also found to be abnormal in human tumor specimens. These include Wnt/ -catenin, Notch, PI3KinaseAkt-mTOR, as well as the Hedgehog signaling pathway. For example, c-Myc, a downstream target of the -catenin signaling pathway, was found to be upregulated in glioma * To whom correspondence should be addressed. Mailing address: Depts. of Surgery and Molecular and Integrative Physiology, TC 2210B, Box 5343, University of Michigan Medical Center, 1500 E. Medical Center Dr., Ann Arbor, MI 48109. Phone: (734) 615-1600. Fax: (734) 936-5830, E-mail: simeone@umich.edu. † Department of Surgery. ‡ Department of Molecular and Integrative Physiology. Chem. Rev. 2009, 109, 3200–3208 3200

Sensitivity of KRAS-Mutant Colorectal Cancers to Combination Therapy That Cotargets MEK and CDK4/6

Purpose: The emerging need for rational combination treatment approaches led us to test the concept that cotargeting MEK and CDK4/6 would prove efficacious in KRAS-mutant (KRASmt) colorectal cancers, where upregulated CDK4 and hyperphosphorylated retinoblastoma (RB) typify the vast majority of tumors. Experimental Design: Initial testing was carried out in the HCT-116 tumor model, which is known to harbor a KRAS mutation. Efficacy studies were then performed with five RB+ patient-derived colorectal xenograft models, genomically diverse with respect to KRAS, BRAF, and PIK3CA mutational status. Tolerance, efficacy, and pharmacodynamic evaluation of target modulation were evaluated in response to daily dosing with either agent alone or concurrent coadministration. Results: Synergy was observed in vitro when HCT-116 cells were treated over a broad range of doses of trametinib and palbociclib. Subsequent in vivo evaluation of this model showed a higher degree of antitumor activity resulting from the combination compared to that achievable with single-agent treatment. Testing of colorectal patient-derived xenograft (PDX) models further showed that combination of trametinib and palbociclib was well tolerated and resulted in objective responses in all KRASmt models tested. Stasis was observed in a KRAS/BRAF wild-type and a BRAFmt model. Conclusions: Combination of trametinib and palbociclib was well tolerated and highly efficacious in all three KRAS-mutant colorectal cancer PDX models tested. Promising preclinical activity seen here supports clinical evaluation of this treatment approach to improve therapeutic outcome for patients with metastatic colorectal cancer. Clin Cancer Res; 22(2); 405–14. ©2015 AACR.

The kinase activity of the Ser/Thr kinase BUB1 promotes TGF-β signaling.

A kinase that controls cell division also promotes the activity of the transforming growth factor–β pathway. Placing BUB1 in the TGF-β Pathway The transforming growth factor–β (TGF-β) pathway regulates cell proliferation and migration, processes involved in development, regeneration, and tumorigenesis. The kinase BUB1, which promotes proper chromosome alignment as cells prepare to divide, also regulates cell proliferation. Nyati et al. connected BUB1 to TGF-β signaling. They found that knocking down BUB1 impaired TGF-β–mediated proliferation of tumor cells—but not by acting at chromosomes. Instead, cytoplasmic BUB1 interacted with TGF-β receptor subunits at the cell surface, promoting the interaction between receptor subunits and between the receptor and downstream signaling proteins. Inhibiting the kinase activity of BUB1 suppressed TGF-β pathway activity in cells in culture and in xenografts. The findings suggest a possible point of crosstalk between the mitotic checkpoint and TGF-β signaling. Transforming growth factor–β (TGF-β) signaling regulates cell proliferation and differentiation, which contributes to development and disease. Upon binding TGF-β, the type I receptor (TGFBRI) binds TGFBRII, leading to the activation of the transcription factors SMAD2 and SMAD3. Using an RNA interference screen of the human kinome and a live-cell reporter for TGFBR activity, we identified the kinase BUB1 (budding uninhibited by benzimidazoles-1) as a key mediator of TGF-β signaling. BUB1 interacted with TGFBRI in the presence of TGF-β and promoted the heterodimerization of TGFBRI and TGFBRII. Additionally, BUB1 interacted with TGFBRII, suggesting the formation of a ternary complex. Knocking down BUB1 prevented the recruitment of SMAD3 to the receptor complex, the phosphorylation of SMAD2 and SMAD3 and their interaction with SMAD4, SMAD-dependent transcription, and TGF-β–mediated changes in cellular phenotype including epithelial-mesenchymal transition (EMT), migration, and invasion. Knockdown of BUB1 also impaired noncanonical TGF-β signaling mediated by the kinases AKT and p38 MAPK (mitogen-activated protein kinase). The ability of BUB1 to promote TGF-β signaling depended on the kinase activity of BUB1. A small-molecule inhibitor of the kinase activity of BUB1 (2OH-BNPP1) and a kinase-deficient mutant of BUB1 suppressed TGF-β signaling and formation of the ternary complex in various normal and cancer cell lines. 2OH-BNPP1 administration to mice bearing lung carcinoma xenografts reduced the amount of phosphorylated SMAD2 in tumor tissue. These findings indicated that BUB1 functions as a kinase in the TGF-β pathway in a role beyond its established function in cell cycle regulation and chromosome cohesion.

Pancreatic Cancer and Hedgehog Pathway Signaling: New Insights

While several aberrant signaling pathways have been attributed to the formation and progression of pancreatic cancer, there is mounting evidence for the increased role of the Hedgehog (Hh) pathway in multiple aspects of pancreatic tumor development. The Hh pathway is a signaling cascade that plays an important role in cell patterning of multiple tissues and organs, including the development of the gastrointestinal system. While normal pancreatic tissue exhibits little Hh pathway activity, patients with pancreatic adenocarcinoma show high levels of Hh pathway signaling in both the tumor epithelia and the surrounding mesenchyme. Several recent studies have focused on this paracrine activation of Hh signaling in the tumor microenvironment and have provided evidence for how activation of this pathway may play roles in mediating cellular proliferation, metastasis, and resistance to therapy. Together, these findings present new insights into how modulation of this pathway may allow us to target multiple aspects of pancreatic tumor biology.

Modeling Selective Elimination of Quiescent Cancer Cells from Bone Marrow

Patients with many types of malignancy commonly harbor quiescent disseminated tumor cells in bone marrow. These cells frequently resist chemotherapy and may persist for years before proliferating as recurrent metastases. To test for compounds that eliminate quiescent cancer cells, we established a new 384-well 3D spheroid model in which small numbers of cancer cells reversibly arrest in G1/G0 phase of the cell cycle when cultured with bone marrow stromal cells. Using dual-color bioluminescence imaging to selectively quantify viability of cancer and stromal cells in the same spheroid, we identified single compounds and combination treatments that preferentially eliminated quiescent breast cancer cells but not stromal cells. A treatment combination effective against malignant cells in spheroids also eliminated breast cancer cells from bone marrow in a mouse xenograft model. This research establishes a novel screening platform for therapies that selectively target quiescent tumor cells, facilitating identification of new drugs to prevent recurrent cancer.

Cancer Stem Cell Marker Phenotypes Are Reversible and Functionally Homogeneous in a Preclinical Model of Pancreatic Cancer

Survival rates associated with pancreatic cancer remain dismal despite advancements in detection and experimental treatment strategies. Genetically engineered mouse models of pancreatic tumorigenesis have gained considerable attention based on their ability to recapitulate key clinical features of human disease including chemotherapeutic resistance and fibrosis. However, it is unclear if transgenic systems exemplified by the Kras(G12D)/Trp53(R172H)/Pdx-1-Cre (KPC) mouse model recapitulate the functional heterogeneity of human pancreatic tumors harboring distinct cells with tumorigenic properties. To facilitate tracking of heterogeneous tumor cell populations, we incorporated a luciferase-based tag into the genetic background of the KPC mouse model. We isolated pancreatic cancer cells from multiple independent tumor lines and found that roughly 1 out of 87 cells exhibited tumorigenic capability. Notably, this frequency is significantly higher than reported for human pancreatic adenocarcinomas. Cancer stem cell (CSC) markers, including CD133, CD24, Sca-1, and functional Aldefluor activity, were unable to discriminate tumorigenic from nontumorigenic cells in syngeneic transplants. Furthermore, three-dimensional spheroid cultures originating from KPC tumors did not enrich for cells with stem-like characteristics and were not significantly more tumorigenic than cells cultured as monolayers. Additionally, we did not observe significant differences in response to gemcitabine or salinomycin in several isolated subpopulations. Taken together, these studies show that the hierarchical organization of CSCs in human disease is not recapitulated in a commonly used mouse model of pancreatic cancer and therefore provide a new view of the phenotypic and functional heterogeneity of tumor cells.

Targeting ADAM17 inhibits human colorectal adenocarcinoma progression and tumor-initiating cell frequency

ADAM17 (a disintegrin and metalloproteinase 17)/TACE (TNFα converting enzyme) has emerged as a potential therapeutic target in colorectal cancer (CRC) and other cancers, due in part to its role in regulating various tumor cell surface proteins and growth factors and cytokines in the tumor microenvironment. The emergence of MEDI3622, a highly potent and specific antibody-based ADAM17 inhibitor, has allowed testing of the concept that targeting ADAM17 may be an important new therapeutic approach for CRC patients. We demonstrate that MEDI3622 is highly efficacious on tumor growth in multiple human CRC PDX models, resulting in improved survival of animals bearing tumor xenografts. MEDI3622 was further found to impact Notch pathway activity and tumor-initiating cells. The promising preclinical activity seen here supports further clinical investigation of this treatment approach to improve therapeutic outcome for patients diagnosed with metastatic CRC, including patients with KRAS-mutant tumors for whom other therapeutic options are currently limited.ADAM17 (a disintegrin and metalloproteinase 17)/TACE (TNFα converting enzyme) has emerged as a potential therapeutic target in colorectal cancer (CRC) and other cancers, due in part to its role in regulating various tumor cell surface proteins and growth factors and cytokines in the tumor microenvironment. The emergence of MEDI3622, a highly potent and specific antibody-based ADAM17 inhibitor, has allowed testing of the concept that targeting ADAM17 may be an important new therapeutic approach for CRC patients. We demonstrate that MEDI3622 is highly efficacious on tumor growth in multiple human CRC PDX models, resulting in improved survival of animals bearing tumor xenografts. MEDI3622 was further found to impact Notch pathway activity and tumor-initiating cells. The promising preclinical activity seen here supports further clinical investigation of this treatment approach to improve therapeutic outcome for patients diagnosed with metastatic CRC, including patients with KRAS-mutant tumors for whom other therapeutic options are currently limited.

Imaging Reporters for Proteasome Activity Identify Tumor- and Metastasis-Initiating Cells.

Tumor-initiating cells, also designated as cancer stem cells, are proposed to constitute a subpopulation of malignant cells central to tumorigenesis, metastasis, and treatment resistance. We analyzed the activity of the proteasome, the primary organelle for targeted protein degradation, as a marker of tumor- and metastasis-initiating cells. Using human and mouse breast cancer cells expressing a validated fluorescent reporter, we found a small subpopulation of cells with low proteasome activity that divided asymmetrically to produce daughter cells with low or high proteasome activity. Breast cancer cells with low proteasome activity had greater local tumor formation and metastasis in immunocompromised and immunocompetent mice. To allow flexible labeling of cells, we also developed a new proteasome substrate based on HaloTag technology. Patient-derived glioblastoma cells with low proteasome activity measured by the HaloTag reporter show key phenotypes associated with tumor-initiating cells, including expression of a stem cell transcription factor, reconstitution of the original starting population, and enhanced neurosphere formation. We also show that patient-derived glioblastoma cells with low proteasome activity have higher frequency of tumor formation in mouse xenografts. These studies support proteasome function as a tool to investigate tumor- and metastasis-initiating cancer cells and a potential biomarker for outcomes in patients with several different cancers.