Signal strength

Associate Professor Brendan Jenkins is helping to uncover the cellular signals involved in stomach cancer, which may one day be used as biomarkers or as new targets for treatment.

Stomach cancer, after lung, breast and colorectal cancer, is the fourth most commonly diagnosed form of cancer worldwide and is one of the leading causes of cancer mortality, particularly in east Asia. Because there is no routine screen for stomach cancer, and the early symptoms are easily overlooked or mistaken for regular dyspepsia, it is often diagnosed late after it has set in and become aggressive.

Treatment often includes surgery, including a total or partial gastrectomy, including multiple cycles of chemotherapy. But now there is new hope for better treatments for stomach cancer and other diseases emerging from cutting-edge research conducted by Associate Professor Brendan Jenkins at the Monash Institute of Medical Research (MIMR) in Melbourne that might aid in the diagnosis, monitoring and possibly also targeted treatment of these diseases.

Jenkins has spent his research career trying to understand how some of the cellular signalling pathways that are activated by cytokines - proteins that modulate the immune system - contribute to human disease. Around seven years ago, Jenkins joined MIMR in Melbourne to continue this research. Here his focus is the interleukin 6 (IL-6) family of cytokines and how their deregulation plays a role in inflammatory and malignant diseases of the lung and stomach.

“We have known for well over a decade that uncontrolled signalling by members of the IL-6 family, particularly IL-6 and IL-11, plays a part in inflammatory diseases, such as Crohn’s disease and arthritis, and in many cancers including stomach, lung, pancreatic and colon,” said Jenkins. “Indeed, about 35% of all cancers have an obvious inflammatory component, but the mechanisms by which inflammation leads to cancer are poorly understood.”

His research aims to identify genes that could be used as biomarkers for the early detection or monitoring of such diseases and as novel targets for more personalised and thus effective treatment strategies.

Jenkins’ group at MIMR uses numerous molecular biological and genetic approaches in their research, together with translational studies using clinical samples. One of their main tools of trade, and the linchpin of their recent exciting findings, is a novel knock-in mouse model of gastric cancer. These mice have a defect engineered into their IL-6/IL-11 activation pathways that leads to gastric cancer.

For the translational research, Jenkins’ group is closely aligned with clinicians at the nearby Monash Medical Centre and with clinical researchers in Singapore and Japan who have a large collection of gastric cancer biopsies. “These clinical biopsies are a very important resource for validating findings from our mouse model, such as identifying a particular mechanism that could be promoting stomach or lung cancer. It allows us to immediately realise the significance of our findings for potential clinical translation, and push through on those results.”

The TLR2 story

One such set of very promising results was recently published in the prestigious journal Cancer Cell and will be the subject of Jenkins’ presentation at the Lorne Infection and Immunity meeting in February. They centre on a cytokine signalling pathway activated by IL-11 and involving other key cancer-related molecules, namely one of the toll-like cell-surface receptors, TLR2, and a downstream intracellular regulator of transcription called signal transducer activator of transcription-3, or STAT3.

Jenkins’ interest in TLRs was piqued several years ago when their critical role of driving inflammation in response to pathogenic insult was demonstrated. “Having worked on gastric cancer and cytokines for about 10 years, and knowing the very strong inflammatory component to these types of cancer, I started thinking: maybe cytokines such as the IL-11 cytokine that we work with could somehow be up-regulating the TLR system to the point that the whole inflammatory response goes into overdrive with tumour formation as the result.”

With Jenkins’ hypothesis driving the work, his team started with the basics. “We took tumours from our gastric cancer model mice and another gastric cancer model called ‘Gan’ from colleagues in Japan and, using real-time PCR, we looked at the gene expression levels of all the TLRs,” says Jenkins. “And there was this one TLR gene that was always up-regulated in the mouse tumours: TLR2.”

They then used a modified mouse model, in which the stomach tumour phenotype had been rescued by lowering the level of STAT3 activation, to show that mice with no tumour formation had normal levels of TLR2 gene expression.

The next step was based on two other premises: earlier research by the group showing that STAT3 is actually a potent pro-inflammatory and oncogenic factor in its own right, albeit by an unknown mechanism; and the realisation that one of the main downstream signalling molecules from the IL-11 cytokine is STAT3. The connection was getting stronger.

The group then demonstrated very clearly that STAT3 directly up-regulates the expression of TLR2, and that this IL-11-linked event occurs only in the stomach. This single finding is easy to say, but the path to proving that TLR2 is in fact a novel STAT3 target gene involved an awful lot of hard work and PhD student angst to achieve.

However, it was a key part of the puzzle, especially given that in stomach cancer, IL-11 is often up-regulated and STAT3 is over-activated in about 50% of cases, so the clinical link between IL-11 and STAT3 in gastric malignancies was already strong.

Getting definitive

It was time for the definitive mouse experiments, as Jenkins explains. “We crossed our model mice that develop the tumours - the ones with high TLR2 expression - with mice genetically engineered to have no TLR2 gene expression, and the progeny mice also showed no TLR2 expression.

Strikingly, the crossed mice also showed a 50% reduction in size of their tumours and whole stomach. The beauty of this result was that it mimicked the rescue experiment mice, which had reduced STAT3 activity. “We therefore had further proof that STAT3 up-regulates TLR2 at the gene expression level in the mice tumours, and that it is all driven by IL-11,” says Jenkins.

Interestingly, this set of experiments also showed that the TLR2 activation only promoted tumour cell growth and not inflammation, which was present in both the presence and absence of TLR2 expression. This was an unexpected finding of the study, especially given the strong inflammatory component of gastric cancer.

Finally, they used an antibody-mediated therapeutic approach, in which the mice with the stomach tumours were treated with a TLR2-blocking antibody for two to three months. The treated mice showed no further tumour growth compared to untreated controls, and this cemented the critical role of TLR2 in promoting the cancer’s progression.

It just remained for the team to validate the exciting mouse findings in a clinically based experimental system, and this is where the biopsy samples from their overseas colleagues came in. From the Singapore set of stomach cancer samples, around 40% had elevated levels of the TLR2 gene and, even more importantly, those patients with both high STAT3 activation and high TLR2 gene expression had a poor prognosis based on overall patient survival data (five-year rates) compared to patients with low TLR2 expression and low STAT3 activation.

“This was really nice clinical data demonstrating that our mouse disease model findings were also represented in the human condition.”

Gene signature

From here, Jenkins and his team would like to move their results into human xenograft models of gastric cancer using a swag of cell lines already in the lab as well as lines derived from patient cells. The aim now is to establish xenografts in mice from those cells and then start testing the efficacy of this blocking TLR2 antibody, which is made by a Dublin-based pharmaceutical company, Opsona Therapeutics, with whom the group has established close ties.

Indeed, Opsona has recently developed a humanised version of the antibody, which could form the basis of a potential as first- or second-line adjuvant to chemotherapy for patients with stomach cancer. Because this antibody is already entering Phase I/II trials for another indication, Jenkins is hopeful that the TLR2 blocking treatment could be trialled for gastric cancer within a few years.

“The other main part of our gastric cancer work is towards early detection and screening, and that is when you really start thinking about biomarkers. We are now in the process of trying to identify a TLR2-regulated ‘gene signature’ that could potentially translate into cancer biomarkers.

“The ideal candidates would be small secreted proteins that appear in the bloodstream in affected patients and therefore can be used in a simple blood test-based screening program.”

There is clearly a need to catch this disease in the early stages because most patients with stomach tumours do not know about it until the cancer is too well established for any treatment to be effective.

In this context, the TLR2 system identified by Jenkins and his team is very promising because it seems to be activated in such a large subset of gastric cancer patients. They also have a new NHMRC grant to follow up other promising STAT3-regulated immune system genes that might be driving gastric tumour formation in the same way as TLR2.

With emerging evidence that such genes may be playing an important role in other inflammation-type cancers such as colon and liver, the findings from Jenkins’ lab promise hope to the many people worldwide with these lethal cancers.

Associate Professor Brendan Jenkins’ lab is in the Centre for Innate Immunity and Infectious Diseases at MIMR. Following PhD studies at the Hanson Institute in Adelaide, Jenkins continued his research interest on the roles of cytokines in disease at the Fred Hutchinson Cancer Research Centre in Seattle, USA. In 1999 he returned to Australia to join the Ludwig Institute for Cancer Research before taking up his MIMR appointment in early 2006.

Image credit ©iStockphoto.com/Krzysztof Zmij