Targeting EGFL6 Protein Halts Growth and Spread of Ovarian Cancer

Dr. Ronald J. Buckanovich, professor of hematology/oncology and gynecologic oncology at the University of Michigan Medical School, and his colleagues have identified a protein that help ovarian cancer cells multiply and spread to other organs.  When he and his coworkers inhibited this protein with an antibody they were able to stop the spread of ovarian cancer cold.

The EGFL6 or epidermal growth factor like 6 precursor protein, which is also known as MAEG, maps to human Xp22 chromosome.  The EGFL6 protein is expressed primarily in fetal tissues and during early development (see Yeung G., et al., (1999) Genomics 62, 304307; and Buchner G., et al., (2000) Genomics 65, 1623).  The expression of MAEG has also been detected in several tissues, including the dermis of the trunk, hair follicles, and the mesenchyme of the cranio-facial region (see Buchner G., and others, (2000) Mech. Dev. 98, 179182).  EGFL6 protein has been proposed as a possible biomarker in ovarian cancer (Buckanovich R. J., and others, (2007) J. Clin. Oncol. 25, 852861).

In this paper, which appeared in Cancer Research, Buckanovich and others amplified the expression of EGFL6 in ovarian cancer cells.  Increased EGFL6 expression stimulated cancer growth some two-three times.  This effect was observed in cultured ovarian cancer cells and in a mouse model of ovarian cancer.  Conversely, elimination of EGFL6 greatly reduced ovarian cancer growth, decreasing the rate of growth some four-fold.

EGFL6 specifically acts in cancer stem cells.  To review, in tumors, not all cancer cells are the same.  Inside malignant tumors or even among circulating cancerous cells (as in the case of leukemia) there are usually a variety of different types of cancer cells.  The stem cell theory of cancer proposes that among cancerous cells, a small population among them act as stem cells that reproduce themselves and sustain the cancer.  Cancer stem cells, therefore, are like normal stem cells that renew and sustain our organs and tissues.  Therefore, cancer cells that are not stem cells can certainly adversely affect health, but they cannot sustain the cancer long-term.  Therefore, cancer stem cells fuel the growth and spread of cancers and also are often resistant to chemotherapy and radiation treatments.

Further experiments by Buckanovich and his colleagues showed that EGFL6 cause cancer stem cells to divide asymmetrically so that the one of the daughter cells remains a cancer stem cell while the other daughter cell is a cancer cell that can affect the patient but cannot sustain the cancer. This asymmetric cell division also generates a good deal of diversity among cancer cells.

Buckanovich noted: “What this means is that the stem cell population remains stable.  But the daughter cells, which can have a burst of growth, multiply, and allow the cancer to grow.”.

EGFL6 does more than just promote cancer cell proliferation.  EGFL6 is also a promoter of cancer stem cell migration.  When blood vessels were engineered to express EGFL6, tumor metastasis (spread) was even more robust.

Treatment of tumor-afflicted mice with an antibody that specifically binds to EGFL6 and inactivates it caused a 35% reduction in cancer stem cells and significantly reduced overall tumor growth.  Additionally, the antibody also prevented tumor metastasis.

Buckanovich thinks that targeting EGFL6 might be a potential therapy for women with stage 3 ovarian cancer.  Such a treatment might control the growth and spread of ovarian cancers.

Dr. Buckanovich added: “The bigger implication is for women at high risk of ovarian cancer.  These patients could be treated before cancer develops, potentially blocking cancer from developing or preventing it from spreading.  If cancer did develop, it could be diagnosed at an early stage, which would improve patient outcomes.”.

The next step for Buckanovich and his colleagues is developing an antibody that can properly work in human cancer patients.

Vitamin A as a Treatment For Colon Cancer

A new study that was published in the journal Cancer Cell, has introduced a way to treat colon, which is not only a leading cause of cancer deaths worldwide, but is notoriously resistant to treatment.

This collaboration between Swiss and Japanese scientists has identified an anticancer mechanism that includes vitamin A that can be tapped to inhibit colon cancer.

Colon cancer patients are typically treated with chemotherapy, which kills off most of the cancer cells, but leaves a few resistant cells that then aggressively grow back to form another deadlier tumor that can readily spread throughout the body. Chemotherapy ultimately fails because there are a core of rouge stem cells that divide uncontrollably called cancer stem cells that drives the growth of the cancer. These cancer stem cells are what is driving the growth of the tumor and if these cancer stem cells are not eliminated, the tumor will simply come back after chemotherapy. When a colon cancer patient receives treatment such as chemotherapy, most of the cancer cells die off.

Joerg Huelsken, Ph.D., of Ecole Polytechnique Federale de Lausanne (EPFL) led his research team to understand how stem cells populations in the colon give rise to new colon cells to replace dead, dying or cells that have been sloughed off. In mice and in tissue samples from human patients, a protein called HOXA5 kept asserting itself. HOXA5 turns out to be an integral part of the machinery of the cell that ensures that the cells of the colon properly differentiate after they are born from colon stem cells.

Huelsken’s team showed that in the colon, HOXA5 helps restrict the number of stem cells. Cancerous stem cells, however, block the expression of HOXA5 and prevent it from restricting stem cell numbers. HOXA5 is part of a signaling pathway that activates aspects of the cellular machinery that negatively controls cell growth. By blocking expression of the HOXA5 gene, these cancerous stem cells in the colon can grow uncontrollably and spread, thereby causing relapses and metastasis or spread of the colon cancer.

Huelsken and his team, in collaboration with Japanese researchers from Kyoto University investigated ways to unblock the expression of HOXA5 in colon cancer stem cells. The answer came from an unexpected corner – vitamin A. Vitamin A is a member of the retinoid family of molecules and has been known for some time to be able to induce differentiation of skin-based stem cells. Huelsken’s group showed that retinoids like vitamin A can upregulate HOXA5 and antagonize the mechanism in colon cancer stem cells that staunch its expression.

In a mouse colon cancer model system, treatment with retinoids not only blocked progression of the tumors, but normalized the tissue. The activation of the expression of the HOXA5 gene eliminated cancer stem cells and prevented metastasis in live animals. These results were then faithfully recapitulated in samples from actual patients.

From this study, it seems that screening tumors for the absence of HOXA5 expression is a relatively easy way to determine if a patient’s colon cancer will respond to treatment with vitamin A. Treatment with vitamin A or other retinoids might not only prove effective against colon cancer, but also as a preventive measure in high-risk patients.

Pancreatic Cancer Stem Cells Could Be “Suffocated” by an Anti-Diabetic Drug

A new study by researchers from Queen Mary University of London’s Barts Cancer Institute and the Spanish National Cancer Research Centre (CNIO) in Madrid shows that pancreatic cancer stem cells (PancSCs) are very dependent on oxygen-based metabolism, and can be “suffocated” with a drug that is already in use to treat diabetes.

Cancer cells typically rely on glycolysis; a metabolic pathway that degrades glucose without using oxygen. However, it turns out that not all cancer cells are alike when it comes to their metabolism.

PancSCs use another metabolic process called oxidative phosphorylation or OXPHOS to completely oxidize glucose all the way to carbon dioxide and water. OXPHOS uses large quantities of oxygen and occurs in the mitochondria. However, this is precisely the process that is inhibited by the anti-diabetic drug, metformin.

Ever crafty, some PancSCs manage to adapt to such a treatment by varying their metabolism, and this leads to a recurrence of the cancer. However, this English/Spanish team thinks that they have discovered a way to prevent such resistance and compel all PancSCs to keep using OXPHOS. This new discovery might open the door to new treatments that stop cancer stem cells using oxygen and prevent cancers from returning after conventional treatments. A clinical trial is planned for later next year.

Dr Patricia Sancho, first author of the research paper, said: “We might be able to exploit this reliance on oxygen by targeting the stem cells with drugs that are already available, killing the cancer by cutting off its energy supply. In the long-term, this could mean that pancreatic cancer patients have more treatment options available to them, including a reduced risk of recurrence following surgery and other treatments.”

PancSCs become resistant to metformin by suppressing a protein called MYC and increasing the activity of a protein called PGC-1α. However, this resistance mechanism of PancSCs can be abolished if a drug called menadione is given. Menadione increases the amount of reactive oxygen species in mitochondria. Additionally, resistance to metformin can be prevented or even reversed if the MYC protein is inhibited by genetic or pharmacological means. Therefore, the specific metabolic features of pancreatic Cancer Stem Cells are amendable to therapeutic intervention and can provide the basis for developing more effective therapies to combat this lethal cancer.

Pancreatic cancer is still one of the most difficult cancer types to treat. It rarely causes symptoms early on and does not usually trigger diagnosis until its later and more advanced stages. Unfortunately, many patients do not live longer than a year after being diagnosed. These cancers are also becoming more common due to obesity, which increases the patient’s risk for metabolic syndrome and diabetes, which are pancreatic cancer risk factors. Limited treatment options and a failure to improve survival rates mean that finding new treatment strategies is a priority.

PancSCs could be an important but as yet overlooked piece of this puzzle, since they compose only a small proportion of the tumor. PancSCs also have the potential to make new tumors, even if all the other cells are killed, and are prone to spreading around the body (metastasis). Therefore killing these PancSCs is a better way to treat such dangerous cancers.

Two Genes Control Breast Cancer Stem Cell Proliferation and Tumor Properties

When mothers breastfeed their babies, they depend upon a unique interaction of genes and hormones to produce milk and deliver it to their hungry little tyke. Unfortunately, this same cocktail of genes and hormones can also lead to breast cancer, especially if the mother has her first pregnancy after age 30.

A medical research group at the Medical College of Georgia at Georgia Regents University has established that a gene called DNMT1 plays a central role in the maintenance of the breast (mammary gland) stem cells that enable normal rapid growth of the breasts during pregnancy. This same gene, however, can also maintain those cancer stem cells that enable breast cancer. According to their work, the DNMT1 gene is highly expressed in the most common types of breast cancer.

Also, another gene called ISL1, which encodes a protein that puts the brakes on the growth of breast stem cells, is nearly silent in the breasts during pregnancy and in breast cancer stem cells.

Dr. Muthusamy Thangaraju, a biochemist at the Medical College of Georgia, who is the corresponding author of this study, which was published in the journal Nature Communications, said, “DNMT1 directly regulates ISL1. If the DNMT1 expression is high, this ISL1 gene is low.” Thangaraju and his team first observed the connection between DNMT1 and ISL1 when they knocked out the DNMT1 gene mice and noted an increase in the expression of ISL1. These results inspired them to examine the relationship of these two genes in human breast cancer cells.

Thangaraju and his co-workers discovered that ISL1 is silent in most human breast cancers. Furthermore, they demonstrated that restoring higher levels of ISL1 to human breast cancer cells dramatically reduced cancer stem cell populations, the growth of these cells, and their ability to spread throughout the tissue; all of which are hallmarks of cancer.

Conversely, Thangaraju and his team knocked out the DNMT1 gene in a breast-cancer mouse model, the breast will not develop as well. However, according to Thangaraju, this same deletion will also prevent the formation of about 80 percent of breast tumors. In fact, DNMT1 also down-graded super-aggressive, triple-negative breast cancers, which are negative for the estrogen receptor (ER-), progesterone receptor (PR-), and HER2 (HER2-).

The findings from this work also point toward new therapeutic targets for breast cancer and new strategies to diagnose early breast cancer. For example, a blood test for ISL1 might provide a marker for the presence of early breast cancer. Additionally, the anti-seizure medication valproic acid is presently being used in combination with chemotherapy to treat breast cancer, and this drug increases the expression of ISL1. This might explain why valproic acid works for these patients, according to Thangaraju. Workers in Thangaraju’s laboratory are already screening other small molecules that might work as well or better than valproic acid.

Mammary stem cells maintain the breasts during puberty as well as pregnancy, which are both periods of dynamic breast cell growth. During pregnancy, breasts may generate 300 times more cells as they prepare for milk production. Unfortunately, these increased levels of cell growth might also include the production of tumor cells, and the mutations that lead to breast cancer increase in frequency with age. If the developing fetus dies before she comes to term, immature breast cells that were destined to become mature mammary gland cells can more easily become cancer, according to Rajneesh Pathania, a GRU graduate student who is the first author of this study.

DNMT1 is essential for maintaining a variety of stem cell types, such as hematopoietic stem cells, which produce all types of blood cells. However, the role of DNMT1 in the regulation of breast-specific stem cells that make mammary gland tissue and may enable breast cancer has not been studied to this point.

For reasons that unclear, there is an increased risk of breast cancer if the first pregnancy occurs after age 30 or if mothers lose their baby during pregnancy or have an abortion. Women who never have children also are at increased risk, but multiple term pregnancies further decrease the risk, according to data compiled and analyzed by the American Cancer Society.

Theories to explain these phenomena include the coupling of the hormone-induced maturation of breast cells that occurs during pregnancy with an increase in the potential to produce breast cancer stem cells. Most breast cancers thrive on estrogen and progesterone, which are both highly expressed during pregnancy and help fuel stem cell growth. During pregnancy, stem cells also divide extensively and as their population increases, DNMT1 levels also increase.

In five different types of human breast cancer, researchers found high levels of DNMT1 and ISL1 turned off. Even in a laboratory dish, when they reestablished normal expression levels of ISL1, human breast cancer cells and stem cell activity were much reduced, Thangaraju said.

Promising New Drug Attacks Cancer Stem Cells in Mesothelioma

The anticancer drug defactnib is currently the subject of clinical trials being conducted in multiple countries. Data from this trial in patients with a type of cancer called mesothelioma have shown that defactnib is potentially quite effective in the treatment of these cancers. Defactnib, which is being marketed by biopharmaceutical company Verastem, Inc.

Mesothelioma attacks the lining of the lung, abdomen and, in some cases, the heart. It has only one known cause; the ingestion or inhalation of asbestos fibers.

This trial is currently in its second phase, and it involves 180 patients in 13 countries. The goal of this trial is to evaluate the ability of Defactnib to kill cancer stem cells that are the main cause of the spread of the tumors and their recurrence. Even though the main focus of this trial has been on treating mesothelioma, positive results have also been achieved by using defactnib to treat other types of cancer as well.

In this trial, mesothelioma patients were treated with a combination of defactnib and pemetrexed for 12 days prior to undergoing surgery. Pemetrexed has been approved by the U.S. Food and Drug Administration to treat mesothelioma. Tumors shrunk in 70 percent of the treated mesothelioma patients. Defactnib is designed to inhibit the activity of a protein called the Focal Adhesion Kinase or FAK. FAK is very important for cancer stem cell function and without it, cancer stem cells cannot grow.

“These and other exciting developments continue to build belief that there may be an end to this horrible disease in sight,” said Russell Budd, president and managing shareholder of the mesothelioma law firm Baron and Budd. “It is extremely encouraging to see signs of progress occur on a regular basis.”

Stem Cells Lurk in Tumors and Can Resist Treatment

Regenerative medicine seeks to train stem cells to transform into nearly any kind of cell type. Unfortunately, this ability that makes stem cells so useful also is cause for concern in cancer treatments. Malignant tumors contain resident stem cells, which prompts worries among cancer experts that the cells’ transformative powers help cancers escape treatment.

Data from new research shows that the threat posed by cancer stem cells is more prevalent than previously thought. Until now, stem cells had been identified only in aggressive, fast-growing tumors. However, a mouse study at Washington University School of Medicine in St. Louis has revealed that slow-growing tumors also have treatment-resistant stem cells.

Brain tumor stem cells (orange) in mice express a stem cell marker (green). Researchers at Washington University School of Medicine in St. Louis are studying how cancer stem cells make tumors harder to kill and are looking for ways to eradicate these treatment-resistant cells. Credit: Yi-Hsien Chen
Brain tumor stem cells (orange) in mice express a stem cell marker (green). Researchers at Washington University School of Medicine in St. Louis are studying how cancer stem cells make tumors harder to kill and are looking for ways to eradicate these treatment-resistant cells. Credit: Yi-Hsien Chen

In mice, low-grade brain cancer stem cells were less sensitive to anticancer drugs. When compared to healthy stem cells, tumor-based stem cells from brain tumors, revealed the reasons behind their resistance to treatments, which points to new therapeutic strategies.

“At the very least, we’re going to have to use different drugs and different, likely higher dosages to make sure we kill these tumor stem cells,” said senior author David H. Gutmann, MD, PhD, the Donald O. Schnuck Family Professor of Neurology.  Their data were published in the March 12 edition of Cell Reports.

First author Yi-Hsien Chen, who is a senior postdoctoral research associate in Gutmann’s laboratory, used a mouse model of neurofibromatosis type 1 (NF1), which forms low-grade brain tumors, to identify cancer stem cells and demonstrate that they could form tumors when transplanted into normal, cancer-free mice.

Neurofibromatosis type I is caused by mutations in the NF1 genes, and such mutations affect about 1 in every 2,500 babies. Neurofibromatosis type I can cause an array of physical problems, including brain tumors, impaired vision, learning disabilities, behavioral problems, heart defects and bone deformities.

In children with NF1 mutations, the most common brain tumor is optic gliomas. Treatment for NF1-related optic gliomas usually includes drugs that inhibit a cell growth pathway originally identified by Gutmann. In laboratory tests conducted as part of the new research, it took 10 times the dosage of these drugs to kill the low-grade cancer stem cells.

Compared with healthy stem cells from the brain, cancer stem cells made multiple copies of a protein called Abcg1 that helps those cells survive stress.

“This protein blocks a signal from inside the cells that should make them more vulnerable to treatment,” Gutmann explained. “If we can identify a drug that disables this protein, it would make some cancer stem cells easier to kill.”

Even though these laboratory mice were bred to model NF1 optic gliomas, Gutmann and others said that their findings could be applied more broadly to other brain tumors.

“Because stem cells haven’t differentiated into specialized cells, they can easily activate genes to turn on new developmental programs that allow the cells to survive cancer treatments,” said Gutmann, who directs the Washington University Neurofibromatosis Center. “Based on these new findings, we will have to develop additional strategies to keep these tumors from evading our best treatments.”

Inhibiting Leukemic Stem Cells

Blood cancers, also known as leukemias, are, in many ways, a disease of stem cells. A core of cancerous stem cells divide and produce progeny that overpopulate, overwhelm, and in some cases invade and destroy other tissues. However many cancer treatments are designed to specifically attack the progeny of the cancer stem cells and not the leukemic stem cells. Therefore, the disease is destined to relapse, since the main driving entities of the leukemia are left to flourish while their progeny have been killed off.

Acute myeloid leukemia or AML is largely a disease of older adults, and even though the survival rates have increased, only about one out of every four adult patients survives for five years after the AML has been diagnosed. The mean survival time for this disease, which predominantly occurs in the elderly, is less than a year for patients over 65 years. Younger adults tend to do much better than older people. For example, more than half (50%) of the people under 45 diagnosed with AML will live for at least 5 years, and some will be cured. But in others, however, the AML will return and there is no way to tell in advance who has been cured and who will relapse. In people over 65 years of age the outlook tends not to be so good and around 12 out of 100 people (12%) are alive for more than 5 years.

Relapses and treatment failure in AML is almost certainly due to leukaemic stem cells, which cannot be completely eliminated during treatment. However, researcher Dr. Marin Ruthardt from the Hematology Department of the Medical Clinic II and Dr. Jessica Roos, Prof. Diester Steinhilber and Prof. Thorsten Jürgen Maier from the Institute for Pharmaceutical Chemistry has discovered a chink in the armor of leukemic stem cells. They report their data in the current edition of the journal “Cancer Research.”

AML stem cells require an enzyme called 5-lipoxygenase or 5-LO in order to survive. 5-LO plays a very important role in inflammatory diseases like asthma. Ruthardt, Roos, Steinhilber, and Maier and the members of their research teams showed that the leukemic stem cells in a subgroup of AML could be selectively and efficiently attacked by chemical inhibitors of 5-LO. These inhibitors killed the leukemic stem cells in cell culture model systems and in leukemia mouse models.

“These results provide the basis for the potential implementation of 5-LO-inhibitors as stem cell therapeutic agents for a sustained AML cure, although this must be investigated further in preclinical and clinical studies in humans,” explains Dr. Ruthardt.

Prof. Maier continued: “In addition, there are plans for further molecular biological studies with the objective of understanding exactly how the 5-LO inhibitors act on the leukemic cells.”

Hopefully these inhibitors can be fast-tracked for Phase I studies in human patients, and if they prove safe under clinical conditions, then maybe, if all seems well, they can be used to treat AML patients with aggressive cancers that do not respond well to more traditional cancer treatments.