Posts Tagged ‘protein’

How premalignant cells can sense oncogenesis, halt growth

Since the 1980s, scientists have known that mutations in a human gene called RAS are capable of setting cells on a path to cancer. Today, a team at Cold Spring Harbor Laboratory (CSHL) publishes experiments showing how cells can respond to an activated RAS gene by entering a quiescent state, called senescence.

CSHL Professor Nicholas Tonks and Benoit Boivin, now a University of Montreal Assistant Professor, co-led a team that traced the process in exquisite detail. They began by confirming that activation of mutant, oncogenic H-RAS, one of the human RAS oncogene variants, spurs cells to generate hydrogen peroxide (H2O2), a form of reactive oxygen species, or ROS. “Most people, when they think about ROS, think about the great damage they can do at high concentrations,” says Tonks. “But this research exemplifies how the controlled production of ROS in cells can play a beneficial role.”

The team showed how the production of ROS in response to oncogenic H-RAS enables cells to fine-tune signaling pathways, leading them to enter a senescent state. A key part of this process is the impact of ROS on a protein called PTP1B. Tonks discovered PTP1B some 25 years ago. It is an enzyme — one in a family of protein tyrosine phosphatases (PTPs), of which there are 105 in humans — that performs the essential biochemical task of removing phosphate groups from amino acids called tyrosines in other proteins. Adding and removing phosphates is one of the principal means by which signals are sent among proteins.

In cells with oncogenic H-RAS, ROS is produced in small quantities, sufficient to render PTP1B inactive. The team found that with the phosphate-removing enzyme unable to do its usual job, a key protein called AGO2 remains phosphorylated — with the consequence that it can no longer do what it normally does, which is engage the cell’s RNA interference machinery. In normal cells, the RNAi machinery represses a gene called p21. But in this specific condition — with H-RAS oncogenically activated, PTP1B inactivated by ROS, and RNAi disabled — p21 proteins begin to accumulate unnaturally, the team discovered.

“This is the key step — accumulation of p21 proteins effectively halts the cell cycle and enables the cell to enter the senescent state,” explains Ming Yang, a doctoral student in the Tonks lab. She and Astrid Haase, Ph.D., a postdoctoral investigator in the laboratory of CSHL Professor Greg Hannon, are the first two authors, respectively, on the team’s paper, published in Molecular Cell.

“This is confirmation of a hypothesis we presented five years ago,” Tonks says. “We knew that oncogenic RAS induced the production of ROS. We proposed that this would lead to regulation of PTPs, and using the example of PTP1B this is precisely what the team discovered in this work — showing also how inactivation of this PTP is part of a complex signaling cascade that can culminate in the induction of senescence.”

ROS have been linked to the pathogenesis of several diseases including Alzheimer’s, diabetes and heart failure. “By showing that PTP1B inactivation by oxidation prevents AGO2 from doing its job, we make a clear link between ROS and gene silencing which could also be observed in other pathologies” says Boivin. Hence, the role of PTP1B in keeping the RNAi machinery active could have important ramifications.

Entering senescence is not enough to arrest oncogenesis completely. Oncogenic mutations typically multiply as cancers evolve to promote their survival and proliferation. But the current work does show the potential importance of knowing the genetic background of a cancer patient, for there are windows of time — narrow though they may be — in which naturally occurring processes induce pauses in growth.

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New tool to probe cancer’s molecular make-up

Researchers from the Cancer Research UK Manchester Institute based at The University of Manchester — part of the Manchester Cancer Research Centre — and the Institute of Cancer Research, London, looked at protein kinases, molecules that control various aspects of cellular function.

The study, funded by a Biotechnology and Biological Sciences Research Council (BBSRC)/Pfizer CASE studentship and CRUK,was published in Nature Methods this week (24 August).

Earlier work has shown that mutations or increases in a range of protein kinases are linked to tumour growth, and for several decades researchers have looked to develop drugs that target and prevent this activity in order to kill cancer cells. Ten types of drugs which reduce the activity have so far been approved for cancer treatment in patients.

Dr Claus Jørgensen, who led the study as team leader in the Division of Cancer Biology at The Institute of Cancer Research, London, before taking up a new post as head of the Systems Oncology group at the Cancer Research UK Manchester Institute, said: “Protein kinases regulate how cells communicate. When these molecules are deregulated it corresponds to cells “hearing voices” with a resulting change in their behaviour. Doctors need a way to spot changes in kinase levels in individual tumours so they can see how they respond to treatments and match patients to the treatment that works best for them.”

The team investigated the make-up of over 200 protein kinases. They used a technique known as mass spectrometry to develop a method that can both identify and measure the amount of various kinases in a biological sample — for example from a part of a tumour removed in surgery. “Our new method can correctly measure the amount of protein kinases in a sample. It means we can monitor cancer cell behaviour and also how tumours respond to different therapy in cancer patients,” added Dr Jørgensen.

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Cellular biology of colorectal cancer: New Insight

Lead author Kristi Neufeld, associate professor in the Department of Molecular Biosciences and co-leader of the Cancer Biology program at the KU Cancer Center, has spent the better part of her career trying to understand the various activities of APC, a protein whose functional loss is thought to initiate roughly 80 percent of all colon polyps, a precursor to colon cancer. Neufeld, along with her postdoctoral fellow Maged Zeineldin, undergraduate student Mathew Miller and veterinary pathologist Ruth Sullivan, now reports that APC found in a particular subcellular compartment, the nucleus, protects from inflammation as well as tumor development associated with chronic colitis.

Whether APC reaches the nucleus may well affect the ability of intestinal stem cells to produce differentiated cells with specialized functions, Neufeld said.

“It’s not widely appreciated, but there is still plenty of cell growth going on in adults, with the colon being a good example,” she said. “On average, we shed and replace about 70 pounds of intestinal tissue annually, so you can imagine that this process requires exquisite control to prevent tumor formation.”

Regular renewal of the colon lining occurs through stem cells that are capable of constantly dividing. These cells produce descendants that take up specific roles: By secreting mucin, for instance, goblet cells generate a mucus layer that serves as the colon’s physical barrier against its many microbial tenants. But if APC can’t find its way to the nucleus, Neufeld and her team have noted far fewer goblet cells as one outcome.

“We introduced a specific APC mutation into mice that took away the nuclear zip code, so to speak, leaving APC stuck in the cytoplasm,” Neufeld said. The researchers studied this mouse model under conditions that induce ulcerative colitis, a form of inflammatory bowel disease that can be a prelude to colon cancer.

Observing significantly more colon tumors in these mice compared to those with normal APC in the same disease setting, they hypothesized that functional nuclear APC might somehow guard against inflammation and its downstream effects, including tumor development. Now, Neufeld thinks she and her team may have a clue as to how this happens.

“The drop in goblet cell numbers we observed was striking,” she said. “We then examined one of the proteins found in mucus, called Muc2, and found that its RNA levels were greatly decreased. If there are fewer goblet cells as a result of APC being unable to reach the nucleus, there will also be less mucus, which could increase the colon’s sensitivity to bacteria and other microorganisms in the gut that are capable of promoting inflammation.”

Neufeld said while there are still no quick fixes for mutant genes, perhaps tools could be developed to synthetically replace this less-than-ideally thick mucus layer in affected people.

One known function of APC is that it halts cell proliferation: by muzzling the canonical arm of the Wnt signaling pathway, which otherwise instructs cells to go forth and multiply. Neufeld and her group have already shown, using the same mouse model, that APC stationed in the nucleus is necessary to suppress Wnt and its signaling partners — particularly β-catenin, a key target of normal APC. With a role for nuclear APC in controlling goblet cell differentiation now supported, the researchers are probing possible mechanisms to learn if and how Wnt pathway members might be involved.

Comprising 2,843 amino acids, APC is a large protein.

“Rather than being a simple, single-function protein, APC is more like a complex set of moving parts, each doing something different and most still poorly understood,” Neufeld said. “I think if the sole purpose of this protein was to target β-catenin for destruction, it wouldn’t need to be this large. Our next job is to figure out exactly how goblet cell differentiation is controlled by one or more of APC’s many components.”

Beyond a slew of mechanistic details, the bigger picture that Neufeld and her group will keep exploring is that some colon cancers could arise from an inflammatory response to bacterial penetration of a thinner-than-normal mucus layer in the gut, resulting from defective APC. The possibilities of just what APC does and doesn’t do, and how to compensate for any intestinal glitches related to loss of APC function, present a challenging mystery but also a plentiful harvest for scientists like Neufeld to reap going forward.

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