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.

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?”

Myriad Genetics Hordes Breast Cancer Data


Kathleen Sloan the president of the National Organization of Women has a troubling article at the Center for Bioethics and Culture website. It tells the story of a biotechnology company called Myriad Genetics and it BRCA1 & 2 test.

What the heck is BRCA1 & 2?  BRCA stands for “breast cancer” and mutations in BRCA 1 or 2 predispose females to breast and ovarian cancer. Mutations in BRCA genes also increase the risk of colon, prostate and pancreatic cancer.  Approximately 7% of breast cancer and 11 – 15% of ovarian cancer cases are caused by mutations in the BRCA genes.  If someone carries a mutation in either BRCA 1 or 2, they have a syndrome called Hereditary Breast and Ovarian Cancer (HBOC) syndrome.

The BRCA genes encode proteins that help repair DNA when it is damaged. Even though BRCA 1 & 2 work with several other proteins to accomplish this repair, mutations in the BRCA genes that compromise the quality of the proteins they encode can diminish the ability of cells to repair their DNA. Loss of efficient DNA repair systems leads to greater numbers of mutations in cells, some of which cause either loss of tumor suppress genes that normally put the brakes of cell proliferation, or activation of proto-oncogenes, which encode proteins that promote cell proliferation. Loss of tumor suppressor genes and activation of proto-oncogenes produces a cancer cell, and mutations in BRCA 1 or 2 and accelerate the onset of cancer cell formation (this is a highly simplified explanation and I apologize to the aficionados out there, but I am trying put the cookies on a nice low shelf).

Myriad Genetics came along and developed a genetic test for cancer-causing mutations in BRCA 1 & 2. This is good news, but Myriad Genetics is presently with holding their data from patients. This is not good news. Myriad Genetics wants to generate a database of mutations found in BRCA 1 and 2 genes from women all over the world. Some of these mutations do not affect the function of the encoded protein and do not predispose the patient to breast cancer, but some do. Which ones are harmful and which ones are not?

At this point things get sticky. Myriad has complied its sequence data on BRCA in order to construct a “variants of unknown significance” or VUS. Such a compilation would be invaluable, since it would help physicians correctly interpret the results of a breast cancer test. According to its present data archive, Myriad Genetics claims that only 3% of its tests fall into the VUS unknown category. However, other testing services report a 20% VUS rate. Who’s right? hard to say, given that Myriad Genetics will not release its data. Apparently they feel that their data has commercial value.

The problem is that lots of outfits that provided data to Myriad Genetics free of charge in order for them to develop their test. These other outfits have all their data available on public databases. What about Myriad Genetics – nope.

According to Ms. Sloan, “Myriad Genetics, producer of the world’s biggest-selling gene test for breast and ovarian cancers, has become synonymous with corporate greed. In an egregious breach of bioethics, the company refuses to share groundbreaking knowledge that could benefit cancer patients.”

Myriad worked hard to develop this test – I do not think anyone is contesting that. Myriad Genetics has every right to make money off their test, but when they start hoarding potentially life-saving data, I think Ms. Sloan is right that they have crossed the line.

Myriad Genetics is also being sued because of their attempts to patent the BRCA genes. An impressive consortium of researchers, genetic counselors, women patients, cancer survivors, breast cancer and women’s health groups, and scientific associations representing 150,000 geneticists, pathologists and laboratory professionals are all plaintiffs in this lawsuit against the U.S. Patent Office, Myriad Genetics and the University of Utah Research Foundation, which hold the patents on the genes.

The lawsuit avers that patents on human genes violate the First Amendment because genes are “products of nature.” Therefore, such things cannot be patented. Such an argument has a strong intuitive appeal, and is almost certainly correct.

Read Ms. Sloan’s article here and see what you think.

Some Mesenchymal Stem Cells Inhibit Tumor Growth But Other Types of Mesenchymal Stem Cells Enhance Tumor Growth and Metastasis


Mesenchymal stem cells (MSCs) have the ability to home to growing tumors, and for this reason, many researchers have examined the possibility of using MSCs to treat various types of cancers. However, there is a genuine safety concern with using MSCs in cancer patients, because in laboratory animals, MSCs can form blood vessels that help tumors grow and spread. Consider the following publications:

1. Klopp AH, Gupta A, Spaeth E, Andreeff M, Marini F 3rd (2011) Concise review: Dissecting a discrepancy in the literature: do mesenchymal stem cells support or suppress tumor growth? Stem Cells 29: 11–19.
2. Kidd S, Spaeth E, Klopp A, Andreeff M, Hall B, et al. (2008) The (in) auspicious role of mesenchymal stromal cells in cancer: be it friend or foe. Cytotherapy 10: 657–667.
3. Coffelt SB, Marini FC, Watson K, Zwezdaryk KJ, Dembinski JL, et al. (2009) The pro-inflammatory peptide LL-37 promotes ovarian tumor progression through recruitment of multipotent mesenchymal stromal cells. Proc Natl Acad Sci U S A 106: 3806–3811.
4. Karnoub AE, Dash AB, Vo AP, Sullivan A, Brooks MW, et al. (2007) Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature 449: 557–563.

Because MSCs are multipotental (that is they differentiate into several different adult tissues), they can serve the tumor as a source of blood vessels that augment tumor metastasis and growth. However, several pre-clinical studies with genetically engineered MSCs that deliver chemotherapuetic agents to tumors have proven quite successful (see Waterman RS, Betancourt AM (2012) The role of mesenchymal stem cells in the tumor microenvironment: InTech). So what are we to believe? After MSCs good or bad as tumor treatments?

In 2010, Aline M. Betancourt and colleagues at Tulane University, New Orleans, Louisiana defined two distinct MSC subypes in a MSC population. They referred to these subtypes as MSC1 and MSC2. When challenged with molecules normally found in invading microorganisms, MSC1 populations tend to promote the immune response, where as MSC2 populations tend to suppress the immune response. This simple priming experiment provided a way to distinguish between the two MSC subtypes, but it also gave stem cell scientists a reason why experiments with MSCs tend to give conflicting results in different laboratories – because the two labs were probably working with populations that consisted of different MSC subtypes. See Waterman RS, Tomchuck SL, Henkle SL, Betancourt AM (2010) A New Mesenchymal Stem Cell (MSC) Paradigm: Polarization into a Pro-Inflammatory MSC1 or an Immunosuppressive MSC2 Phenotype. PLoS ONE 5(4): e10088. doi:10.1371/journal.pone.0010088.

With this in mind, Betancourt and co-workers examined the ability of the distinct MSC subtypes to interact with cancers. When grown in culture with several different types of tumor-causing cell lines, they discovered that MSC1 do not support tumor growth but MSC2 robustly support tumors growth. MSC2 also increased the ability of the tumors to invade other tissues and migrate in culture whereas MSC1 supported neither tumor invasion nor tumor migration.

Other features were different as well. For example, MSC1 recruited a completely different cadre of white blood cells to the tumor when compared to MSC2. Also, the molecules deposited in the vicinity of the tumor by MSC1 and MSC2 differed greatly. By providing a bed of molecules upon which tumors cell like to move and grow, MSC2s promoted tumor cell activity, but the materials laid down by MSC1 were not nearly as attractive to the tumor cells.

These show why MSCs can promote the growth of particular tumors in some experiments but not others. Furthermore, it shows that there is a relatively simple test to separate these two MSC subtypes. All further pre-clinical experiments with MSCs, should account for these distinct MSC subtypes and determine if one MSC subtype is a better candidate for an anticancer treatment regime than the other.

See Waterman RS, Henkle SL, Betancourt AM (2012) Mesenchymal Stem Cell 1 (MSC1)-Based Therapy Attenuates Tumor Growth Whereas MSC2-Treatment Promotes Tumor Growth and Metastasis. PLoS ONE 7(9): e45590. doi:10.1371/journal.pone.0045590.