Mechanisms of Resilience

Understanding how malignant cells survive our attempts to kill them is critical for improving cancer therapy

BY DARRELL E. WARD

Arnab Chakravarti 2More than 1.6 million Americans were expected to develop cancer in 2012, and more than half of them were likely treated with radiation therapy. “Glioblastoma and certain other cancers are highly radiation resistant, while others such as neuroblastoma and certain lymphomas are inherently sensitive to radiation,” says Timothy Lautenschlaeger, MD, an American Board of Radiology B. Leonard Holman Scholar in Radiation Oncology at The Ohio State University Comprehensive Cancer Center – Arthur G. James Cancer Hospital and Richard J. Solove Research Institute (OSUCCC – James).

“At this point, we don’t really know why some types of cancer are so sensitive and others so resistant,” Lautenschlaeger says. “It isn’t simply a mutation or change in a particular gene that determines radiosensitivity in general; these differences are cancer-specific and dramatic.”

The failure of radiation therapy – which uses high-energy X-rays to kill tumor cells – is a significant cause of the 577,000 deaths from cancer that were expected in 2012. “Overcoming the mechanisms of therapeutic resistance is key to improving cancer treatment,” says Arnab Chakravarti, MD, chair of Radiation Oncology and co-director of the Brain Tumor Program at the OSUCCC – James.

Radiation resistance has multiple components that involve DNA repair, hypoxia, activation of pro-survival pathways, cancer stem cells, and changes that lead to invasion and metastasis, he says. Chakravarti has recruited Lautenschlaeger and other leaders in the field to the OSUCCC – James in an effort to understand and overcome radiation resistance.

DNA Repair


Fen XiaRadiation kills cancer cells mainly by causing breaks in both strands of the DNA helix. “Failure to repair double-strand breaks triggers cell-death processes, including apoptosis, whereas tumors adept at repairing double-strand breaks tend to be radiation resistant,” says Fen Xia, MD, PhD, associate professor of Radiation Oncology and an OSUCCC – James researcher who specializes in DNA damage response and repair in radiation resistance.

Xia wants to understand how cells sense DNA damage and initiate the repair response, and how these actions differ between tumor and normal cells. “We want to interrupt this process and kill tumor cells while preserving normal cells,” she says.

To overcome radiation resistance, Xia is exploring two strategies: protecting normal cells from the lethal effects of radiation while concurrently enhancing radiation-induced tumor cell death.

BRCA1 and PARP1 Inhibition


An example of the second strategy is Xia’s work to make cancer cells with functional BRCA1 protein susceptible to PARP1 inhibition. PARP1 – or poly ADP-ribose polymerase – is a molecular complex that quickly repairs breaks in one of DNA’s two strands.

DNA single-strand breaks can lead to double-strand breaks during replication. Unrepaired double-strand breaks are lethal to proliferating cancer cells. Dysfunction in the repair of both single-strand breaks and double-strand breaks will be synthetically lethal.

BRCA1 – infamous, along with BRCA2, for raising breast-cancer risk when mutated – is a DNA-repair protein that corrects double-strand
breaks. Xia, in a 2012 study published in the journal Cancer Research, and others have shown that the PARP1 inhibitor olaparib is highly selective in killing BRCA1-mutated familial breast tumors.

“Unfortunately, over 90 percent of patients who develop sporadic breast cancer carry functional BRCA1 and BRCA2 proteins and are proficient in repair of double-strand breaks, precluding them from this potent therapy,” Xia says. “An important goal of our work is to make PARP inhibitors available to the majority of patients, those with working BRCA1 genes.”

In earlier work, Xia demonstrated that BRCA1’s repair function occurs in the nucleus, but that the BRCA1 protein shuttles between the nucleus and cytoplasm. In the cytoplasm, it loses its DNA-repair function and instead facilitates apoptosis. “We show that if the BRCA1 protein moves to the cytoplasm, cells die much more quickly and efficiently,” Xia says.

Xia and her collaborators believe that if they can force BRCA1 out of the nucleus, it could make tumors that have functional DNA repair susceptible to PARP-inhibition therapy. The same strategy could be used to sensitize tumors to radiation-induced DNA damage, she says.

Hypoxia


Nicholas DenkoWhen tumors outgrow their blood supply, they develop new blood vessels that are abnormal and leaky. These poorly perfused tumors have pockets of low oxygen and necrosis; that is, they are hypoxic.

Hypoxia within tumors reduces the effectiveness of radiation therapy. When therapeutic ionizing radiation strikes cellular molecules, it generates ions, free electrons and hydroxyl radicals. Some of these reactive elements are captured by the cell’s natural buffering system, but many strike its DNA and cause double-strand breaks that overwhelm the repair system and kill the cell.

“The presence of oxygen enhances the fixation of this DNA damage and the lack of oxygen inhibits it,” says Nicholas Denko, MD, PhD, associate professor of Radiation Oncology at the OSUCCC – James and an authority on hypoxia and radiation therapy.

The absence of oxygen in tissue can decrease the effective radiation dose by a factor of three – defined as the oxygen enhancement ratio. That is, it can take three times the radiation dose to kill seriously hypoxic cells versus well-oxygenated cells, he says.

But there is little latitude for raising a patient’s radiation dose, which is chosen primarily to kill tumor cells and is about the maximum possible without causing complications.

“Most tumor types have areas of hypoxia,” Denko says. Studies have been done in patients with head and neck cancer, cervical cancer or soft-tissue sarcomas. “We calculate that in these tumors even a small fraction of hypoxic tumor cells can have a big effect on patient outcome,” he says.

“People have understood the oxygen enhancement ratio for 50 years, but no one has figured out how to use it clinically,” he explains. Investigators have tried to deliver more oxygen to the tumor. They’ve transfused patients with red blood cells, given patients erythropoietin to grow more red blood cells and had patients breathe pure oxygen to get more oxygen to the tumor. “These have all had disappointing clinical effectiveness,” he says.

Denko and his colleagues are approaching the problem differently. “Rather than trying to increase the tumor’s oxygen supply, we want to reduce the tumor’s oxygen demand,” he says.

The researchers are evaluating drugs that reduce oxygen consumption by mitochondria, the main oxygen sink in cells. “Mitochondria produce ATP, which powers everything we do,” Denko says. “There are drugs that inhibit mitochondria as a side effect. If we can use them in tumors to reduce oxygen demand by mitochondria, we might improve the tumor’s response to radiation therapy.”

Pro-Survival Pathways


Research by Ohio State’s Arnab Chakravarti shows that radiation therapy can activate critical pro-survival signal-transduction pathways and shut down cell-death pathways, enhancing cancer-cell survival and radiation resistance.

In a Journal of Clinical Oncology study, Chakravarti reported that activation of the PI3 kinase/AKT pathway is associated with adverse outcomes in malignant glioma patients. These pro-survival molecules were activated far more often in higher-grade gliomas. “Glioblastomas had the highest activation of PI3 kinase family members compared with grade III tumors or grade II tumors, and the degree of activation was strongly associated with adverse clinical outcome and radiation resistance,” Chakravarti says. As such, the degree of pathway activation in glioblastoma is an independent prognostic marker over and beyond tumor grade.

In a 2011 New England Journal of Medicine paper, Chakravarti collaborated with Marcus Bredel, MD, an associate professor at the University of Alabama Birmingham and an adjunct associate professor of Radiation Oncology at the OSUCCC – James, and showed that loss of a gene called NF-kBIA promotes the growth of glioblastoma multiforme, the most common and deadly form of brain cancer. The findings suggested that therapies that stabilize this gene might improve survival for certain glioblastoma patients.

“The NF-kB pathway is thought to be related to the PI3 kinase/AKT pathway, so it’s interesting that an NF-kBIA deletion is also strongly associated with therapeutic resistance,” Chakravarti says. “Overall, this shows that these pro-survival pathways play a major role in mediating radiation resistance.”

Low-Grade Gliomas


Erica Hlavin Bell, PhD, research assistant professor of Radiation Oncology at the OSUCCC – James, works closely with Chakravarti to identify biomarkers of radiation resistance in low-grade gliomas and prostate cancer.

“We want to learn whether grade 2 and grade 3 gliomas respond differently to treatment compared with grade 4 tumors (i.e., glioblastoma multiforme),” Bell says.

“Most studies of brain tumors have been completed with varying grades, and patients with different grades are often treated in a very similar fashion,” she adds. “We are asking whether lower grade tumors need the same treatment as glioblastoma. We’ve learned that, at the molecular level, lower-grade brain tumors are very different from grade 4 tumors. Because of this, we believe that their treatment response is probably also different. We want to understand that in detail.”

Bell’s investigations use tumor samples obtained through two Radiation Therapy Oncology Group (RTOG) trials: RTOG-9813 (ClinicalTrials.gov identifier NCT00004259) and RTOG-9802 (ClinicalTrials.gov identifier NCT00003375). She is looking at low-grade glioma tissue for new and known biomarkers and comparing them with patient outcomes.

“This tissue is precious,” she says, “and we want to acquire as much information as possible from each specimen.”

Glioblastoma and Melanoma


People with grade-4 melanoma of the skin or with grade-4 glioma (glioblastoma) in the brain survive about one year after diagnosis. Both malignancies are highly resistant to chemotherapy and radiation. “We are trying to identify the cause of that resistance,” says Kamalakannan Palanichamy, PhD, research assistant professor of Radiation Oncology at the OSUCCC – James.

In both malignancies, Palanichamy and his lab are combining genomic, transcriptomic, epigenomic, proteomic and metabolomic analyses to identify subsets of patients who will better benefit from a particular treatment.

In addition, Palanichamy is investigating the role of self-renewing populations of cancer cells, also called cancer stem cells (CSCs), in radiation resistance. They isolate CSCs from glioblastoma tumors, culture them and expose them to chemotherapy, small-molecule inhibitors and radiation alone and in combination.

Recurrent Bladder Cancer


Some 73,500 Americans were expected to develop bladder cancer in 2012, and an estimated 14,900 people died from the disease. Three quarters of newly diagnosed bladder-cancer cases present with superficial, noninvasive tumors. These are treated by minimally invasive surgery through the urethra, sometimes with chemotherapy. There is a high rate of prolonged survival.

For invasive bladder cancer, the bladder is frequently removed. In many cases, however, it is possible to preserve the bladder with transurethral surgery, radiation and chemotherapy, says Lautenschlaeger, who is studying radiation therapy for bladder cancer.

“Bladder removal versus bladder preservation is also important when superficial bladder cancer recurs as muscle-invasive cancer,” Lautenschlaeger says.

Lautenschlaeger is working to identify biomarkers of recurrence that will enable urologists to identify patients who are better candidates for surgery or for bladder-preservation therapy in collaboration with a team led by William U. Shipley, MD, at the Massachusetts General Hospital (MGH)/Harvard Medical School. Together with MGH investigators who originally pioneered the concept of bladder-preservation therapy, Lautenschlaeger leads a correlative study for markers of treatment resistance that is part of a phase II randomized study for patients with muscle-invasive bladder cancer evaluating transurethral surgery and concomitant chemoradiation (Clinical trials.gov identifier NCT00777491).

“Ideally, we will one day be able to tell patients whether or not they are good candidates for bladder preservation,” he says. “Patients with markers indicating bladder preservation can avoid the quality-of-life changes associated with bladder loss, and patients with markers indicating a high risk of recurrence can avoid the side effects of chemotherapy and radiation.”

Fueling Resistance


Deliang Guo, PhD, assistant professor of Radiation Oncology, is investigating links between tumor-cell metabolism cells and radiation and chemotherapy resistance. “Cancer cells have altered and enhanced metabolism, and oncogenes are involved in that metabolic reprogramming,” Guo says. “We want to unravel the links between oncogenic signaling pathways and cancer metabolism.”

Additionally, Guo hypothesizes that radiation therapy can alter glucose metabolism in cancer cells in ways that contribute to radiation resistance. He reasons that radiation can boost the production of ATP and of nucleic acids, lipids and amino acids, which cancer cells need for growth and proliferation.

“We are now investigating whether the irradiation of cancer cells directly increases glucose uptake and ATP production in cancer cells,” he says.

Guo, whose specialty is oncogene signaling and metabolic pathways, is collaborating on this work with Chakravarti, a specialist in radiation therapy. “We’ve combined our expertise to learn whether radiation causes this metabolic change, and whether we can interrupt this interaction and reduce radiation resistance,” Guo says.

The Future


Research is improving radiation therapy, Lautenschlaeger says. “We’re learning, for example, that changing the fractionation schedule might improve treatment outcomes in certain tumors. The traditional schedule of low daily doses works well and is safe, but some cancers might be more sensitive to one or just a few high doses of radiation.

“There are now protocols for lung cancer that use only a few fractions of very high doses of radiation,” he says. “This was first tried in patients with advanced, inoperable disease. Now, trials using radiation without surgery are available to patients who are operable.

“The day is coming when we will do molecular profiling of tumors and understand that the tumor has a particular array of gene changes, and that information will enable us to determine the optimal treatment regimen,” he says. “Radiation therapy for cancer will become much more personalized and even more effective.”

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