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.

Destroying Brain Tumors With Engineered Stem Cells

Khalid Shah from the Harvard Stem Cell Institute (HSCI) has designed a new way to genetically engineered stem cells to secrete tumor-killing toxins. In collaboration with colleagues from the HSCI and Massachusetts General Hospital, Shah and his team have shown that their toxin-secreting stem cells can be used to eradicate cancer cells in the brains of mice after the main tumor has been removed.

Shah and his coworkers used the toxin produced by a bacterium called “Pseudomonas.” This soil bacterium secretes a powerful exotoxin that blocks protein synthesis in cells. This Pseudomonas exotoxin or PE has the capability to kill tumor cells. Unfortunately, this powerful toxin will also kill any other cell it comes into contact with. Therefore, the administration of PE is a very delicate and precise enterprise. Therefore, Shah and his collaborators modified PE so that it bound to specific receptors that are found on the surfaces of particular brain tumors. This way. the toxin was only taken up by cells that expressed these particular receptors. Shah and others also engineered the stem cells to harbor a mutation in the gene that encodes the target for PE (EF-2) that renders this target protein resistant to the effects of PE (for those who are interested, a G‐to‐A transition in the first nucleotide of
codon 717 that is known to confer toxin resistance).

Next, Shah and others embedded the toxin-secreting stem cells into biodegradable hydrogels that were implanted at the site of the tumor. This way, the toxin-secreting cells are near the site of the tumor and only secrete the toxin where it is needed. Any toxin that escapes from this site undergoes rapid degradation, which prevents it from causing any systemic side effects or toxicities.

Shah and his colleagues are pursuing FDA approval to bring this and other stem cell-based technologies to clinical trials.

“A few years ago recognized hat stem cells could be used to continuously deliver these therapeutic toxins in the brain, but first we needed to genetically engineer the stem cells that could resist being killed themselves by the toxins,” said Shah.

Shah continued: “Cancer-killing toxins have been used with great success in a variety of blood cancers, but they didn’t work as well in solid tumors because the cancers aren’t as accessible and the toxins have a short half-life.”

“We tested these stem cells in a clinically relevant mouse model of brain cancer, where you resect the tumors [from patients] and implant the stem cells encapsulated in a gel into the resection cavity. After doing all of the molecular analysis and imaging to track the inhibition of protein synthesis within brain tumors, we do see the toxins kill the cancer cells and eventually prolong the survival in animal models of resected brain tumors.”

Shah next plans to combine his toxin-secreting stem cells with several different therapeutic stem cells developed by his team to further enhance their positive results in mouse models of glioblastoma, which is the most common brain tumor in human adults. Shah hopes that he will bring these therapies into clinical trials within the next five years.

Using Fat Stem Cells to Treat a Deadly Cancer

Johns Hopkins University researchers have reported the successful use of stem cells derived from human body fat to deliver biological treatments directly to the brains of mice suffering from the most common and aggressive form of brain tumor. Such treatments significantly extended the lives of these cancer-stricken animals.

These experiments offer proof-of-principle that such a technique would work in human patients after surgical removal of brain cancers called glioblastomas. This technique provides a way to find and destroy any remaining cancer cells in those areas of the brain that are difficult to reach. Glioblastoma cells represent a challenge for cancer treatments, since they are quite sprightly, and can migrate across the entire brain, hide out and establish new tumors. Consequently, the cure rates for glioblastoma are notoriously low.

In the mouse experiments conducted by the Johns Hopkins group, investigators used mesenchymal stromal cells (MSCs) from fat tissue. Fat-based MSCs have a mysterious ability to sniff out cancer and other damaged cells. After genetically modifying the MSCs so that they secreted a protein called bone morphogenetic protein 4 (BMP4), these MSCs were injected into the brains of mice that suffered from glioblastomas. BMP-4 is a small, secreted protein that plays essential regulatory roles in embryonic development, but also has a demonstrated tumor suppression function.

Study leader Alfredo Quinones-Hinojosa, M.D., a professor of neurosurgery, oncology and neuroscience at the Johns Hopkins University School of Medicine and his colleagues published the results of this experiment in the journal Clinical Cancer Research. According to their results, those mice that were treated with the BMP-4-secreting fat-based MSCs had significantly less tumor growth and spread. In general the cancers in these animals were less aggressive and had fewer migratory cancer cells compared to mice that didn’t get the treatment. Also, the stem cell-treated mice survived significantly longer (an average of 76 days, compared to 52 days in the untreated mice).

“These modified mesenchymal stem cells are like a Trojan horse, in that they successfully make it to the tumor without being detected and then release their therapeutic contents to attack the cancer cells.”

Standard treatments for glioblastoma include chemotherapy, radiation and surgery. Unfortunately, even a combination of all three rarely leads to more than 18 months of survival after diagnosis. Discovering new ways to seek and destroy straggling glioblastoma cells that other treatments can’t get is a long-sought goal, says Quinones-Hinojosa. However, he also cautions that years of additional studies are needed before human trials of fat-derived MSC therapies could begin.

Quinones-Hinojosa also treated brain cancer patients at Johns Hopkins Kimmel Cancer Center, and he and his co-workers were greatly encouraged that the genetically-engineered stem cells let loose into the brain in his experiments did not transform themselves into new tumors.

These latest findings build on research published in March 2013 by Quinones-Hinojosa and his team, which demonstrated that harvesting MSCs from fat was much less invasive and less expensive than getting them from bone marrow (PLoS One, March 2013).

Ideally, he says, if MSCs work as a cancer treatment, a patient with a glioblastoma would have some adipose tissue (fat) removed from any number of locations in the body a short time before surgery. Afterwards, these fat-derived MSCs would be isolated and manipulated in the laboratory so that they would secrete BMP4. Then, after surgeons removed whatever parts of the brain tumor they could get to, they would deposit these BMP-secreting cells into the brain in the hopes that they would seek out and destroy the left-over cancer cells.


Radio Interview About my New Book

I was interviewed by the campus radio station (89.3 The Message) about my recently published book, The Stem Cell Epistles,

Stem Cell Epistles

It has been archived here. Enjoy.

Stem Cell-Promoting Gene Also Promotes the Growth of Head and Neck Cancer

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.

Nanog function during development
Nanog function during development

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.

Bmi1 - Nanog interaction

“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.”

Stem Cell Gene Provides Target for Cancer Treatment

A gene called SALL4 encodes a zinc finger transcription factor protein that helps stem cells maintain their undifferentiated state and continue dividing. Cells tend to only express SALL4 during embryonic development, but in almost all cases of acute myeloid leukemia, and in 10-30% of liver, gastric, ovarian, endometrial, and breast cancers, SALL4 is re-expressed. This is solid evidence that SALL4 plays a central role in tumor formation.

Harvard Stem Cell Institute (HSCI)-affiliated labs in Singapore and Boston have shown that knocking out the SALL4 gene in mouse tumors leads to a cessation of tumor growth. Additionally, designing small molecules that inhibit SALL4 activity also treat the cancer and cause cessation of tumor growth and shrinkage of the tumor.

“Our paper is about liver cancer, but it is likely true about lung cancer, breast cancer, ovarian cancer, many, many cancers,” said HSCI Blood Diseases Program leader Daniel Tenen, who also directs a laboratory at the Cancer Science Institute of Singapore (CSI Singapore). “SALL4 is a marker, so if we had a small molecule drug blocking SALL4 function, we could also predict which patients would be responsive.”

Studying the therapeutic potential of a transcription factor is unusual in the field of cancer research. Transcription factors are typically avoided because of the difficulty of developing drugs that safely interfere with genetic targets. Most cancer researchers focus their attention on kinases (enzymes that attach phosphate groups to other molecules).

However, inquiry into the basic biology of the SALL4 gene by HSCI researchers has shown that there is another way to interfere with its activity in cancer cells. The SALL4 protein turns off a tumor suppressor gene, and this causes the cell to divide uncontrollably. By targeting the SALL4 protein with synthetic molecules that inhibit its activity, they could halt the growth of the tumors.

“The pharmaceutical companies decided that if it is not a kinase, and it is not a cell surface molecule, then it is ‘undruggable,'” said Tenen. “To me, if you say anything in ‘undoable,’ you are limiting yourself as a biomedical scientist.”

Earlier this year, Tenen’s co-author, HSCI-affiliated faculty member Li Chau, assistant professor of pathology at Harvard Medical School and Brigham and Woman’s Hospital, published a report that synthetic SALL4 inhibitors have treatment potential in leukemia cells.

Chai took blood samples from patients with acute myeloid leukemia, and treated the leukemia cells with this synthetic inhibitor and then transplanted that blood back into the leukemic mice. The cancer showed gradual regression.

“I am excited about being on the front line of this new drug development,” said Chai. “As a physician-scientist, if I can find a new class of drug that has very low toxicity to normal tissues, my patients can have a better quality of life.”

Chai and Tenen are working with HSCI Executive Committee member Lee Rubin from the Harvard Institute of Chemistry and Biology, and James Bradner from the Dana Farber Cancer Institute (another HSCI-affiliated faculty member), to help them with the drug development part of their project. Demonstrating the potential of SALL4-interfering compounds is labor intensive, but might also be efficacious for the treatment of other cancers.

“I think as academics, we seek to engage drug companies because they can do these types of things better than we can,” said Tenen. “But, also as an academic, I want to go after the important biologic targets that are not being sought after by the typical drug company – because if we do not, who will?”

The Nooks and Crannies in Bone Marrow that Nurture Stem Cells

Stems cells in our bodies often require a specific environment to maximize their survival and efficiency. These specialized locations that nurture stem cells is called a stem cell niche. Finding the right niche for a stem cell population can go a long way toward growing more stem cells in culture and increasing their potency.

To that end, a recent discovery has identified the distinct niches that exist in bone marrow for hematopoietic stem cells (HSCs), which form the blood cells in our bodies.

A research team from Washington university School of Medicine in St. Louis has shown that stem niches in bone marrow can be targeted, which may potentially improve bone marrow transplants and cancer chemotherapy. Drugs that support particular niches could encourage stem cells to establish themselves in the bone marrow, which would greatly increase the success rate of bone marrow transplants. Alternatively, tumor cells are known to hide in stem cell niches, and if drugs could disrupt such niches, then the tumor cells would be driven from the niches and become more susceptible to chemotherapeutic agents.

Daniel Link, the Alan A. and Edith L. Wolff Professor of Medicine at Washington University, said, “Our results offer hope for targeting these niches to treat specific cancers or to impress the success of stem cell transplants. Already, we and others are leading clinical trials to evaluate whether it is possible to disrupt these niches in patients with leukemia or multiple myeloma.”

Working in mice, Link and his colleagues deleted a gene called CXCL12, only in “candidate niche stromal cell populations.” CXCL12 which encodes a receptor protein known to be crucial for maintaining HSC function, including retaining HSCs in the bone marrow, controlling  HSCs activity, and repopulating the bone marrow with HSCs after injury.

CXCL12 crystal structure
CXCL12 crystal structure

CXCL12 signaling pathways

In bone marrow, HSCs are surrounded by a whole host of cells, and it is difficult to precisely identify which type of cells serve as the niche cells. These bone marrow cells are known collectively as “stroma,” but there are several different types of cells in stroma. Cells that have been implicated in the HSC niche include endosteal osteoblasts (osteoblasts are bone-making cells and the endosteum in the layer of connective tissue that lines the inner cavity of the bone), perivascular stromal cells (cells that hang out around blood vessels), CXCL12-abundant reticular cells, leptin-receptor-positive stromal cells, and nestin–positive mesenchymal progenitors. Basically, there are a lot of cells in the stroma and figuring out which one is the HSC niche is a big deal.

bone marrow stromal cells

When HSCs divide, they form two cells, one of which replaces the HSC that just divided and a new cells called a hematopoietic progenitor cell (HPC), which can divide and differentiate into either a lymphoid progenitor or a myeloid progenitor. The lymphoid progenitor differentiates into either a B or T lymphocyte and the myeloid progenitor differentiates into a red blood cell, or other types of white blood cells (neutrophil, basophil, macrophage, platelet or eosinophil). As the cells become more differentiated, they lose their capacity to divide.

HSC differentiation

Deleting CXCL12 from mineralizing osteoblasts (bone making cells) did nothing to the HSCs or those cells that form lymphocytes (lymphoid progenitors). Deletion of Cxcl12 from osterix-expressing stromal cells, which include CXCL12-abundant reticular cells and osteoblasts, causes mobilization of hematopoietic progenitor cells (HPCs) from the bone marrow into the bloodstream, and loss of B-lymphoid progenitors, but HSC function is normal. Cxcl12 deletion from blood vessel cells causes a modest loss of long-term repopulating activity. Deletion of Cxcl12 from nestin-negative mesenchymal progenitors causes a marked loss of HSCs, long-term repopulating activity, and lymphoid progenitors. All of these data suggest that osterix-expressing stromal cells comprise a distinct niche that supports B-lymphoid progenitors and retains HPCs in the bone marrow. Also, the expression of CXCL12 from stromal cells in the perivascular region, including endothelial cells and mesenchymal progenitors, supports HSCs.

Link summarized his results this way: “What we found was rather surprising. There’s not just one niche for developing blood cells in the bone marrow. There’s a distinct niche for stem cells, which have the ability to become any blood cell in the body, and a separate niche for infection-fighting cells that are destined to become T cells and B cells.”

These data provide the foundation for future investigations whether disrupting these niches can improve the effectiveness of cancer chemotherapy.

In a phase 2 study at Washington University, led by oncologist Geoffrey Uy, assistant professor of medicine, Link and his team are evaluating whether the drug G-CSF (granulocyte colony stimulating growth factor) can alter the stem cell niche in patients with acute lymphoblastic leukemia and whose disease is resistant to chemotherapy or has recurred. The FDA approved this drug more than 20 years ago to stimulate the production of white blood cells in patients undergoing chemotherapy, who have often weakened immune systems and are prone to infections.

Uy and his colleagues want to evaluate G-CSF if it is given prior to chemotherapy. Patients enrolled at the Siteman Cancer Center will receive G-CSF for five days before starting chemotherapy, and the investigators will determine whether it can disrupt the protective environment of the bone marrow and make cancer cells more sensitive to chemotherapy.

This trial is ongoing, and the results are not yet in, but Link’s work has received a welcome corroboration of his work. A companion paper was published in the same issue of Nature by Sean Morrison, the director of the Children’s Medical Center Research Institute at the University of Texas Southwestern Medical Center in Dallas. Morrison and his team used similar methods as Link and his colleagues and came to very similar conclusions.

Link said, “There’s a lot of interest right now in trying to understand these niches. Both of these studies add new information that will be important as we move forward. Next, we hope to understand how stem cells niches can be manipulated to help patients undergoing stem cell transplants.”