Even though my previous posts about cancer stem cells have generated very little interest, understanding cancer as a stem cell-based disease has profound implications for how we treat cancer. If the vast majority of the cells in a tumor are slow-growing and not dangerous but only a small minority of the cells are rapidly growing and providing the growth the most of the tumor, then treatments that shave off large numbers of cells might shrink the tumor, but not solve the problem, because the cancer stem cells that are supplying the tumor are still there. However, if the treatment attacks the cancer stem cells specifically, then the tumor’s cell supply is cut off and the tumor will wither and die.
In the case of breast cancer, the tumors return after treatment and spread to other parts of the body because radiation and current chemotherapy treatments do not kill the cancer stem cells.
This premise constitutes the foundation of a clinical trial operating from the University of Michigan Comprehensive Cancer Center and two other sites. This clinical trial will examine a drug that specifically attacks breast cancer stem cells. The drug, reparixin, will be used in combination with standard chemotherapy.
Dr. Anne Schott, an associate professor of internal medicine at the University of Michigan and principal investigator of this clinical trial, said: “This is one of only a few trials testing stem cell directed therapies in combination with chemotherapy in breast cancer. Combining chemotherapy in breast cancer has the potential to lengthen remission for women with advanced breast cancer.”
Cancer stem cells are the small number of cells in a tumor that fuel its growth and are responsible for metastasis of the tumor. This phase 1b study will test reparixin, which is given orally, with a drug called paclitaxel in women who have HER2-negative metastatic breast cancer. This study is primarily designed to test how well patients tolerate this particular drug combination. However, researchers will also examine how well reparixin appears to affect various cancer stem cells indicators and signs of inflammation. The study will also examine how well this drug combination controls the cancer and affects patient survival.
This clinical trial emerged from laboratory work at the University of Michigan that showed that breast cancer stem cells expressed a receptor on their cell surfaces called CXCR1. CXCR1 triggers the growth of cancer stem cells in response to inflammation and tissue damage. Adding reparixin to cultured cancer stem cells killed them and reparixin works by blocking CXCR1.
Mice treated with reparixin or the combination of reparixin and paclitaxel had significantly fewer (dramatically actually) cancer stem cells that those treated with paclitaxel alone. Also, riparixin-treated mice developed significantly fewer metastases that mice treated with chemotherapy alone (see Ginestier C,, et al., J Clin Invest. 2010, 120(2):485-97).
Nanog is a very funny name for a gene, but the Nanog gene is an essential part of the cellular machinery that keeps embryonic stem cells from differentiating and maintains them in a pluripotent state. Unfortunately, Nanog also has other roles if it is mis-expressed and that includes in the genesis of cancers of the head and neck.
This study emerged from work done by researchers at the Ohio State University Comprehensive Cancer Center – Arthur G. James Cancer Hospital, and Richard J. Solove Research Institute or OSUCCC-James. Since Nanog has been studied in some depth, understanding Nanog activity might provide vital clues in the design of targeted drugs and reagents for treating particular cancers.
“This study defines a signaling axis that is essential for head and neck cancer progression, and our findings show that this axis may be disrupted at three key steps,” said Quintin Pan, associate professor of otolaryngology at OSUCCC-James and principal investigator in this research effort. “Targeted drugs that are designed to inhibit any or all of these three steps might greatly improve the treatment of head and neck cancer.”
What kind of signaling axis is Dr. Pan referring to? An enzyme called protein kinase C-epsilon or PKC-epsilon can place phosphate groups on the Nanog protein. Phosphate groups are negatively charged and are also quite bulky. Attaching such chemical groups to a protein can effectively change its structure and function. In the case of Nanog, phosphorylation of stabilizes it and activates it.
Phosphorylated Nanog proteins can bind together to form a dimer, which attracts a third protein to it; p300. This third protein, p300, in combination with the paired Nanog proteins acts as a potent activator of gene expression of particular genes, in particular a gene called Bmi1. When expressed at high levels, Bmi1 stimulates the proliferation of cells in an uncontrolled fashion.
“Our work shows that the PKCepsilon/Nanog/Bmi1 signaling axis is essential to promote head and neck cancer,” Pan said. “And it provides initial evidence that the development of inhibitors that block critical points in this axis might yield a potent collection of targeted anti-cancer therapeutics that could be valuable for the treatment of head and neck cancer.”
Female mammary glands are home to a remarkable population of stem cells that grow in culture as small balls of cells called “mammospheres.” Clayton and others were able to identify these stem cells in 2004 (Clayton, Titley, and Vivanco, Exp Cell Res 297 (2004): 444-60), and Max Wicha’s laboratory at the University of Michigan showed that a signaling molecule called Sonic Hedgehog and a Polycomb nuclear factor called Bmi-1 are necessary for the self-renewal of normal and cancerous mammary gland stem cells (Lui, et al., Cancer Res June 15, 2006 66; 606). The biggest problem with mammary gland stem cells is isolating them from the rest of the mammary tissue.
Mammary gland stem cells or MaSCs are very important for mammary gland development and during the induction of breast cancer. Getting cultures of MsSCs is really tough because the MaSCs share cell surface markers with normal cells and they are also quite few in number.
Gregory Hannon and his co-workers at Cold Spring Harbor Laboratory used a mouse model to identify a novel cell surface protein specific to MaSCs. By exploiting this unusual marker, Hannon and his team were able to isolate MaSCs from mouse mammary glands of rather high purity.
Camila Do Santos, the paper’s first author, said that “We are describing a marker called Cd1d.” Cd1d is also found on the surfaces of cells of the immune system, but is specific to MaSCs in mammary tissue. Additionally, MaSCs divide slower than the surrounding cells. Do Santos and her colleagues used this feature to visually isolate MaSCs from cultured mammary cells.
They used a mouse strain that expresses a green glowing protein in its cells and then made primary mammary cultures from these green glowing mice. After shutting of the expression of the green glowing protein with doxycycline, the cultured cells divided, and diluted the quantity of green glow protein in the cells. This caused them to glow less intensely. However, the slow-growing MaSCs divided much more slowly and glowed much more intensely. Selecting out the most intensely glowing cells allowed Dos Santos and her colleagues to enrich the culture for MaSCs.
“The beauty of this is that by stopping GFP expression, you can directly measure the number of cell divisions that have happened since the GFP was turned off,” said Dos Santos. She continued: “The cells that divide the least will carry GFP the longest and are the ones we characterized.”
Using this strategy, Dos Santos and others selected stem cells from the mammary glands in order to examine their gene expression signature. They also confirmed that by exploiting Cd1d expression in the MaSCS, in combination with other techniques, they could enhance the purity of the cultures several fold.
Hannon added, “With this advancement, we are now able to profile normal and cancer stem cells at a very high degree of purity, and perhaps point out which genes should be investigated as the next breast cancer drug targets.”
Will we be able to use these cell for therapeutic purposes some day? Possibly, but at this time, more must be known about them and MaSCs must be better characterized.
Eighty-five percent of all tumors are carcinomas, which are tumors that form in layers of cells that line surfaces. Such cell layers are known as an epithelium. When carcinomas form, they undergo an “epithelial-mesenchymal” transformation” or EMT. EMT means that cells go from being closely aligned and tightly bound to each other in a an organized layer to cells that have little to do with each other and grow in unorganized clumps. Is there a molecule that unites the carcinomas and if so is this molecule a potential drug target for cancer treatments?
Researchers at the University of Texas MD Anderson Cancer Center have identified a protein that seems to play a pivotal role in EMT. This protein, FOXC2, may lay at the nexus of why some carcinomas resist chemotherapy and grow uncontrollably and spread. FOXC2 could, conceivably represent a novel drug target for chemotherapy.
Sendurai Mani, assistant professor of Translational Molecular Pathology and co-director of the Metastasis Research Center at MD Anderson, said, “We found that FOXC2 lies at the crossroads of the cellular properties of cancer stem cells and cells that have undergone EMT, a process of cellular change associated with generating cancer stem cells.”
Cancer stem cells are fewer in number than other tumor cells, yet research has tied them to cancer progression and resistance to treatment. Abnormal activation of EMT can actually create cancer stem cells, according to Mani.
Mani continued, “There are multiple molecular pathways that activate EMT. We found many of these pathways also activate FOXC2 expression to launch this transition, making FOXC2 a potentially efficient check point to block EMT from occurring. ” Mani’s research group used experiments with cultured cells and mice to discover these concepts, but the next step will require assessing the levels of FOXC2 expression in human tumors samples.
In the meantime, these new data from Mani’s research team may have profound implication for the treatment of particular types of carcinomas that have proven to be remarkably stubborn. Breast cancers, for example, are typically carcinomas of the mammary gland ductal system. A specific group of breasts cancers are very notoriously resistant to treatment, and FOXC2 seems to be at the center of such breast cancers.
The anti-cancer drug sunitinib, which is marketed under the trade name Sutent, has been approved by the US Food and Drug Administration (US FDA) for three different types of cancers. In this study, sunitinib proved effective against these particularly stubborn types of breast cancer; the so-called “triple-negative, claudin-low” breast cancers.
Mani explained why such breast cancers are so resistant to treatment: “FOXC2 is a transcription factor, a protein that binds to DNA in the promoter region of genes to activate them. For a variety of reasons, transcription factors are hard to target with drugs.”
However, sunitinib seems to target these triple-negative breast cancers. When mice with triple-negative breast cancer were treated with sunitinib, the treated mice had smaller primary tumors, longer survival, and fewer incidences of metastasis. The cancer cells also showed a marked decreased in their ability to form “mammospheres,” or balls of cancer stem cells (this is an earmark of cancer stem cells). Thus sunitinib seem to attack cancer stem cells.
As it turns out, FOXC2 activates the expression of the platelet-derived growth factor receptor-beta (PDGFRc-beta). Activation of PDGFRc-beta drives cell proliferation in FOXC2-positive cells, and sunitinib inhibits PDGFRc-beta and inhibits cells that have active FOXC2 and undergoing EMT.
Triple-negative breast cancer cells lack receptors that are used by the most common anti-cancer drugs. These deficiencies are responsible for the resistance of these cancers to treatment. Such cancer cells also tend to under go EMT because they lack the protein claudin, which binds epithelial cells together. Without claudin, these cancer cells become extremely aggressive.
Since cells undergoing EMT are heavily expressing FOXC2, Mani and his colleagues used a small RNA molecule that makes a short hairpin and inhibits FOXC2 synthesis. Unfortunately, blocking FOXC2 had no effect on cell growth, but it did alter the physical appearance of the cells and reduced their expression of genes associated with EMT and increased the expression of E-cadherin, a protein necessary for epithelial cell organization. Breast cancer cells also became less invasive when FOXC2 was inhibited, and they down-regulated CD44 and CD24, which are markers of cancer stem cells.. Additionally, triple-negative breast cancer cells that had FOXC2 inhibited had a reduced ability to make mammospheres. Thus, FOXC2 expression is elevated in cancer stem cells, and inhibition of FOXC2 decreased the ability of the cancer stem cells to behave as cancer stem cells.
Mani’s group also approached these experiments from another approach by overexpressing FOXC2 in malignant mammary epithelial cells. This forced FOXC2 expression drove cells to undergo EMT and become much more aggressive and metastatic (the cancer spread to the liver, hind leg, lungs, and brain). Breast cancer cells without forced FOXC2 overexpression showed no tendency to metastasize.
Finally, Mani’s group examined metastatic mammary tumors that were highly aggressive when implanted into nude mice (mice that cannot reject transplants). Two of the tumors were claudin-negative and both of these tumors showed elevated FOXC2 expression. When FOXC2 expression was blocked by Mani’s hairpin RNA, the claudin-negative tumors became less aggressive and grew more as mesenchymal cells. The cells that underwent EMT also showed high levels of PDGF-RC-beta expression.
Mani said of these data: “We thought PDGF-B might be a drugable target in these FOXC2-expressing cells.” Mani’s group also showed that suppressing FOXC2 reduced the expression of PDGFRC-Beta. Thus, this small molecule might be an effective therapeutic strategy for treating these hard-to-treat breast cancers.
MD Anderson has filed a patent application connected to this study.
See Hollier B.G., Tinnirello A.A., Werden S.J., Evans K.W., Taube J.H., Sarkar T.R., Sphyris N., Shariati M., Kumar S.V., Battula V.L., Herschkowitz J.I., Guerra R., Chang J.T., Miura N., Rosen J.M., and Mani S.A.,. FOXC2 expression links epithelial-mesenchymal transition and stem cell properties in breast cancer. Cancer Research. e-Pub 2/2013.
Benjamin Dekel, the head of the Pediatric Stem Cell Research Institute in Tel Aviv, Israel, and his team have isolated cancer stem cells from tumors found in the kidneys of some children. Wilms’ tumor is an inherited for of cancer that is found in the kidneys of particular children at a young age. Fortunately, these tumors are easily removed, but these children are at risk for other cancers throughout their lives.
Accord to Professor Dekel, “In earlier studies, cancer stem cells were isolation from adult cancers of the breast, pancreas, and brain, but so far much less is known about stem cells in pediatric cancers.” Professor Dekel continued, “Cancer stem cells contain the complete genetic machinery necessary to start, sustain and propagate tumor growth and they are often referred to as cancer initiating cells. As such, they not only represent a useful system to study cancer development but they also serve as a way to study new drug targets and potential treatments designed to stop the growth and spread of different types of cancer. We have demonstrated for the first time the presence of cancer stem cells in a type of tumor that is often found in children.”
Wilm’s tumors represent the most frequent type of kidney tumor found in children, and while children do usually respond well if the tumors are removed early surgically and if the patients are given chemotherapy, recurrences are possible and they can spread to other tissues.
Conventional chemotherapy is toxic to all cells in the body and if given to children may lead to the development of secondary cancers when they become adults. Thus, scientists would like to target tumor cells in as specific a manner as possible.
Researchers were able to remove parts of the tumors of cancer patients and graft them into mice. This procedure allowed researchers to test for the presence of cancer stem cells, since only the cancer stem cells could propagate the tumor from one animal to another. In the case of Wilms’ tumor, it was clear that cancer stem cells were present and could even be isolated from the rest of the tumor cells.
An international research team led by researchers at the University of California, San Diego School of Medicine has identified an enzyme that plays a key role in cancer stem cell reprogramming in a blood-based cancer known as chronic myeloid leukemia (CML).
CML treatment has received a tremendous boost by the discovery and development of chemotherapuetic agents known as tyrosine kinase inhibitors. Tyrosine kinase inhibitors attack a very specific group of signaling molecules that go awry in CML, and because of their high degree of specificity, these drugs are well tolerated and rather effective.
Tyrosine kinase inhibitors or TKIs block receptors called receptor tyrosine kinases that bind growth factors. Such receptors include molecules like Epidermal Growth Factor Receptor, which binds the growth factor EGF (Epidermal Growth Factor), Plate-Derived Growth Factor Receptor, which binds Platelet-Derived Growth Factor, and several others. Receptor tyrosine kinases are proteins that are embedded in the membrane of cells and when they are engaged by a specific growth factor, they pair up with another molecule and bind the growth factor tightly. Because this binding of their growth factor targets pairs two receptor tyrosine kinases together, the portion of the receptor protein that sticks toward the inside of the cell is activated. This internal portion of the receptor has “kinase” activity. Kinases are enzymes that stick phosphate groups on other molecules. Kinases place their phosphate groups on very specific targets. In the case of receptor tyrosine kinases, the target is the amino acid tyrosine. It just so happens that the internal piece of receptor tyrosine kinases contains several tyrosine residues and the paired receptors molecules tag each other with several phosphate groups on their tyrosines.
Phosphotyrosine acts as a signal to the inside of the cell, because specific protein contain pieces that can bind to phosphotyrosine. These phosphotyrosine-binding proteins (SH2-domain proteins for those who care about such things) drag powerful signaling molecules to the cell membrane. These signaling molecules are activated and the cell undergoes changes that cause it to move, growth, divide, or do other types of things.
In the case of blood cells, activation of particular receptor tyrosine kinases induces cells to grow and divide. Because there are exquisite controls on the signals set in motion by tyrosine, these signals cells divide and then stop. However, if the genes that encode these receptor tyrosine kinases undergo mutations that allow the receptors to pair up without binding growth factors, then the receptors will activate themselves at will without being dependent on the availability of growth factors. Cell will grow uncontrollably and fill up the bone marrow and blood.
At this point, we can see how TKIs work. These small molecules bind to the kinase part of receptor tyrosine kinases and gum them up. Because cells do not receive the signal to grow, they stop growing uncontrollably and this send the cancer into remission. TKIs include such famous drugs as Gleevec (imatinib), which was one of the first TKIs and has provent very successful against CML. However, after long periods of time on Gleevec, tumor cells can become resistant to it, and the physician must change drugs. Other TKIs include gefitinib (Iressa), and erlotinib (Tarceva), which inhibit Epidermal Growth Factor Receptor, Lapatinib (Tykerb), which is a dual inhibitor of EGFR and a subclass called Human EGFR type 2, and Sunitinib (Sutent) which is multi-targeted drug that inhibits Platelet-Derived Growth Factor Receptor and Vascular Endothelial Growth Factor Receptor.
There are also other most specialized TKIs such as sorafenib (Nexavar), which targets a complex pathway that leads to a kinase signaling cascade, and nilotinib (Tasinga) which inhibits the fusion protein bcr-abl and is typically prescribed when a patient has shown resistance to imatinib (Gleevex).
Well, with all these new drugs, what’s the problem? The problem is that blood cancers, leukemias, can find ways around these treatments. Therefore, we must learn more these cancers in order to improve treatment of them. Leukemias are definitely tumors that emerge from cancer stem cells. Therefore, if you kill the cancer stem cells, you kill the tumor.
Principle investigator of this research, Catriona Jamieson, associate professor of medicine at UC San Diego, in collaboration with colleagues from Canada and Italy reported that inflammation, a phenomenon long associated with the development of cancer, increases the activity of the enzyme ADAR1 or adenosine deamiinase 1.
ADAR1 is expressed during embryonic development and it is essential in blood cell development. After embryonic development, ADAR1 switches off, but is reactivated by viral infections. Its role during viral infections is to protect blood cell-making stem cells from viral attacks. In leukemia stem cells, however, ADAR1 enhances the abnormal processing of RNA molecules. This causes enhanced cell renewal and resistance of malignant stem cells to chemotherapy.
Jamieson has already studied the link between cancer stem cell instability and abnormal RNA processing. She said, “People normally think about DNA instability in cancer, but in this case, it’s how the RNA is edited by enzymes that really matters in terms of cancer stem cell generation and resistance to conventional therapy.”
Because this RNA processing process is basic to cells and occurs in closely related organisms as well, studying it should be possible in model systems. It also represents a novel target for new therapies. According to Jamieson, inflammation is “an essential driver of cancer relapse and therapeutic resistance.”
Jamieson continued, “ADAR1 is an enzyme that we may be able to specifically target with a small molecule inhibitor, an approach we have already used effectively with other inhibitors. If we can block the capacity of leukemia stem cells to use ADAR1, if we can knock down that pathway, maybe we can put stem cells back on the right track and stop malignant cloning.”
The initiation of CML requires a mutation that fuses two genes together. One of these genes, BCR, fuses to a protein tyrosine kinase called ABL to generate the BCR-ABL gene that makes a fusion protein with uninhibited activity. The white blood cells that contain the BCR-ABL fusion protein expand slowly. The slowness with which this leukemia expands makes it difficult to diagnose early, and diagnosis is only possible once there are large numbers of precursors and malignant cells throughout the bloodstream and bone marrow. The median age at which CML is diagnosed is 66, and despite the advances in chemotherapeutic treatments, the vast majority of patients relapse if therapy is discontinued, since the cancer stem cells are dormant and resistant to treatment. If ADAR1 is addressed as a target, then perhaps treatments that target ADAR1 will overcome cancer stem cell resistance and prevent relapse.
In the United States alone, there are an estimated 70,00 people with CML and as the population ages, the prevalence of this disease is projected to jump to approximately 181,000 by 2050.
See “ADAR1 promotes malignant progenitor reprogramming in chronic myeloid leukemia.” Qingfei Jianga et al., PNAS DOI: 10.1073/pnas.2123021110.
John C. Morris and colleagues from the University of Cincinnati Cancer Institute have succeeded in isolating lung cancer stem cells and growing them in the laboratory. These findings should provide scientists with a new model system for testing therapeutic strategies that target cancer stem cells.
According to Morris, “Increasing evidence supports the idea that cancerous tumors have a population of stem cells, also called cancer-initiating cells that continually regenerate and fuel cancer growth. These cancer stem cells may also have the highest potential to spread to other organs.”
We normally think of cancer as a disease in which almost all the cells of a tumor have capacity to propagate the tumor. Treating cancer with drugs that attack dividing cells in general is very different that treating a small subset of cells in the cancer that propagate the tumor. Also, new therapies focus on the interaction between the immune system and the cancer. If the only target that the immune needs to recognize is the cancer stem cells, then the therapeutic strategy changes substantially.
Morris and his colleagues used a technique called the “tumor-sphere” assay. This assay evaluates floating non-adherent cell aggregates that form in cultures of cancer cells, when grown under conditions that do not promote cell adhesion. Such clumps of cells are a surrogate for tumors, since they appear to mirror the cellular architecture and composition and behavior of in tumors. Additionally, these clumps are enriched for cancer stem cells.
Morris said: “Studying these unique cells could greatly improve our understanding of lung cancer’s origins and lead to the novel therapeutics targeting these cells and help to more effectively eradicate this disease.” Morris continued: “Immunotherapy is the future of cancer treatment. We are hopeful that this new method will accelerate our investigation of immunotherapies to specifically target cancer stem cells.”
Morris’ group is interested in how cancer stem cells escape the body’s immune system in order to develop more effective therapies that target stem cells.
“One of the hypotheses behind why cancer therapies fail is that the drug only kills cells deemed to be ‘bad’ (because of certain molecular characteristics), but leaves behind stem cells to repopulate the tumor,” said Morris. “Stem cells are not frequently dividing, so they are much less sensitive to existing chemotherapies used to eliminate cells deemed abnormal.”