Mark Hynes

Aldehyde Dehydrogenase 1 Is a Marker for Normal and Malignant Human Colonic Stem Cells (SC) and Tracks SC Overpopulation during Colon Tumorigenesis

Although the concept that cancers originate from stem cells (SC) is becoming scientifically accepted, mechanisms by which SC contribute to tumor initiation and progression are largely unknown. For colorectal cancer (CRC), investigation of this problem has been hindered by a paucity of specific markers for identification and isolation of SC from normal and malignant colon. Accordingly, aldehyde dehydrogenase 1 (ALDH1) was investigated as a possible marker for identifying colonic SC and for tracking them during cancer progression. Immunostaining showed that ALDH1(+) cells are sparse and limited to the normal crypt bottom, where SCs reside. During progression from normal epithelium to mutant (APC) epithelium to adenoma, ALDH1(+) cells increased in number and became distributed farther up the crypt. CD133(+) and CD44(+) cells, which are more numerous and broadly distributed in normal crypts, showed similar changes during tumorigenesis. Flow cytometric isolation of cancer cells based on enzymatic activity of ALDH (Aldefluor assay) and implantation of these cells in nonobese diabetic-severe combined immunodeficient mice (a) generated xenograft tumors (Aldefluor(-) cells did not), (b) generated them after implanting as few as 25 cells, and (c) generated them dose dependently. Further isolation of cancer cells using a second marker (CD44(+) or CD133(+) serially) only modestly increased enrichment based on tumor-initiating ability. Thus, ALDH1 seems to be a specific marker for identifying, isolating, and tracking human colonic SC during CRC development. These findings also support our original hypothesis, derived previously from mathematical modeling of crypt dynamics, that progressive colonic SC overpopulation occurs during colon tumorigenesis and drives CRC development.

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.

Aldehyde Dehydrogenase–Expressing Colon Stem Cells Contribute to Tumorigenesis in the Transition from Colitis to Cancer

Patients with chronic ulcerative colitis are at increased risk of developing colorectal cancer. Although current hypotheses suggest that sporadic colorectal cancer is due to inability to control cancer stem cells, the cancer stem cell hypothesis has not yet been validated in colitis-associated cancer. Furthermore, the identification of the colitis to cancer transition is challenging. We recently showed that epithelial cells with the increased expression of aldehyde dehydrogenase in sporadic colon cancer correlate closely with tumor-initiating ability. We sought to determine whether ALDH can be used as a marker to isolate tumor-initiating populations from patients with chronic ulcerative colitis. We used fluorescence-activated cell sorting to identify precursor colon cancer stem cells from colitis patients and report both their transition to cancerous stem cells in xenografting studies as well as their ability to generate spheres in vitro. Similar to sporadic colon cancer, these colitis-derived tumors were capable of propagation as sphere cultures. However, unlike the origins of sporadic colon cancer, the primary colitic tissues did not express any histologic evidence of dysplasia. To elucidate a potential mechanism for our findings, we compared the stroma of these different environments and determined that at least one paracrine factor is up-regulated in the inflammatory and malignant stroma compared with resting, normal stroma. These data link colitis and cancer identifying potential tumor-initiating cells from colitic patients, suggesting that sphere and/or xenograft formation will be useful to survey colitic patients at risk of developing cancer.

The Notch pathway is important in maintaining the cancer stem cell population in pancreatic cancer.

Background Pancreatic cancer stem cells (CSCs) represent a small subpopulation of pancreatic cancer cells that have the capacity to initiate and propagate tumor formation. However, the mechanisms by which pancreatic CSCs are maintained are not well understood or characterized. Methods Expression of Notch receptors, ligands, and Notch signaling target genes was quantitated in the CSC and non-CSC populations from 8 primary human pancreatic xenografts. A gamma secretase inhibitor (GSI) that inhibits the Notch pathway and a shRNA targeting the Notch target gene Hes1 were used to assess the role of the Notch pathway in CSC population maintenance and pancreatic tumor growth. Results Notch pathway components were found to be upregulated in pancreatic CSCs. Inhibition of the Notch pathway using either a gamma secretase inhibitor or Hes1 shRNA in pancreatic cancer cells reduced the percentage of CSCs and tumorsphere formation. Conversely, activation of the Notch pathway with an exogenous Notch peptide ligand increased the percentage of CSCs as well as tumorsphere formation. In vivo treatment of orthotopic pancreatic tumors in NOD/SCID mice with GSI blocked tumor growth and reduced the CSC population. Conclusion The Notch signaling pathway is important in maintaining the pancreatic CSC population and is a potential therapeutic target in pancreatic cancer.

Anti-DLL4 Has Broad Spectrum Activity in Pancreatic Cancer Dependent on Targeting DLL4-Notch Signaling in Both Tumor and Vasculature Cells

Purpose: We previously showed that targeting Delta-like ligand 4 (DLL4) in colon and breast tumors inhibited tumor growth and reduced tumor initiating cell frequency. In this report, we have extended these studies to pancreatic cancer and probed the mechanism of action in tumor and stromal cells involved in antitumor efficacy. Experimental Design: Patient-derived pancreatic xenograft tumor models were used to evaluate the antitumor effect of anti-DLL4. To investigate the mechanism of action, we compared the activity of targeting DLL4 in tumor cells with an anti-human DLL4 antibody (anti-hDLL4) and in the host stroma/vasculature with an anti-mouse DLL4 antibody (anti-mDLL4). The effect of these antibodies on cancer stem cell frequency was examined by in vivo limiting dilution assays. Results: The combination of anti-hDLL4 and anti-mDLL4 was efficacious in a broad spectrum of pancreatic tumor xenografts and showed additive antitumor activity together with gemcitabine. Treatment with either anti-hDLL4 or anti-mDLL4 delayed pancreatic tumor recurrence following termination of gemcitabine treatment, and the two together produced an additive effect. Anti-hDLL4 had a pronounced effect in reducing the tumorigenicity of pancreatic cancer cells based on serial transplantation and tumorsphere assays. In contrast, disruption of tumor angiogenesis with anti-mDLL4 alone or with anti-VEGF had minimal effects on tumorigenicity. Gene expression analyses indicated that anti-DLL4 treatment regulated genes that participate in Notch signaling, pancreatic differentiation, and epithelial-to-mesenchymal transition. Conclusions: Our findings suggest a novel therapeutic approach for pancreatic cancer treatment through antagonism of DLL4/Notch signaling. Clin Cancer Res; 18(19); 5374–86. ©2012 AACR.

Novel Neutralizing Hedgehog Antibody MEDI-5304 Exhibits Antitumor Activity by Inhibiting Paracrine Hedgehog Signaling

The hedgehog pathway has been implicated in the tumorigenesis, tumor progression, and metastasis of numerous human cancers. We generated the first fully human hedgehog antibody MEDI-5304 and characterized its antitumor activity and preclinical toxicology. MEDI-5304 bound sonic hedgehog (SHH) and Indian hedgehog (IHH) with low picomolar affinity and neutralized SHH and IHH activity in cellular mGLI1 reporter assays. The antibody inhibited transcription of hedgehog target genes and osteoblast differentiation of C3H10T1/2 cells. We evaluated the activity of MEDI-5304 in vivo in model systems that allowed us to evaluate two primary hypotheses of hedgehog function in human cancer, paracrine signaling between tumor and stromal cells and cancer stem cell (CSC) self-renewal. MEDI-5304 displayed robust pharmacodynamic effects in stromal cells that translated to antitumor efficacy as a single agent in an HT-29/MEF coimplantation model of paracrine hedgehog signaling. MEDI-5304 also improved responses to carboplatin in the HT-29/MEF model. The antibody, however, had no effect as a single agent or in combination with gemcitabine on the CSC frequency or growth of several primary pancreatic cancer explant models. These findings support the conclusion that hedgehog contributes to tumor biology via paracrine tumor-stromal signaling but not via CSC maintenance or propagation. Finally, the only safety study finding associated with MEDI-5304 was ondontodysplasia in rats. Thus, MEDI-5304 represents a potent dual hedgehog inhibitor suitable for continued development to evaluate efficacy and safety in human patients with tumors harboring elevated levels of SHH or IHH. Mol Cancer Ther; 13(2); 386–98. ©2013 AACR.

Review Paper: Implications of the “Cancer Stem Cell” Hypothesis on Murine Models of Colon Cancer and Colitis-associated Cancer

The use of murine models to investigate human diseases has been an invaluable tool. In the areas of inflammation and oncogenesis, such models have provided unique insights into pathogenesis and mechanisms to evaluate potential therapy. As such, one facet of these disease processes links inflammation and cancer. Inflammation is associated with at least 15% of the worlds malignancies. One example of this relationship is documented in the association between colitis and colorectal cancer. To date, the precise molecular events linking inflammation and cancer remain unclear. A new paradigm that may bridge these processes includes the cancer stem cell hypothesis. In this review, murine models of colitis, colon cancer, and colitis-associated cancer are discussed in reference to the potential of this paradigm to clarify the relationship of these devastating diseases.

M1199 Atorvastatin Induces Apoptosis In Vitro and Slows Growth of Tumor Xenografts but Does Not Reduce Polyp Initiation

endoscopy. CRLs were categorized into adenomatous and serrated lesions. Results A total of 2,310 patients (mean age 58.4 yrs, range 18-93 yrs and 46.1% males) were included. Seventy-nine % of patients were referred due to symptoms, while 20.7% for screening or surveillance indications. In the total population, 37% (n=855) had diverticular disease, of which 77% (n=658) left-sided, 2% (n=16) right-sided and 21% (n=181) generalized. Endoscopic signs of diverticulitis were found in 9.0% (n=77) of the patients with diverticular disease. Of all patients, 27% (n=619) had at least one adenoma and 13% (n=307) at least one serrated lesion. In patients with diverticular disease vs. those without, adenomas were found in 26.8% vs. 17.5% (p<0.001), serrated lesions in 9.5% vs. 6.2% (p=0.004) and both lesions combined in 7.0% vs. 5.2% (p=0.08) of patients, respectively. Multiple logistic regression analysis with interactions, showed that the relationship between diverticular disease and CRLs was affected by age (p<0.001). Presence of diverticular disease was associated with increased risk for CRLs in patients aged <70 yrs (OR 2.2, 95% CI 1.7-3.0, p<0.001), while this was not found in patients aged ≥70 yrs. Noteworthy, the association between diverticular disease and risk for CRLs gradually increased with younger age, as follows: OR 3.0 (95% CI 2.1-4.3, p<0.001) for age <60 yrs, OR 4.5 (95% CI 2.8-7.0, p<0.001) for age <50 yrs and OR 6.6 (95% CI 3.8-11.6, p<0.001) for age <40 yrs. Conclusion We found an age-dependent association between diverticular disease and colorectal lesions. In younger patients diverticular disease is an independent risk factor for simultaneous presence of colorectal lesions, while this is not the case in patients aged 70 yrs or more.

Abstract LB-257: c-Met: a new cancer stem cell marker and therapeutic target for pancreatic cancer

Pancreatic adenocarcinoma is a highly aggressive disease which is usually diagnosed in an advanced state and for which there are little/no effective therapies. The growth of many cancers depends on a subpopulation of self-renewing cells, termed cancer stem cells (CSC). We have previously identified such a CSC population in human pancreatic cancers which possess the cell surface marker phenotype CD44 + CD24 + ESA + . C-Met, a member of the receptor tyrosine kinases (RTKs) family and receptor for the hepatocyte growth factor (HGF) ligand, has been recently described as a marker of normal mouse pancreatic stem/progenitor cells. We hypothesized that c-Met may function as a cell surface marker and therapeutic target for human pancreatic CSCs. Studies were performed in low passage primary human pancreatic adenocarcinoma xenografts (n = 3) established in NOD-SCID mice. Self-renewal capacity of c-Met high pancreatic cancer cells was assessed using in vitro sphere assays and in vivo tumorigenicity of c-Met high pancreatic cancer cells was evaluated by cell implantation in NOD/SCID mice. In vitro studies demonstrated that c-Met high cells formed spheres in non-adherent cultures, while c-Met − cells did not. Use of the c-Met inhibitor XL184 or c-Met knockdown with shRNA significantly inhibited tumor sphere formation. Pancreatic cancer cells expressing high c-Met had increased tumorigenic potential in vivo and cells expressing both CD44 and c-Met high , representing 0.5-5% of pancreatic cancer cells, demonstrated the highest tumorigenic potential of all populations studied to date. C-Met high CD44+ expressing pancreatic cancer cells demonstrated the capacity for self-renewal and production of differentiated progeny, properties that define CSCs. Further, c-Met inhibition of established primary human pancreatic tumors in NOD/SCID mice significantly inhibited tumor growth and depleted the pancreatic CSC population, when used alone or in combination with gemcitabine. Inhibition of c-Met also prevented the developmental of metastases using an intracardiac injection model. In this report, we define c-Met as a new marker for pancreatic CSC and define c-Met hi g h CD44 + cells as a highly tumorigenic pancreatic CSC population. Our data also indicate that targeting c-Met may be useful in eradicating a CSC population and inhibiting the development of metastases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-257.