In the fight against cancer, doctors dish out combination-blows of surgery, chemotherapy and other drugs to beat back a merciless foe. Now, scientists have taken early steps toward adding a stinging punch to clinicians’ repertoire.

With a novel targeted therapy researchers at the Georgia Institute of Technology have purged ovarian tumors in limited, in vivo tests in mice. “The dramatic effect we see is the massive reduction or complete eradication of the tumor, when the ‘nanohydrogel’ treatment is given in combination with existing chemotherapy,” said chief researcher John McDonald.

That nanohydrogel, a type of nanoparticle, is a minute gel pellet that honed in on malignant cells with a payload of an RNA strand. The RNA entered the cell, where it knocked down a protein gone awry that is involved in many forms of cancer.

In trials on mice, it put the brakes on ovarian cancer growth and broke down resistance to chemotherapy. That allowed a common chemotherapy drug, cisplatin, to drastically reduce or eliminate large carcinomas, with very similar speed and manner. The successful results treating four mice with the combination of siRNA and cisplatin showed little variance.

Chink in the armor

The therapeutic short interfering RNA (siRNA) developed by McDonald and Georgia Tech researchers Minati Satpathy and Roman Mezencev, thwarted cancer-causing overproduction of cell structures called epidermal growth factor receptors (EGFRs), which extend out of the wall of certain cell types. EGFR overproduction is associated with aggressive cancers.

The researchers from Georgia Tech’s School of Biological Sciences published their results on Monday, November 7, 2016, in the journal Scientific Reports. Research was funded by the National Institutes of Health’s IMAT Program, the Ovarian Cancer Institute, the Deborah Nash Endowment Fund, the Curci Foundation and the Markel Foundation.

The new treatment has not been tested on humans, and research would be required by science and by law to demonstrate consistent results – efficacy – among other things, before preliminary human trials could become possible.

The current in vivo success strengthens the idea that knocking out EGFR at the RNA level may be a worthy goal to explore in the fight against carcinomas in general. The same patented nanohydrogel packed with other types of therapeutic RNA is currently being tested for the treatment of other types cancers.

Helper turned killer

EGFRs are receptors found in epithelial cells, which line organs throughout the body: Lungs, mouth, throat, intestines and others. In women, it also lines reproductive organs: Ovaries, uterus and cervix.

They are long proteins that poke through the cell membrane, connecting the cell’s interior with the outside. They look like squiggly worms with tiny mouths on the outside that take up a messenger protein.

In a healthy cell, those messenger molecules cause EGFRs to trigger long chains of biochemical reactions that lead to the activation of genes involved in a variety of cellular functions. In carcinoma cells, the number of EGFRs present typically skyrockets.

“In many cancers, EGFR is overexpressed,” said McDonald, who heads Georgia Tech's Integrated Cancer Research Center. “The problem is that because of this overexpression, many cellular functions, including cell replication and resistance to certain chemotherapy drugs, are dramatically cranked up.”

The cell goes haywire, metabolizes too much sugar, divides too much, and resists chemotherapy. The cancer grows into a tumor and can spread through the body.

An overabundance of EGFRs found in a biopsy is usually a sign that cancer patient prognosis is poor. “In 70 percent of ovarian cancer patients, EGFR is overexpressed at very high levels,” McDonald said.

Cell suicide: apoptosis

EGFR overexpression also makes cancer cells resistant to chemotherapy by thwarting a natural defense mechanism.

“The platinum-based chemotherapies used to treat ovarian cancers cause DNA damage, which switches on apoptosis,” McDonald said. Apoptosis is cell suicide. When cells can’t repair DNA damage, they’re programmed to kill themselves to keep the damaged cells from spreading.

The primary chemotherapy used to treat ovarian cancer works by coaxing cancer cells to trigger the suicide program, but having too many epidermal growth factor receptors gets in the way.

“EGFR overexpression hinders apoptosis; they won’t die. By knocking down EGFR, we make the cell hypersensitive to the drug. Apoptosis is reactivated,” McDonald said.

Existing EGFR targeted drugs called tyrosine-kinase inhibitors disrupt an EGFR function, but their success in treating ovarian cancer has been limited. “Clinicians have tried EGFR inhibitors to treat ovarian cancers for some years, and they only get about 20% of patients responding to it,” McDonald said. “Apparently, the particular EGFR function inhibited by these drugs is not critical to ovarian cancer.”

Guided brass knuckles

The short interfering (si) RNA designed by the Georgia Tech researchers attacks the cancer much closer to its root.

To make the protein for EGFR, RNA has to transfer its genetic code from DNA. The researchers’ siRNA binds to the cell’s RNA and stops it from working.

“We’re knocking down EGFR at the RNA level,” he said. “Since EGFR is multi-functional, it’s not exactly clear which malfunctions contribute to ovarian cancer growth. By completely knocking out its production in ovarian cancer cells, all EGFR functions are blocked.”

The nanohydrogel that delivers the siRNA to the cancer cells is a colloid ball of a common, compact organic molecule and about 98 percent water. Another molecule is added to the surface of the nanohydrogel as a guide. It makes the pellets adhere to the cancer cells like sticky cluster bombs.

Cancerous tissue may also be aiding the nanohydrogel in targeting it. “When you get into a tumor, there are a lot of blood vessels, and many are broken,” McDonald said. “This may help the nanoparticles get passively trapped in the neighborhood of tumorous tissues.”

In the in vivo trials, the siRNA, which contained a fluorescent tag, allowed researchers to observe nanoparticles successfully honing in on the cancer cells.

Fortuitous victory

“We originally selected to target the EGFR gene because its activity is easily measured, and we wanted to use it simply as an indicator that our nanoparticle siRNA delivery system was working,” McDonald said. “The fact that the EGFR knockdown so dramatically sensitized the cells to standard chemotherapy came as a bit of a surprise.”

At first, his team observed how the tumors responded to chemotherapy alone. Then they combined it with the nanoparticle treatment.

“When we gave the chemotherapy alone, the response was moderate, but with the addition of the nanoparticles, the tumor was either significantly reduced or completely gone,” McDonald said.

But he tempered enthusiasm with caution. “Further work will be required to see if the treatment completely destroyed every trace of cancer cells in the tumors that disappeared, or if future recurrence is possible.”

If the researchers’ continuing studies further prove to be consistent, the combination of the nanohydrogel with other therapeutic RNAs could represent a significant advancement in the treatment of a wide spectrum of cancers.

Georgia Tech’s Lijuan Wang and Dr. Benedict Benigno from Atlanta’s Northside Hospital coauthored the paper. Research was funded by the National Institutes of Health’s Program for Innovative Molecular Analysis Technologies Program (grant 1R21CA155479-01), the Ovarian Cancer Institute at Northside Hospital, the Deborah Nash Endowment Fund, the Curci Foundation, and the Markel Foundation. Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the sponsoring agencies.

Luis Miguel Rodriguez-Rojas graduated with a Ph.D. in Bioinformatics with a minor in Biomedical Engineering. He came to Georgia Tech with an M.S. in Biological Sciences from Universidad de Los Andes, in Bogota, Colombia; an M.S. in Applied Informatics from Université Montpellier 2 (currently Université de Montpellier), in Montpellier, France; and B.S. in Biology from Universidad Nacional de Colombia, in Bogota. He is off to a postdoctoral position in Georgia Tech’s School of Civil and Environmental Engineering.

What attracted you to study in Georgia Tech? How did Georgia Tech meet your expectations?

The main reason was my advisor, Dr. Kostas Konstantinidis. I read some of his work while I was an undergraduate and was fascinated by his research. While studying for my master’s degree, I had the privilege of visiting his lab for two weeks. During this period, I became convinced that I wanted to work in microbial ecology, and he offered to receive me as a Ph.D. student. Once I started the program, I quickly realized that Georgia Tech exceeded my expectations, offering a far richer campus life than I had anticipated.

What is the most important thing you learned while at Georgia Tech?

Balancing work and academic life with other activities. I became involved in social dancing, a hobby I’ve cultivated and enjoyed for over three years now, learning salsa, bachata, zouk, and tango. I discovered in Georgia Tech the importance of this balance in carrying out a productive and happy academic life.

What surprised you the most at Georgia Tech? What disappointed you the most?

I was surprised by the variety of cultural activities. Having a stereotypical image of a technology institute in mind, I was pleasantly surprised by poetry recitals, concerts, dance and theater performances, and many more activities on campus. After the success of the BVN Youth Poetry Slam semifinals at Georgia Tech in summer 2015, it was a disappointment that Tech didn’t continue to build on promoting slam poetry.

Which professor(s) or class(es) made a big impact on you? Why?

Certainly my advisor, Dr. Konstantinidis. Not only did I learn about microbial ecology from him, but also his frequent encouragement to critically discuss ideas has prepared me for scholastic discussion outside of Tech.

What is your most vivid memory of your time at Georgia Tech?

I cherish with particular warmth my memories of the Salsa Club, first as a regular member and later as a board member and an instructor.

On the basis of your experience, what advice would you give to incoming new graduate students at Georgia Tech?

Learn to say no and value your free time.

Learning to say no is hard, but as graduate students we often get bombarded with options and our first instinct is to try and cover them all. Some diversity in research topics is highly desirable, but it’s important to find a balance in which, at the end, a consistent story can be told in the dissertation.

Another area in which balance is hard to find is time management. We tend to err on the side of too much academic involvement and little or no personal life. Hobbies are important, they keep us healthy, happy, and productive, and it’s our own job to cultivate them and devote some time to them.

What feedback would you give to Georgia Tech leaders, faculty, and/or staff to improve the Georgia Tech experience for future students?

I would encourage more curricular freedom for graduate students. I was fortunate enough to be in the Ph.D. in Bioinformatics, a program with great latitude on the courses I could (or should) take. And yet, even in this program, I was never presented with the possibility of attending classes outside of the main program areas, while most advisors explicitly discourage this. For example, Georgia Tech offers very interesting courses in the humanities that are never mentioned to graduate students in the sciences or engineering.

Where are you headed after graduation? How did your Georgia Tech education prepare you for this next step?

I’ll stay in Georgia Tech for a short-term postdoctoral position in the School of Civil and Environmental Engineering. I plan on continuing in an academic career, for which Georgia Tech has prepared me with valuable practical experience in research, collaborations with faculty and students from other laboratories, and proposals of novel research ideas and projects.

Genetic mitochondrial disease is present in about 1 out of every 5,000 babies, who face insurmountable odds from the moment they are born. That’s because at present, there is no cure for these conditions. But a new assisted reproductive technology that prevents the transmission of mitochondrial disease from mother to child holds great promise.

Mitochondrial replacement (MR) therapy combines the nuclear DNA from the mother with healthy mitochondria from a donor egg to create a healthy new egg that can be fertilized with the father’s sperm, thereby yielding a “three-person baby.” Last year, the world’s first three-person baby resulting from this method was delivered by U.S. doctors in Mexico, where there are no laws prohibiting the procedure.

The healthy newborn got about 0.1 percent of his DNA from the donor, and the vast majority of his genetic code – specifying eye color, hair, etc. – from his mom and dad.

Mitochondrial DNA comprises just a small percentage of our total DNA, containing just 37 of the 20,000 to 25,000 protein-coding genes in our body. And while nuclear DNA comes from both parents, “our mitochondrial DNA comes directly from our mothers, so my mitochondrial genome will be exactly like my mother’s, yours will be like your mother’s, and so on,” says Lavanya Rishishwar, former grad student in the lab of Petit Institute researcher King Jordan and team lead for Applied Bioinformatics Laboratory (ABiL, a public-private partnership between Georgia Tech and IHRC Inc.).

While the method hasn’t been green lighted in the U.S. yet, the United Kingdom gave the go-ahead for MR therapy in December. This announcement came in the wake of concerns about the safety of MR therapy that were raised by evolutionary biologists, who argue that nuclear and mitochondrial genomes evolved concurrently, and therefore mitochondria from one person or population may not be compatible with nuclear material from another.

In support of the evolutionary biologists’ nuclear-mitochondrial mismatch hypothesis, a number of previous studies on model organisms have provided evidence for incompatibility between nuclear and mitochondrial genomes from divergent populations of the same species. But a recent study by Jordan and Rishishwar published in BMC Genomics lays those fears to rest.

“The alarm was raised based on work that was done on model systems,” says Jordan, associate professor in the School of Biological Sciences and director of the Bioinformatics Graduate Program. “They didn’t work with humans, they worked with fruit flies, with mice, and those experiments resulted in a host of different problems for the resulting offspring. The key is, those were artificial experiments. Meanwhile, there’s been an ongoing natural experiment that has been conducted over millennia in human populations.”

So Jordan and Rishishwar tested the nuclear-mitochondrial mismatch hypothesis for humans by observing the source: humanity. They used data from the 1,000 Genomes Project and the Human Genome Diversity Project, studying the incidents of nuclear- mitochondrial DNA mismatch seen in more than 3,500 people from about 60 populations on five continents.

“We’ve been working for some years on human population genomics and remain interested in admixed American populations,” Jordan says. “The trajectory of modern human evolution for the past 50,000 to 100,000 years starts with the journey out of Africa, followed by a long period when populations were geographically isolated for the most part.  During that time, human populations genetically diverged since they were physically isolated.”

But over the past 500 years or so, since Columbus came to the new world from Europe, “that process of isolation and divergence got flipped upside down,” Jordan notes. “Over a very short evolutionary time, you had populations from the Americas, Europe, and shortly thereafter, Africa because of the transatlantic slave trade, that were all brought together.”

Hence, in the Americas we’ve seen the creation of genome sequences that are evolutionarily novel in the history of humanity, in that they contain combinations of variants that had never existed together before. Jordan and his team have been studying this for a while, and understood that healthy individuals can bear combinations of variants that had different ancestral sources within the same genomic background.

“We knew that at a very intuitive level because of our own research,” says Jordan, who stumbled on a paper in Nature expressing the grave concerns of evolutionary biologists and thought, “instead of relying on artificial experiment systems, why don’t we just try to read the results of this long, ongoing experiment of human evolution and see what it tells us.”

They found that even people with very similar nuclear DNA (nDNA) genomes can have highly divergent mitochondrial DNA (mtDNA) and vice versa. Ultimately, their results showed that mitochondrial and nuclear genomes from divergent human populations can co-exist in healthy individuals, indicating that mismatched nDNA-mtDNA combinations are basically harmless and not likely to jeopardize the safety of MR therapy.

“We tend to think that the story of our evolution is the story of migration, physical isolation, and genetic diversification,” Jordan says. “But all throughout that process, there was admixture along the way. It’s not like there was a linear, onward march. It confirms and underscores the fact that humans are a relatively evolutionarily young species, and from the genetic perspective, there is complete compatibility between human populations.”

Drexel University and Georgia Institute of Technology researchers have discovered how the Rad52 protein is a crucial player in RNA-dependent DNA repair. The results of their study, published June 8 in the journal Molecular Cell, uncover a surprising function of the homologous recombination protein Rad52. They also may help to identify new therapeutic targets for cancer treatment.

Radiation and chemotherapy can cause a DNA double-strand break, one of the most harmful types of DNA damage. The process of homologous recombination — which involves the exchange of genetic information between two DNA molecules — plays an important role in DNA repair, but certain gene mutations can destabilize a genome. For example, mutations in the tumor suppressor BRCA2, which is involved in DNA repair by homologous recombination, can cause the deadliest form of breast and ovarian cancer. 

Alexander Mazin, a professor at Drexel University’s College of Medicine, and Francesca Storici, an associate professor at Georgia Tech’s School of Biological Sciences, have dedicated their research to studying mechanisms and proteins that promote DNA repair. 

In 2014, Storici and Mazin made a major breakthrough when they discovered that RNA can serve as a template for the repair of a DNA double-strand break in budding yeast, and Rad52, a member of the homologous recombination pathway, is an important player in that process. 

“We provided evidence that RNA can be used as a donor template to repair DNA and that the protein Rad52 is involved in the process,” said Mazin. “But we did not know exactly how the protein is involved.”

In their current study, the research team uncovered the unusual, important role of Rad52: It promotes “inverse strand exchange” between double-stranded DNA and RNA, meaning that the protein has a novel ability to bring together homologous DNA and RNA molecules. In this RNA-DNA hybrid, RNA can then be used as a template for accurate DNA repair. 

It appeared that this ability of Rad52 is unique in eukaryotes, as otherwise similar proteins do not possess it. 

“Strikingly, such inverse strand exchange activity of Rad52 with RNA does not require extensive processing of the broken DNA ends, suggesting that RNA-templated repair could be a relatively fast mechanism to seal breaks in DNA,” Storici said. 

As a next step, the researchers hope to explore the role of Rad52 in human cells. 

“DNA breaks play a role in many degenerative diseases of humans, including cancer,” Storici added. “We need to understand how cells keep their genomes stable. These findings help bring us closer to a detailed understanding of the complex DNA repair mechanisms.”

The research was supported by the National Institutes of Health, the National Science Foundation and the Howard Hughes Medical Institute.

These results offer a new perspective on the multifaceted relationship between RNA, DNA and genome stability. They also may help to identify new therapeutic targets for cancer treatment. It is known that active Rad52 is required for proliferation of BRCA-deficient breast cancer cells. Targeting this protein with small molecule inhibitors is a promising anticancer strategy.  However, the critical activity of Rad52 required for cancer proliferation is currently unknown.

This new Rad52 activity in DNA repair, discovered by Mazin, Storici and their team, may represent this critical protein activity that can be targeted with inhibitors to develop more specific — and less toxic — anti-cancer drugs. Understanding of the mechanisms of RNA-directed DNA repair may also lead to development of new RNA-based mechanisms of genome engineering. 

This research was supported by the National Institute of General Medical Sciences (NIGMS) of the NIH (grant GM115927), the National Science Foundation (grant 1615335), and the Howard Hughes Medical Institute Faculty Scholar Program (grant 55108574). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the sponsoring agencies.

Written by Drexel University.

CITATION: Olga M. Mazina, Havva Keskin, Kritika Hanamshet, Francesca Storici,
Alexander V. Mazin, “Rad52 Inverse Strand Exchange Drives RNA Templated
DNA Double-Strand Break Repair,” (Molecular Cell, 2017). http://dx.doi.org/10.1016/j.molcel.2017.05.019

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