Patients with stage II and III colorectal cancer whose tumors possessed more stem cell–like properties had worse 5-year disease-free survival rates than individuals with more differentiated tumors, 6.9% compared to 93.1%, according to a new study. The presence of the CDX2 transcription factor in tumors, which drives differentiation of intestinal cells, statistically correlated with the least aggressive cancers and highest rates of survival in the validation set. The research, which appeared in the New England Journal of Medicine in February, was conducted by Michael Clarke, M.D., Karel H. and Avice N. Beekhuis Professor of Cancer Biology at Stanford University in Palo Alto, Calif., and colleagues ( N. Engl. J. Med . 2016;374:211–22; doi: 10.1056/NEJMoa1506597).
Michael Clarke, M.D.
“This study, which indicates that tumors with a stem cell–like signature are more aggressive and is highly predictive of survival, may be the first to demonstrate clinical utility in cancer stem cell research,” Clarke said. “We have known that these cells, in contrast to other cancer cells, are treatment resistant,” he said. Clarke’s discovery “sets the field of cancer stem cell research on firmer footing,” said John Dick, Ph.D., Canada Research Chair in stem cell biology and senior scientist at the Princess Margaret Cancer Centre in Toronto. Dick developed a xenotransplant assay and with it, the first leukemia stem cells (LSCs) in mice in 1994 ( Nature 1994;367:645–8). In research that anticipates Clarke’s study, Dick found that stem cell gene expression programs influence clinical outcome in a mouse model of human acute myeloid leukemia ( Nat. Med. 2011;17:1086–93; doi:10.1038/nm.2415N).
Research into cancer stem cells (CSCs) also called tumor-initiating cells, is booming and controversial. The definition, derivation, and importance of those relatively rare cells, discovered first in blood cancers and later in many solid tumors, is not universally agreed upon. Most if not all do agree that “cancer must arise in cells that can self-renew,” said Max Wicha, M.D., founding director of the University of Michigan–Ann Arbor’s Comprehensive Cancer Center and distinguished professor of oncology and internal medicine, who discovered the first stem cells in solid tumors in 2003. CSCs are also at times quiescent, rendering them difficult to eradicate with chemotherapy. Most concede that the existence of CSCs helps explain why cancers form, become treatment resistant, and ultimately recur. But exactly how these cells arise, what factors influence them, how they generate tumors, and how they are derived remains contentious. Some say that CSCs reprogram themselves in response to cues in the microenvironment, whereas others, such as Wicha, won’t go that far, but say that they dedifferentiate. “We originally thought that cancers started in CSCs, but now we believe that they start in cells that dedifferentiate,” he said. Still others, including scientists at the Ludwig Center for Cancer Stem Cell Research and Medicine at Stanford University in Stanford, Calif., including Clarke, say that CSCs arise from normal stem cells, or precursor cells that normal stem cells produce. They note that normal stem cells are the only cells that reproduce themselves and exist long enough to acquire all the changes needed to produce cancer.
Underlying those issues is the stem cell model of cancer, the notion that cancer is organized in heterogeneous hierarchies, driven primarily by a subpopulation of unique CSCs that reproduce and sustain tumors, produce metastases, cause minimal residual disease, and relapse. The earlier stochastic model proposes that tumors are biologically homogeneous, that all cancer cells have the same potential to grow and divide, and that each cell randomly “chooses” between self-renewal and differentiation. That model holds that tumor heterogeneity occurs because those events are unpredictable factors. People who subscribe to the stem cell theory of cancer agree that it is a model in flux. “In the last 2 years, some of these issues have become more complicated,” Wicha said. “We put cells in immune-suppressed mice but overlooked the role of the immune system’s effects on these cells, which we are now considering,” he said. Whatever the disagreements, researchers hope that more research will translate into new disease markers and the ability to selectively target these resistant cells and reduce disease recurrence.
One hundred early-stage trials are under way, targeting CSCs by using a variety of approaches, such as focusing on developmental pathways such as Hedgehog, WNT, and Notch; regulators of particular CSCs, such as HER2 and AKT in breast cancer; the PI3K–mTOR signaling pathway; and certain microRNAs (miRs). Preclinical research has been promising, but some early trials have disappointed. “The problem is that there’s not a lot of evidence to date that if we knock out these CSCs we will cure cancer,” Wicha said. “There have been no home runs,” he said.
Basic Research Lends Support
A new study has shown that the first tumor cell that appears in a zebrafish model of melanoma possesses stem cell–like activity. Leonard Zon, M.D., Grousbeck Professor of Pediatric Medicine at Harvard Medical School, Howard Hughes Medical Institute investigator, and director of the stem cell program at Children’s Hospital in Boston, made visual, in the transparent zebrafish, cancer’s oncogene-expressing melanocytes reprogrammed into neural crest–like progenitors before becoming invasive tumors ( Science 2016;351:6272; doi:10.1126/science.aad2197). In this model of BRAF v600→E mutation and p53 tumor suppressor gene loss, Zon tracked the first cancer cells, which followed oncogene activation using green fluorescent protein. “We saw that the first [cancer] cell has stem cell activity [and is reprogrammed], but [the study] does not address the heterogeneity issue of the cancer stem cell hypothesis,” Zon said.
Other recent research shows that a particular miR, miR126, regulates the stem cell qualities of human acute myeloid LSCs and may be used as a marker to isolate these cells, as well as for prognosis ( Cancer Cell 2016;29:214–28; doi:10.1016/j.ccell.2015.12.011). LSCs share many characteristics with normal hematopoietic stem cells (HSCs), including the capacity to self-renew and quiescence. miR-126 governs stemness and quiescence of both cell types. Perturbing miR-126 causes different outcomes in LSCs and HSCs, which can be exploited to target LSCs without exerting toxic effects on HSCs, said Dick. Quiescence in LSCs therefore can be targeted by perturbing miR-126 levels, which represses the PI3K–AKT–mTOR signaling pathway. That epigenetic strategy could be combined with other therapies that target cancer cells themselves.
As new basic research like these and other studies emerge, the CSC model is evolving to show that nongenetic determinants, such as developmental pathways and epigenetics, contribute to functional heterogeneity and hierarchically organized tumor tissues “where a subpopulation of self-renewing CSCs sustain long-term maintenance of cancer,” Dick said.
Clinical Research, Inconsistent Results
With strong preclinical research backing early clinical trials, CSC research is dogged by discrepancies between preclinical and clinical results. The Hedgehog pathway is dysregulated in several blood cancers, including acute myeloid leukemia and myeloproliferative disorders such as myelofibrosis. Evidence indicates that this pathway acts on stromal cells in the microenvironment and regulates hematopoietic stem cell behavior, which is confirmed in several myelofibrosis mouse models. But in a recent phase II study using the Hedgehog inhibitor IPI-926, most patients stopped treatment because of side effects or having no response ( J. Clin. Oncol. 2014;7:18; doi:10.1186/1756-8722-7-18).
Defactinib, or VS-6063, which inhibits the focal adhesion kinase (FAK) and PI3K–mTOR signaling pathways, is in many phase I and II studies in non–small-cell lung cancer, pancreatic, ovarian cancer, and mesothelioma, with mixed results. Both pathways are critical in CSC survival and disease progression. FAK inhibition increases cytotoxic T cells, enhances T-cell killing of tumor cells, and reduces stromal density. Principal investigator David Gerber, M.D., associate professor at the UT Southwestern Medical Center in Dallas reported results from a phase II in KRAS-mutant non–small-cell lung cancer at the 16th World Conference on Lung Cancer in Denver in September 2015. Fifty-three heavily pretreated patients with advanced disease received defactinib, and about one-third met the 12-week progression-free survival (PFS) endpoint, with one achieving a partial response. Median PFS was 46 days. About one-quarter had grade 3–5 side effects, including two grade 5 respiratory failures. Gerber said that in KRAS-mutated patients, “the drug showed promising activity.” In September 2015 a trial in mesothelioma as maintenance therapy was halted due to lack of efficacy.
Researchers hope that combining defactinib with chemotherapy and immunotherapy will yield better results. A new trial combining it with pembrolizumab, an immunotherapy that targets the PD1 receptor, and gemcitabine is under way in pancreatic cancer, said Andrea Wang-Gillam M.D., Ph.D., associate professor and clinical director of the gastrointestinal oncology program at Washington University in St. Louis. “In a preclinical model, FAK inhibition slowed tumor progression by reducing fibrosis and levels of tumor-suppressive cells. This sets the stage for immunotherapy to be active,” she said. “Combining defactinib and pembrolizumab was highly effective in mice,” she said. With reduced fibrosis and immunological interference, gemcitabine should be more active against these tumors, she said.
In a recent randomized phase Ib/II study of gemcitabine plus another Hedgehog inhibitor, vismodegib, or placebo in advanced pancreatic cancer patients, median PFS was 4 months and 2.5 months for gemcitabine–vismodegib and gemcitabine–placebo arms, respectively. Median overall survival was 6.9 and 6.1 months, respectively, which is not statistically significant ( J. Clin. Oncol. 2015;33:4284–92; doi:10.1200/JCO.2015.62.8719J). “Hitting one signaling pathway is not enough,” said first author Daniel Catenacci, M.D., assistant professor of medicine at the University of Chicago Medicine. The way forward is combining agents, he said. “In a 2009 preclinical study, combining a different hedgehog inhibitor with gemcitabine depleted the stromal tissue barrier and increased concentration of gemcitabine,” Catenacci said ( Science 2009;324:1457–61; doi:10.1126/science.1171362).
“Hedgehog inhibitors have been extremely successful when treating tumors in which hedgehog is a driver mutation,” Catenacci said. “We know that hedgehog is not a driver mutation in pancreatic cancer.” Targeting CSCs without regard to driver mutations in particular cancers does not make sense, he said. Whether the stemness of CSCs is transitory or present in a well-defined CSC population, one of the major issues in the field, the determinants of stemness are major contributors to therapy failure, Dick said. The hope is that as trials mature, combining immunotherapy and chemotherapy intelligently with CSC-targeting drugs could make a crucial difference.
