Scientists find 'missing link' in important tumor suppression mechanism

Nov. 12, 2013 — RUNX3, a gene that is intensively studied for its function as a tumor suppressor, is likely to be a key and critical component of the body's first line of defense against lung cancer development, according to a recent study by scientists from the Cancer Science Institute of Singapore (CSI Singapore) at the National University of Singapore (NUS), together with their collaborators from Chungbuk National University in Korea.

The team, led by Professor Yoshiaki Ito of CSI Singapore, showed that RUNX3 is a major component in a well-established tumor suppression mechanism involving p53, a tumor suppressor protein that regulates cell proliferation and prevent cancer. In addition, the research team also demonstrated that RUNX3 plays a pivotal role in preventing early tumor formation.

Although the current study focused on lung cancer, the results help to explain the development of other types of human cancers.

These novel findings were published in leading scientific journal Cancer Cell on 11 November 2013.

Implications on early cancer detection and prevention

Mutations of the p53 gene occur in 50 per cent of virtually all cancer types, indicating that p53 inactivation constitutes one of the main drivers of cancer development. p53 has since been shown to exert multiple effects -- be it enhancing DNA repair, arresting cell proliferation or promoting cell death -- to inhibit cancer growth.

"This study uncovered an important missing component of the tumor suppressor pathway that regulates p53 function. The identification of RUNX3 as the key component throws light on why RUNX3 gene inactivation, particularly via epigenetic mechanisms, is so prevalent in cancer," Prof Ito explained.

"From a clinical viewpoint, this finding has applications in early cancer detection and prevention. Cancer-specific epigenetic inactivation of RUNX3 is marked by DNA methylation, which can be readily screened for early detection or prognosis of a wide variety of cancer. It also suggests the exciting possibility that RUNX3 inactivation, if reversed by therapeutic means, may restrain cancer growth. It is therefore likely that many new approaches of cancer treatment or prevention will be generated," he added.

Role of RUNX3 in suppressing tumor formation

Lung cancer is the most frequently occurring cancer worldwide, and it is also the No. 1 killer cancer for both men and women in Asia-Pacific and Singapore. The prevalent type of lung cancer, called adenocarcinoma, is frequently observed to harbour genetic alterations in the oncogene K-Ras.

In normal cells, K-Ras is activated by a short-lived signal to promote cell growth and then rapidly deactivated. However, in lung cancer, a mutated version of K-Ras gene -- K-Ras^G12D -- is expressed. Instead of responding to the growth signal, the mutant K-Ras^G12D constantly stimulates cell growth, causing the uncontrolled and rapid growth of the cancer cells. Confronted with the activation of such oncogenes, normal cells would turn on tumor suppressor pathways to stop cell growth or kill abnormal cells.

Currently, the best studied tumor suppressor is p53, which is frequently inactivated in various human cancers. The activity of p53 is tightly regulated by a gene called MDM2 (which acts to limit the p53 growth-suppressive function) and its inhibitor p14^ARF. While scientists have found that p14^ARF inhibits MDM2 and thus promotes p53 activity, it is unclear how p14^ARF is induced.

To fill this gap in knowledge, the research team studied the molecular mechanism underlying K-Ras-induced lung adenocarcinoma. They found that p14^ARF is induced by the RUNX3 in collaboration with another protein called BRD2, in response to K-Ras^G12D. This suggests that RUNX3 is one of the major components of the well-established tumor suppressor pathway involving p53.

The discovery of the link between RUNX3 and BRD2 will also initiate new directions in studies on complex mechanisms that regulate gene expression, the impairment of which is often associated with cancer development.

RUNX3 as first line of defense against cancer formation

Another important breakthrough achieved by Prof Ito and the team was the discovery of different roles of RUNX3 and p53 in tumor suppression.

Since 2010, it has been known that while p53 can effectively destroy well-developed cancer cells, but it cannot respond to low levels of oncogenic stimuli and prevent the early stages of cancer development.

The research team showed that the sole inactivation of RUNX3 results in the rapid development of pre-cancerous adenoma (a benign tumor that may become malignant over time) in less than four months. On the other hand, inactivation of p53 alone has not been shown to induce adenoma. By showing that RUNX3 prevents adenoma formation, the team contributes to a better understanding of an important aspect of tumor suppression mechanism.

Commenting on the findings, Prof Ito said, "It is very likely that our body is protected by two lines of defense mechanisms against lung cancer development -- RUNX3 forms the first line which protects against adenoma formation while p53 forms the second line in protecting against adenocarcinoma formation. It will be important to examine whether other types of cancer also show two lines of defense mechanisms, since p53 is frequently inactivated in various cancer types."

The next step

p53 is often mutated and it is practically impossible to revert this mutation for the purpose of treatment. By contrast, RUNX3 is inactivated by DNA methylation (ie. epigenetic inactivation), a chemical event that silences the gene without altering its coding information.

"Clinical trials have shown that reactivation of epigenetically inactivated genes is feasible. Indeed, RUNX3 gene can be reactivated by chemicals in cancer cells in laboratory. Therefore, the team's immediate task is to find a way to effectively re-ignite RUNX3 gene activity. Since RUNX3 functions at early stages of cancer, reversal of RUNX3 inactivation could halt cancer development by timely prevention of permanent neoplastic changes," said Prof Ito.

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New insights into the ribosome; important implications for disease

Sep. 19, 2013 — In molecular biology, the ribosome represents the machinery necessary to assemble proteins, the building blocks of life. In this process, information encoded in the genome's DNA is first transcribed to messenger RNA in the nucleus, then transported to the ribosome where protein-assembly instructions are put in motion to translate the code into actual proteins.

But in recent years, it has been demonstrated that the ribosome is far more than just a processing unit; indeed, current research points to an important role for this complex structure in actively regulating biological processes.

Now, in a first-of-its-kind study that broadly examines the composition of the riboproteome, a scientific team led by investigators at Beth Israel Deaconess Medical Center (BIDMC) reveals previously unappreciated components of the ribosome, uncovering a large and dynamic structure that, among other things, can be altered in cancer. Published in today's on-line issue of the journal Cell Reports, the study additionally describes the development of an analytic platform that can be widely applied to numerous biological systems to highlight the functional roles of possible disease genes associated with the riboproteome.

"A primary goal of our lab is to gain a better understanding of translation and its impact on cancer," explains senior author Pier Paolo Pandolfi, MD, PhD, Scientific Director of the Cancer Center at BIDMC and George C. Reisman Professor of Medicine at Harvard Medical School (HMS). "So a key focus of our work has been the role of the ribosome. While the conventional wisdom has been that ribosome composition is absolutely fixed, we have recently pursued the hypothesis that it is, in fact, flexible and dynamic. Moreover, it has become apparent during the course of our investigations that the functional deregulation of the ribosome is implicated in disease initiation and progression, and could serve as a potential target for therapeutic intervention."

In this new study, a scientific team led by Pandolfi lab members John Clohessy, PhD, and Markus Reschke, PhD, examined the ribosome on a large scale to get a clearer picture of the relationship and interactions between ribosomes and the associated proteins required for efficient and correct translation of messenger RNA (mRNA).

"We wanted to find out what was happening in the ribosome on a global scale," explains Clohessy. "So we incorporated into our analysis those proteins that associate with either the ribosome itself or with the mRNA being translated, and which could represent important regulators of translation."

Until recently a lack of high-throughput ribosomal analysis has limited researchers' abilities to characterize the ribosomal proteome, which consists of the ribosome itself and a host of other proteins that interact with this cellular machine to regulate translation. In this new study, the scientists optimized and adapted a SILAC-based mass spectrometry approach to probe this complex microscopic structure through the analysis of far more data than was ever available before.

An acronym for stable isotopic labeling by amino acids in cell culture, SILAC utilizes non-radioactive isotope labeling to detect quantitative differences in protein abundance among cell samples. "A SILAC-based approach offers an elegant way of comparing two different cellular populations," explains Reschke. "The metabolic incorporation into the proteins of amino acids that have been differentially labeled with carbon and nitrogen isotopes results in a mass shift of the corresponding peptides, and it is this shift that can be detected by a mass spectrometer.

"The beauty of this system is that you're keeping the cells in their natural environment, you're simply substituting one amino acid with another," he adds. "This enables you to conduct a direct comparison without other chemical modifications."

Using a panel of cell lines, as well as genetic and pharmacological perturbations, the team embarked on their characterization of the prostate riboproteome. "Mass spectrometry enabled us to look at thousands of proteins at the same time," explains Clohessy. Through SILAC-based mass spec, the investigators were able to gain the first comprehensive overview of proteins within this space and examine how the riboproteome is altered in disease.

"We compared a number of different things," adds Clohessy. "We compared different cancer cell lines. We looked at changes to the riboproteome within a single specific cell line, both with and without use of a pharmacological inhibitor. And, we looked at a cell line in the context of genetic alteration. We essentially got a clear snapshot of which proteins were in this space at any particular time, and saw how they responded to cellular signals and stresses."

The authors then carried out a computational analysis on this extensive dataset to determine if there were specific protein networks or pathways that were highly represented in the data. Their survey revealed a number of exciting and surprising findings -- with important therapeutic implications.

"Among other things, our analysis uncovered a high incidence of genetic alterations to the riboproteome components in cancer, with a distinct bias towards genetic amplification, [whereby cells accumulate multiple copies of genes which can support cancer growth and survival]" explains Reschke, adding that it also revealed the presence of a significant population of RNA binding proteins (RBPs), important regulators of mRNA translation, some of which are also associated with disease.

"Changes in ribosome amounts have been shown to be associated with cancer for more than a century," notes Nahum Sonenberg, PhD, James McGill Professor of Biochemistry at McGill University. "However, the mechanism that links ribosomes and cancer is not well understood. This paper from the Pandolfi laboratory is a very important step towards the understanding of the full gamut of functions of the ribosome and its associated translation mRNA machinery in cancer."

This novel method additionally opens up a new direction in terms of how to study the ribosome in the context of both disease and therapy. "By now, we know a lot about the genes and pathways that converge on translation, and many of them are well-known oncogenes, including MYC and PI3K," explains Pandolfi. 'But the translational dimension we are now describing offers a new perspective on yet another layer of proteins that seem to be amplified in cancer. These findings provide us with a completely new set of proteins that may be used to better understand the process of translation in cancer, and help identify new targets for therapeutic intervention."

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