An illustration of two proteins involved in DNA repair by artist Amy VanDonsel

Using yeast cells, the scientists studied protein molecules that have an important role in homologous recombination, which is one way that cells repair breaks in the DNA double helix. The process in yeast is similar to that in humans and other organisms.

Earlier research had established that a protein molecule named Srs2 regulates homologous recombination by counteracting the work of another protein, Rad51. Reporting in the July 10 issue of the journal Molecular Cell, the research team reveals the mechanism of how Srs2 removes Rad51 from DNA and thereby prevents it from making repairs to broken strands.

“Our findings may make it possible to uncover ways to augment the effect of DNA-damaging agents that are used for cancer chemotherapy,” says senior author Tom Ellenberger, D.V.M, Ph.D., the Raymond H. Wittcoff Professor and head of the Department of Biochemistry and Molecular Biophysics. “Many chemotherapeutic agents work by causing DNA damage in cancer cells, leading to their death, and tumors can become resistant to chemotherapy by using DNA repair mechanisms to keep the cells alive. Drugs that inhibit the DNA repair process could help increase the efficiency of chemotherapeutic agents.”

Ellenberger is also co-director of the Pharmacology Core at Siteman Cancer Center at Barnes-Jewish Hospital and Washington University. The facility aids in the development of anti-cancer agents.

Srs2 is a helicase molecule — a motor protein that’s able to walk or slide along a strand of DNA and remove other proteins from DNA or separate the two strands of the twisted double helix. For studies of Srs2, Ellenberger’s laboratory collaborated with Timothy Lohman, Ph.D., the Marvin A. Brennecke Professor of Biochemistry and Molecular Biophysics, a prominent expert in the biochemistry of motor proteins like Srs2.

Rad51’s job in the cell is to promote the exchange of sequences between two related DNA molecules, which can be used to repair breaks in DNA where both strands of the double helix are compromised. As a DNA matchmaker, Rad51 forms long filaments on DNA. Srs2 can remove these to prevent unwanted exchanges of DNA sequences. Without Srs2, cells lose their ability to maintain the normal structure of chromosomes, and DNA sequences become shuffled.

The biochemists found that Srs2 possesses a small arm that interacts with Rad51 and triggers a chemical reaction within the Rad51 protein causing it to fall off the DNA.

“Scientists had assumed that as Srs2 moved along the DNA strand, it just pushed off everything in its path,” says lead author Edwin Antony, Ph.D., a postdoctoral research associate in biochemistry and molecular biophysics. “This isn’t the case — we showed that Srs2 has a specialized structure that allows it to interact specifically with Rad51.”

This finding shows how a motor protein like Srs2 can perform the specialized task of remodeling a protein-DNA complex without interference by other similar helicases, he adds.

Because they now know more precisely the nature of this interaction between Srs2 and Rad51, the researchers can narrow their search for drugs that will block DNA repair by Rad51. This type of drug could make a lower dose of a DNA-damaging drug effective in treating cancer.

The research team is now trying to identify the Srs2 homologue in human cells and will study its structure in combination with Rad51. That will allow a more rational approach to understanding how cells cope with DNA damage and how some tumors evade cancer therapeutics, they say.

“In the long-term, my laboratory will look for drug-like molecules that influence this interaction,” Ellenberger says. “We are using the Chemical Genetics Screening Center here at the University (http://htc.wustl.edu). It has vast libraries of molecules that may have the activity we want. Edwin’s work on Srs2 and Rad51 will allow us to develop an assay to screen for agents that augment or supersede Srs2’s interference with DNA repair.”

Antony E, Tomko EJ, Xiao Q, Krejci L, Lohman TM, Ellenberger T. Srs2 disassembles Rad51 filaments by a protein-protein interaction triggering ATP turnover and dissociation of Rad51 from DNA. Molecular Cell. 2009;35(1):105-115.

Funding from the National Institutes of Health and the Young Scientist Program at Washington University supported this research.

Washington University School of Medicine’s 2,100 employed and volunteer faculty physicians also are the medical staff of Barnes-Jewish and St. Louis Children’s hospitals. The School of Medicine is one of the leading medical research, teaching and patient care institutions in the nation, currently ranked third in the nation by U.S. News & World Report. Through its affiliations with Barnes-Jewish and St. Louis Children’s hospitals, the School of Medicine is linked to BJC HealthCare.

Siteman Cancer Center is the only federally designated Comprehensive Cancer Center within a 240-mile radius of St. Louis. Siteman Cancer Center is composed of the combined cancer research and treatment programs of Barnes-Jewish Hospital and Washington University School of Medicine. Siteman has satellite locations in West County and St. Peters, in addition to its full-service facility at Washington University Medical Center on South Kingshighway.

By Gwen Ericson – Washington University in St. Louis, School of Medicine

A new prostate cancer “homing device” could improve detection and allow for the first targeted treatment of the disease.

This image depicts transporter molecules carrying therapeutic drugs to PSMA targets on a prostate cancer cell. A Purdue research team designed a molecule that finds and penetrates prostate cancer cells and can transport drugs or imaging agents into the cell. (Image courtesy of Low laboratory)

A team of Purdue University researchers has synthesized a molecule that finds and penetrates prostate cancer cells and has created imaging agents and therapeutic drugs that can link to the molecule and be carried with it as cargo.

A radioimaging application used for body scans is expected to enter clinical trials this fall, and an optical imaging application used to measure prostate cancer cells in blood samples is already in clinical trials.

Philip Low, the Ralph C. Corley Distinguished Professor of Biochemistry who led the team, said a targeted treatment could be much more effective in treating cancer and would greatly reduce the harmful side effects associated with current treatments.

“Currently none of the drugs available to treat prostate cancer are targeted, which means they go everywhere in the body as opposed to only the tumor, and so are quite toxic for the patient,” said Low, who is a member of the Purdue Cancer Center. “By being able to target only the cancer cells, we could eliminate toxic side effects of treatments. In addition, the ability to target only the cancer cells can greatly improve imaging of the cancer to diagnose the disease, determine if it has spread or is responding to treatment.”

Prostate cancer is the most common cancer, other than skin cancers, and is the second leading cause of cancer death in American men, according to the American Cancer Society. It is estimated that about 192,280 new cases will be diagnosed and 27,360 men will die of prostate cancer in the United States this year.

The molecule Low’s team created attaches to prostate-specific membrane antigen, or PSMA, a protein that is found on the membrane of more than 90 percent of all prostate cancers. It also is found on the blood vessels of most solid tumors and could provide a way to cut off the tumor blood supply, Low said.

“A lot of new drugs are being designed to destroy the vasculature of solid tumors, and, if they could be linked to this new targeting molecule, we could have a two-pronged attack for prostate cancer,” he said. “We could not only kill the prostate cancer cells directly, we could also destroy the vasculature that feeds the tumors.”

There also is potential for the targeting molecule to be used to attack the vasculature of solid tumors of other types of cancers, Low said.

Two papers detailing the work of the Purdue team were published in the June 1 issue of Molecular Pharmaceutics. Endocyte Inc. funded the work.

The team’s animal study data shows an ability to eliminate human prostate cancer cells in mice with no evidence of collateral toxicity in normal tissue.

Sumith Kularatne, a graduate student in Purdue’s chemistry department and first author of both papers, compared the targeting molecule to a homing device.

“The molecule acts like a homing device for prostate cancer,” he said. “PSMA, which is found only on prostate cancer cells and tumor blood vessels, acts as the homing signal that the molecule targets. The molecule and its cargo go only to cancerous tissue, leaving healthy tissue unharmed.”

Philip Low, the Ralph C. Corley Distinguished Professor of Biochemistry at Purdue, and graduate student Sumith Kularatne, in foreground, examine the uptake of an imaging agent in prostate cancer cells. Low led a research team that designed a molecule to find and penetrate prostate cancer cells and has created imaging agents and therapeutic drugs that can link to the molecule and be carried with it as cargo. (Purdue University photo/Andrew Hancock)

Once the molecule reaches the PSMA protein, it binds to it. The molecule is designed with a specific shape that fits with the protein like a key to a lock, Kularatne said. The molecule and its cargo are then carried inside the cell with the protein as it goes through its normal cycle.

In 1995 Low developed a similar method to infiltrate cancer cells by attaching treatments to the vitamin folate, which many cancers rapidly consume. This method provided a “Trojan Horse” entry of large treatment molecules that otherwise would not be able to enter cancer cells.

Low was inspired to find a similar way to target prostate cancer, which does not have the same appetite for folate, he said.

A clinical trial of the radioimaging application is expected to begin at the Indiana University Medical Center in the fall through a collaboration between the Purdue Cancer Center and the Indiana University Cancer Center with additional support from Endocyte Inc.

A radioimaging agent linked to the targeting molecule will be injected into prostate cancer patients and pictures will be taken using a special camera that detects radioactivity. The pictures show where the cancer is present to help doctors determine if it has metastasized, or spread, to any other areas of the body. It also will help doctors decide on the best course of treatment, Low said.

There is currently only one radioimaging agent for prostate cancer approved by the Food and Drug Administration.

“The current imaging capabilities available for prostate cancer are very poor,” Low said. “The existing imaging agent is limited because of its large size, which is difficult to get into a solid tumor. Also it seeks out a target located inside the cancer cell and is only able to mark injured cells that are falling apart as opposed to actively growing cancer cells.”

The targeting molecule and radioimaging agent combination designed by Low’s group is more than 150 times smaller than the existing agent and has much easier penetration through a solid tumor to reach all of the cells inside, he said. It also has the advantage of targeting an area of PSMA exposed on the outside of cancer cells.

Already in clinical trials is an optical imaging application that involves attaching a fluorescent dye to the targeting molecule and mixing it with a patient’s blood sample. Circulating prostate cancer cells in the sample fluoresce and are easily measured to help in diagnosing patients with prostate cancer. Researchers also are investigating whether this could be used to evaluate a patient’s response to therapy, Low said.

Low’s research group modeled the targeting molecule after a naturally occurring molecule that strongly binds to PSMA, called DUPA. Several alterations were necessary to create a molecule that fit the needs of a homing device and delivery vehicle, Kularatne said. The team created an area on the molecule that would link to various imaging or therapeutic agents to bring them along as cargo and created a spacer that would stretch the molecule so that its cargo would not keep it from properly fitting into the binding site. The spacer also was designed to improve binding of the targeting molecule to PSMA.

In addition to Low and Kularatne, co-authors of the papers include Endocyte researchers Kevin Wang and Hari-Krishna R. Santhapuram, graduate student in medicinal chemistry Zhigang Zhou, graduate student in chemistry Jun Yang, and professor of medicinal chemistry and molecular pharmacology Carol B. Post.

Low is the chief science officer for Endocyte, a Purdue Research Park-based company that develops receptor-targeted therapeutics for the treatment of cancer and autoimmune diseases. Endocyte holds the license to many of Low’s drug-targeting technologies.

Papers:

Prostate-Specific Membrane Antigen Targeted Imaging and Therapy of Prostate Cancer Using a PSMA Inhibitor as a Homing Ligand
Sumith A. Kularatne, Kevin Wang, Hari-Krishna R. Santhapuram, and Philip S. Low

Design, Synthesis, and Preclinical Evaluation of Prostate-Specific Membrane Antigen Targeted ^99mTc-Radioimaging Agents
Sumith A. Kularatne, Zhigang Zhou, Jun Yang, Carol B. Post, and Philip S. Low

By Elizabeth K. Gardner – Purdue University

June 11, 2009- Scientists at Johns Hopkins have discovered a potential strategy for cancer therapy by focusing on what’s missing in tumors.

The red dots in these photos indicate cancer cells that are dying. The image on the left shows the amount of cell death in a control-treated tumor and, on the right, in a microRNA-treated tumor. Tumor sections were stained with TUNEL, a marker for cells undergoing programmed cell death. * click on the image for high resolution

Noticing the conspicuous absence of single-stranded genetic snippets called microRNAs in cancer cells, a team of researchers from Johns Hopkins and Nationwide Children’s Hospital delivered these tiny regulators of genes to mice with liver cancer and found that tumor cells rapidly died while healthy cells remained unaffected.

Publishing results of the study June 12 in Cell, the researchers say they have provided one of the first demonstrations that microRNA replacement provides an effective therapy in an animal model of human disease.

“This work suggests that microRNA replacement may be a highly effective and nontoxic treatment strategy for some cancers or even other diseases,” says Josh Mendell, M.D., Ph.D., an associate professor in the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine. “We set out to learn whether tumors in a mouse model of liver cancer had reduced levels of specific microRNAs and to determine the effects of restoring normal levels of these microRNAs to these cancer cells. We were very excited to see that the tumors were, in fact, very vulnerable to microRNA replacement.”

His team had considered the possibility that the replacement of a single small RNA might have little if any effect, especially in the setting of all the complex changes that drive the aberrant behavior of a cancer cell. But the tumor cells in the mouse were indeed sensitive to the restoration of the microRNA—so much so that they died, rapidly.

“This concept of replacing microRNAs that are expressed in high levels in normal tissues but lost in diseases hasn’t been explored before,” Mendell says. “Our work raises the possibility of a more general therapeutic approach that is based on restoring microRNAs to diseased tissues.”

The Hopkins team was building on precedent-setting research (published January 2008 in Nature Genetics) showing that in a Petri dish, replacing microRNAs in lymphoma cells stopped the formation of tumors when the cells were injected into mice.

The new study involves animals that develop liver tumors closely resembling the human disease. Researchers chose to target the liver because, according to Mendell, it is a large organ whose function is detoxification and therefore, is a relatively accessible target for the delivery of small molecules, compared to other tissues.

Using a “special delivery” virus that can deliver genes to tissues without causing them any disease or harm, the researchers intravenously injected a fluorescent microRNA-containing virus into one group of mice with aggressive liver cancer, and injected a control virus containing no microRNA into another group. The viral delivery system was developed by Mendell’s father, Jerry Mendell, M.D., director of the Center for Gene Therapy at The Research Institute at Nationwide Children’s Hospital in Columbus, and K. Reed Clark, Ph.D., associate professor and director of the Viral Vector Core Facility at Nationwide Children’s Hospital.

After three weeks, six of eight mice treated with the control virus experienced aggressive disease progression with the majority of their livers replaced by cancerous tissue. In contrast, eight of 10 of animals treated with the microRNA were dramatically protected, exhibiting only small tumors or a complete absence of tumors. Liver body weight ratios were significantly lower in the treated mice, further documenting cancer suppression.

“The livers of the mice that received the microRNA virus glowed fluorescent green, showing that the microRNA ended up where it was supposed to go, and the cancer was largely suppressed,” Mendell said.

On the left are two mouse livers that were treated with the control virus; notice they are engulfed in tumors. On the right are two mouse livers treated with the microRNA virus; they show no tumors or significantly reduced tumors. * click on image for higher resolution

Equally intriguing, he reported, “The tumor cells that received the microRNA were rapidly dying while the normal liver cells were completely spared. These findings, as well as the results of specific tests for liver damage, demonstrated that the microRNA selectively kills the cancer cells without causing any detectable toxic effects on the normal liver or other tissues.”

Mendell points out that the microRNA is normally present at high levels in non-diseased tissues, and especially in the liver. Mendell speculates that this is why healthy cells are very tolerant to therapeutic delivery of even higher levels of this microRNA. However, the sensitivity of tumor cells to this microRNA suggests that loss of this molecule is a critical step as normal cells become cancer cells.

“Since we were able to demonstrate such dramatic therapeutic benefit in this extremely aggressive model of human liver cancer, we are hopeful that similar strategies will be effective for patients with this disease,” says Mendell.

In addition to Joshua Mendell, authors of the paper are Jerry Mendell, K. Reed Clark, Janaiah Kota and Chrystal L. Montgomery, of The Research Institute at Nationwide Children’s Hospital, Columbus, Ohio; and Raghu R. Chivukula, Kathryn A. O’Donnell, Erik A. Wentzel, Hun-Way Hwang, Tsung-Cheng Chang, Perumal Vivekanandan, and Michael Torbenson, all of Johns Hopkins University School of Medicine.

The research was supported by the National Institutes of Health, the Sol Goldman Center for Pancreatic Cancer Research and the Research Institute at Nationwide Children’s Hospital.

Johns Hopkins Medicine, The Johns Hopkins University, The Johns Hopkins Hospital, and Johns Hopkins Health System

In mice, the loss of microRNA miR-31 allows cancer cells to spread to the lungs more easily than cancer cells with miR-31. The edge of the cancer tumors lacking miR-31 are also less defined than tumors containing cells with higher levels of miR-31. Image: Scott Valastyan/ Whitehead Institute

Measuring levels of this so-called microRNA, which is also associated with metastatic breast cancer in humans, may more accurately predict the likelihood of metastasis (which accounts for 90% of cancer-related deaths) and ultimately help determine patient prognoses.

In the study, whose results are reported in the June 12 issue of Cell, Scott Valastyan, a graduate student in Whitehead Member Robert Weinberg’s laboratory, screened patient breast cancer samples for microRNAs with potential roles in metastasis. MicroRNAs are single strands of RNA about 21-23 nucleotides long. Within a cell, a single microRNA can fine-tune the expression of dozens of genes simultaneously. This capability could be particularly important in metastasis, a multi-step process that could be influenced by a single microRNA at several points.

The screened samples were classified as either metastatic cancer or non-metastatic cancer. After analysis, the microRNA miR-31 stood out because of its inverse correlation with metastasis. In samples where a patient’s original tumor had not metastasized, the cancer cells retained high levels of the microRNA. But where the tumor had metastasized, the cancer cells came to possess lower levels of miR-31.

The functional role of miR-31 in metastasis regulation was then confirmed in mice. When Valastyan removed miR-31 from normally non-aggressive breast cancer cells and implanted those cells into mice, the cells formed highly aggressive tumors. Mice injected with the cancer cells lacking miR-31 had 6 to 10 times more cancer cells that metastasized to their lungs than did their counterparts implanted with unmodified cancer cells.

To see how increasing miR-31 levels could affect metastasis, Valastyan introduced miR-31 into breast cancer cells that readily metastasize.  After injecting these altered cells into mice, the mice had four to 40 times fewer metastases than mice injected with the unaltered cells.

Valastyan says that quantifying miR-31 levels in a patient’s cancer cells could one day support a more accurate prognosis. Currently, breast cancers are divided into three major categories, two of which are typically associated with poor prognoses.

“This microRNA seems to be quite unique, in that it seems to provide some prognostic utility across these existing subclassifications [of cancers],” says Valastyan. A better-defined prognosis could help patients determine whether they might benefit from poorly tolerated cancer therapies.

In addition, miR-31 could be a useful target for cancer therapy. Weinberg, who is also a professor of biology at MIT, is cautiously optimistic. “At present, it’s quite difficult to inhibit the action or promote the actions of a microRNA in a whole organism,” he says, “but in the future, microRNAs like this one might prove to be very important in altering the clinical progression of a tumor or causing it to revert to a more benign state.”

This research was supported by the National Institutes of Health (NIH), MIT Ludwig Center for Molecular Oncology, U.S. Department of Defense (DoD), Breast Cancer Research Foundation, Harvard Breast Cancer Specialized Program of Research Excellence (SPORE), and a DoD Breast Cancer Research Program (BCRP) Idea Award.

Robert Weinberg’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also a professor of biology at Massachusetts Institute of Technology.

Full Citation:

“A Pleiotropically Acting microRNA, miR-31, Inhibits Breast Cancer Metastasis”

Cell, June 12, 2009

Scott Valastyan (1,2), Ferenc Reinhardt (1), Nathan Benaich (1,3), Diana Calogrias (4), Attila M. Szász (4), Zhigang C. Wang (5,6), Jane E. Brock (4), Andrea L. Richardson (4), and Robert A. Weinberg (1,2,7).

1.Whitehead Institute, 9 Cambridge Center, Cambridge, MA 02142, USA
2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
3. Department of Biology, Williams College, Williamstown, MA 01267, USA
4. Department of Pathology, Brigham and Women’s Hospital, Boston, MA 02115, USA
5. Department of Surgery, Brigham and Women’s Hospital, Boston, MA 02115, USA
6. Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA
7. MIT Ludwig Center for Molecular Oncology, Cambridge, MA 02139, USA

Written by Nicole Giese – Whitehead Institute for Biomedical Research

Prevention is key Sun-damaged rough patches on the skin — actinic keratoses — can turn into a greater variety of skin cancers than doctors had thought. Credit: NASA

PROVIDENCE, R.I. [Brown University] — Actinic keratoses are sun-damaged rough patches or lesions on the skin — often pink and scaly — that doctors have long believed can turn into a form of skin cancer known as squamous cell carcinoma.

Now researchers at Brown University, the Veterans Administration Medical Centers in Providence and Oklahoma City, and others have determined that actinic keratoses appear responsible for a larger spectrum of skin cancers than previously thought. Their research is highlighted in the current edition of Cancer.

“We found some interesting things,” said Dr. Martin Weinstock, the paper’s lead author. Weinstock, chief of dermatology at the VA Medical Center in Providence, is professor of dermatology and community health at The Warren Alpert Medical School of Brown University. The U.S. Department of Veterans Affairs Office of Research and Development funded the study.

Vincent Criscione, a fourth-year Alpert Medical School student, served as the paper’s first author. Beyond Brown and the VA, researchers from Rhode Island Hospital and Henry Ford Hospital in Detroit also contributed.

To conduct the study, Weinstock and the other researchers looked at 169 patients from the VA Medical Center in Oklahoma City who had a high risk for skin cancers. They, in turn, were among 1,131 patients from multiple cites who took part in a chemotherapy prevention trial conducted previously. Most had at least one actinic keratosis on their body. Combined, they had about 7,784 of the lesions on their faces and ears. There were up to six years of follow-up to quantify the risk of progression of actinic keratoses to cancer.

Among the findings: Two-thirds of the patients who had developed squamous-cell carcinomas, a form of treatable skin cancer, could trace their cancer to actinic keratoses. One-third of patients who ended up with basal cell carcinoma, the most common form of skin cancer in the United States, could trace their cancers to actinic keratoses.

Actinic keratoses The study provided up to six years of follow-up to quantify the risk of progression of actinic keratoses to cancer.

Scientists had previously been able to connect squamous-cell carcinomas to the lesions, but not basal cell. They also found that the actinic keratoses come and go, becoming invisible and resurfacing over time. That was a challenge for doctors because the lesions often were not visible during follow-up.

Thus, the research reinforces the need for skin cancer prevention. Scientists estimate that 40 million people in the United States alone have some form of actinic keratoses, and preventative removal of the lesions costs more than $1 billion annually, Weinstock said.

Before this study, Weinstock said, scientists could rely on one other body of research conducted 20 years ago that found less than 1 in 1,000 instances of actinic keratoses annually turned into squamous cell carcinoma, even though actinic keratoses are commonly removed as a preventative treatment for skin cancer.

Research is underway, Weinstock said, to determine if one of the treatments for actinic keratoses will be effective in preventing skin cancers.

Brown University