CT scan of the chest in a patient with left sided mesothelioma. (Source: wikicommons)

Mesothelioma is a rare form of cancer that most often affects the lining of the pleura (lungs) and peritoneum (abdominal cavity). In almost all cases, pleural and peritoneal mesotheliomas are caused by exposure to asbestos. This aggressive disease remains difficult to diagnose and treat. As a result, most patients are diagnosed in later stages, and the prognosis is unfavorable with average life expectancy of just under a year for most patients.

Research on Mesothelioma Biomarkers

Because early diagnosis is crucial for a more favorable prognosis and wider variety of treatment options, many current studies are focused on mesothelioma biomarkers, a molecule that allows scientists to detect the presence of chemicals associated with this type of cancer. The best available biomarker is a molecule called mesothelin, a glycoprotein that can be found in normal mesothelial cells.

In patients with mesothelioma, mesothelin levels are highly expressed, which may have a connection with tumor metastasis. Recent studies also suggest that testing a patient’s blood for levels of mesothelin can help diagnose mesothelioma at earlier stages, providing patients with better treatment options.

Find out about other work looking to improve mesothelioma diagnosis using Proteintech’s MTAP antibody (11475-1-AP)

Clinical Trials of Chimeric Antibody 

New clinical trials are currently assessing the effectiveness of a mouse-human chimeric monoclonal antibody called MORAb-009. Early research suggests that MORAb-009 inhibits the binding of mesothelin to CA-125, a cancer antigen shed from tumors. It also kills mesothelin-expressing cell lines through creating cellular cytotoxicity. This is promising news for mesothelioma patients because this antibody may inhibit tumor growth and metastasis.

Anthophyllite Asbestos: exposure to this substance is the most common cause of mesothelioma. (Source: wikicommons)

In a Phase I clinical study published in 2010, the antibody was intravenously administered to 24 patients. The goal of this study was to observe the interaction between MORAb-009 and mesothelin as a receptor for CA-125. The study results showed that MORAb-009 prevented the binding of mesothelin and CA-125 in pleural and peritoneal mesothelial cells.

Researchers concluded that this new antibody could be a viable treatment for mesothelioma patients by inhibiting tumor metastasis. However, more clinical trials are still needed to further explore the effect MORAb-009 has on the interaction between mesothelin and CA-125.

In the meantime, further tests on biomarkers are also warranted because of mesothelin’s poor sensitivity.

Guest Blogger Profile: Michelle Y. Llamas is a writer for the Mesothelioma Center. She is committed to generating mesothelioma awareness and providing information regarding breakthroughs in mesothelioma treatment.

Sources:

Hassan, R., Schweizer, C., Lu, K. F., Schuler, B., Remaley, A. T., Weil, S. C., & Pastan, I. (2010). Inhibition of mesothelin-CA-125 interaction in patients with mesothelioma by the anti-mesothelin monoclonal antibody MORAb-009: Implications for cancer therapy. Lung Cancer. 68(3), 455–459. doi: 10.1016/j.lungcan.2009.07.016. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2864325/?tool=pubmed

Hollovet, K., Reitsma, J. B., Creaney, J., Grigoriu, B. D., Robinson, B. W., Scherpereel, A., Cristaudo, A., …, & van Meerbeeck, J. P. (2011). Serum mesothelin for diagnosing malignant pleural mesothelioma: An individual patient data meta-analysis. Journal of Clinical Oncology. Retrieved from http://jco.ascopubs.org/content/early/2012/03/06/JCO.2011.39.6671.abstract

Pass, H. I., Vogelzang, N. J., & Carbone, M. (Eds.). (2005). Malignant mesothelioma: Advances in pathogenesis, diagnosis, and translational therapies. New York, NY: Springer.

U.S. National Institutes of Health. (2007). Understanding clinical trials. Retrieved from http://clinicaltrials.gov/ct2/info/understand#Q01

A mammographic image to detect breast cancer. Source: Wikimedia Commons via University of Copehnage University Post

On April 18, the results of a breast cancer study widely recognized as the largest study of its kind were published online in Nature.

In the past, breast cancer has largely been viewed as one single disease, with the addition of a few different classes in recent years. However, as well as identifying several  novel breast cancer driver genes, the Nature paper describes an entirely new way of classifying and categorizing the disease based on genetic profiling. Through the findings, the Cancer Research UK scientists who conducted the study now propose there are 10 distinct subtypes of breast cancer, each with their own unique set of genetic drivers.

One putative breast cancer gene identified by the Cancer Research study was the MTAP gene. Deletions in this gene are now considered to contribut to the development of breast cancer.

Read about work published with Proteintech’s anti-MTAP antibody

Up to 2,000 individual breast cancer samples had their entire DNA and RNA expression profiles analyzed for the study. A unique aspect of this analysis was that for each tumor sample examined, information on the clinical outcome of their treatment was available for the Cancer Research team to match up with the genetics they found. This led them to identifying individual subgroups with distinct clinical outcomes.

Potentially, the impact of this data could be great; it could be used for better treatment of patients using tailored individualized treatment plans. It could even mean doing away with more aggressive forms of therapies – such as radiotherapy – in the case of certain subgroups.

Learn more about Proteintech products relevant to this work

The team based at the Cambridge Cancer Research Institute originally set out to further understand the nature of breast cancer heterogeneity. Speaking on BBC Radio Four recently, Carlos Caldas Professor of Cancer Medicine at CRUK and lead author on the paper said of the group’s initial research aims: “We knew that breast cancer was heterogeneous but [had] no idea how heterogeneous it was.”

Caldas also explained the future implications of the research in terms of clinical practice:

 “The molecular anatomy of these subtypes is completely different in the biological sense, one day we will be able to tailor a treatment to each one that is a best fit for each of the ten subtypes.”

As to whether more subtypes of the disease remain to be discovered he used an analogy inspired by geography:

“I think we’ve identified the continents…in each continent there will be rivers and mountains and cities…there will be some finer granularity within [them]. We have really grasped…the 10 major subtypes of disease in the breast and that 10 is probably a very reasonable number.”

He said of other human cancers:

 “Whether there will be as many subtypes in other common human tumors remains to be seen…”

Catch the entire BBC Radio Four interview with Carlos Caldas which featured in its Monday 23 April  Material World program here.

Deletions in the MTAP gene identify it as novel breast cancer gene…

MTAP - 11475-1-AP – rabbit polyclonal – ELISA, WB, IHC

CCND1 and PPP2R1B were identified as two subtype specific drivers of breast cancer in the 11q13/14 cis-acting subgroup known as “IntClust 2″…

CCND1 – 60186-1-Ig – mouse monoclonal – ELISA, WB

PPP2R1B – 12621-1-AP – rabbit polyclonal – ELISA, WB

An ER-positive subgroup composed of 11q13/14 cis-acting luminal tumors showed several known and putative driver genes in this chromosomal region; those available in the Proteintech catalog…

CCND1 - as above

PAK1 – 51137-1-AP - rabbit polyclonal – ELISA, WB, IHC

Immunohistochemistry of paraffin-embedded stomach cancer using PAK1 antibody 51137-1-AP (under 40x lens)

Numerous signalling molecules, transcription factors and cell division genes were found associated with a subset of basal cancers harboring chromosome 5q deletions, including alterations in…

AURKB – 60098-1-Ig - mouse monoclonal – ELISA, WB

BCL2 – 12789-1-AP – rabbit polyclonal - ELISA, WB, IHC

BCL2 – 60178-1-Ig – mouse monoclonal – ELISA, WB

CDC20 – 10252-1-AP - rabbit polyclonal – ELISA, WB

CDC45 – 15678-1-AP - rabbit polyclonal -  ELISA, WB

CHEK1 – 65016-1-Ig - mouse monoclonal  - ELISA, WB, IHC

FOXM1 – 13147-1-AP - mouse monoclonal – ELISA, WB

HDAC2 – 12922-3-AP - rabbit polyclonal  - ELISA, WB, IHC, IF

IGF1R – 20254-1-AP - rabbit polyclonal - ELISA, WB, IP

KIFC1 - 20790-1-AP - rabbit polyclonal - ELISA, WB, IF

Immunofluorescence analysis of Hela cells using KIFC1 antibody 20790-1-AP at 1:25 dilution and Rhodamine-labeled goat anti-rabbit IgG (red). Blue pseudocolor = DAPI (fluorescent DNA dye).

MTHFD1L - 16113-1-AP - rabbit polyclonal - ELISA, WB, IHC

RAD51AP1 - 11255-1-AP - rabbit polyclonal – ELISA, WB

UBE2C - 12134-2-AP - rabbit polyclonal - ELISA, WB, IHC

“IntClust 5″ consist of the ERBB2-amplified cancers, composed of HER2-enriched (ER-negative) cases and luminal (ER-positive) cases…

ERBB2 (HER2) p185-specific – 18299-1-AP - ELISA, WB, IHC

ESR1 - 21244-1-AP - rabbit polyclonal – ELISA, WB

ESR2 - 60197-1-Ig - mouse monclonal – ELISA, WB

ESR2 - 14007-1-AP - rabbit polyclonal – ELISA, WB, IHC

rare but potentially significant events were found including amplifications in…

IGF1R – as above

KRAS - 12063-1-AP - rabbit polyclonal – ELISA, WB, IHC

EGFR - 18986-1-AP - rabbit polyclonal – ELISA, WB, IF

WB result using anti-EGFR (18986-1-AP) and loading different cell lysates.

…And homozygous deletions in…

BRCA2 - 19791-1-AP - rabbit polyclonal – ELISA, WB

RB1 - 10048-2-Ig - rabbit polyclonal – ELISA, WB, IHC

SMAD4 - 10231-1-AP - rabbit polyclonal – ELISA, WB

SMAD4 - 60182-1-Ig -mouse monoclonal – ELISA, WB

NCOR1 - 20018-1-AP - rabbit polyclonal – ELISA, WB, IHC

To view the entire Proteintech catalog visit www.ptglab.com

*Speech bubbles do not contain direct quotes from the referenced manuscript, they are brief summaries of several of its findings.

Malignant pleural mesothelioma (MPM) is an aggressive malignant growth of the mesothelium that lines the pleural cavity. It is often a diagnostic minefield for pathologists due to other thoracic malignancies, but even more so because of benign pleural diseases which can mimic MPM.

These benign mesothelial cell proliferations can imitate malignancy due to high cell density and atypical appearance, and are considered ‘reactive proliferations’ (RPs). Conversely, some MPMs can appear morphologically benign.

Researchers in the Department of Pathology at the University Hospital of Copenhagen, Denmark, are trying to identify novel protein markers for MPM to better aid pathologists in their identification of this often indistinct disease.

Previous independent research had shown that the MTAP gene was deleted in 12 of 13 cytological MPM specimens; in contrast, none of 19 benign cytological samples were found to have MTAP deleted.

Immunohistochemistry of paraffin-embedded colon cancer using Proteintech anti-MTAP antibody 11475-1-AP (viewed using 10x lens).

The Copenhagen-based researchers then built on the above study, using IHC to detect MTAP in 99 MPM samples with the Proteintech 11475-1-AP MTAP antibody;  they compared these with those of 39 benign RP cases using the median MTAP levels of the latter to set a threshold for healthy MTAP expression.

First author Zarah Glad Zimling and supervisors Anne Jørgensen and Eric Santoni-Rugiu found that 65% of the MPM samples investigated showed decreased MTAP expression, whereas only 23% of control RP sample showed any sort of reduction in this protein.

The researchers further evaluated MTAP expression in a cohort of pleural effusion samples from 14 MPM patients and 20 patients with RP. For these experiments, the team utilized a double-staining technique with Wilms tumour 1 (WT1) as positive marker for mesothelioma. In these samples, decreased MTAP expression diagnosed MPM with a sensitivity of 71% and a specificity of 90%.

In conclusion the researchers proffer that assessing the presence of MTAP – strictly in combination with several other potential markers - in suspected cases of MPM might be useful for diagnosing malignancy.

Related antibodies

MTAP rabbit polyclonal antibody 11475-1-AP

WT1 rabbit polyclonal antibody 12609-1-AP

Related publications

Zimling ZG, Jørgensen A and Santoni-Rugiu E, The diagnostic value of immunohistochemically detected methylthioadenosine phosphorylase deficiency in malignant pleural mesotheliomas, Histopathology (May 2012);60(6B):E96-E105.

Kim J, Kim MA, Min SY et al., Downregulation of methylthioadenosin phosphorylase by homozygous deletion in gastric carcinoma, Genes Chromosomes & Cancer (June 2011) ;50(6):421-33.

Sommer J, Itani DM, Homlar KC et al., Methylthioadenosine phosphorylase and activated insulin-like growth factor-1 receptor/insulin receptor: potential therapeutic targets in chordoma, J Pathol. (Apr 2010);220(5)608-17.

Huang HY, Li SH, Yu SC, et al., Homozygous deletion of MTAP gene as a poor prognosticator in gastrointestinal stromal tumors, Clin Cancer Res (November 2009);1515(22)6963-72.

Professor Richard Marais (Source: paterson.man.ac.uk)

This month our resident blogger interviewed Professor Richard Marais, skin cancer expert and recently appointed Director of The Paterson Institute for Cancer Research in Manchester. Preceding this, Marais was Professor of Oncology at The Institute of Cancer Research, where his work with the B-RAF protein uncovered a novel signaling paradigm in cancer… 

Professor Richard Marais’ lab is well-known for its work concerning cell communication in melanoma – the most serious form of skin cancer. A major focus of its research is the B-RAF protein, which is a component of the MAPK/ERK signaling cascade controlling cell growth, proliferation and differentiation.

B-RAF is mutated in more than half of melanoma cases and the lab’s work on this protein has paved the way for new therapeutic drugs targeting the disease; namely the B-RAF inhibitor vemurafenib. Since the availability of such drugs, the Marais lab has helped uncover a very interesting aspect of B-RAF’s signaling capabilities – a prime example of how one drug does not fit all…

Earlier work with B-RAF

In 2001 Marais received a phone call from Michael Stratton, joint-head of the Cancer Genome Project at the Wellcome Trust Sanger Institute: “Mike mentioned the Sanger team had found mutations in B-RAF but not in its isoform C-RAF.  This immediately made a lot of sense; we knew BRAF was primed for activation and we also knew that C-RAF wasn’t.”

The Marais lab then turned its focus to B-RAF.  “We’d already been working on B-RAF a little and we had begun to understand the difference between B-RAF and C-RAF signaling in cancer – it was very easy for us to get involved.”

The lab’s initial involvement was to conduct functional studies of the mutations identified by the Sanger Institute team. Initial work was published in Nature along with the original  findings from Sanger.

“The Stratton data told us B-RAF was mutated in cancer; it was a heavily smoking gun – but it did not tell us what B-RAF was really doing.”

The lab then characterized the functions of 22 of over 30 mutations from the original Nature paper, including the most common: V600E – accounting for about 80% of B-RAF mutations.

“We found the mutations broke down into three different classes. There are those that behave like the V600E mutation, which is an activating mutation causing increased kinase activity; there are those causing intermediate activation of the protein (yet still above the levels of wild-type activation) and, most intriguingly, those that are kinase-dead with no activity.”

“The discovery of those rare, loss-of-function mutations begged the question: ‘How do they work?’”

Another interesting, collective feature of these mutations was their location: “When we looked at the position of each mutation we saw two hotspots: one in the glycine-rich loop and the other in the activation segment. All three types of mutation could be found in these two regions – so we realized the best way to work out what they were doing was to solve the crystal structure.”

Locked into action: How B-RAF causes cancer

Working with David Barford at The Institute of Cancer Research, the lab solved the structures of both wild-type B-RAF and oncogenic V600E B-RAF and (along with the functional data above) published them in Cell:

“The structural data showed us that normally the glycine-rich loop and activation segment interact in such a way that they trap B-RAF in an inactivated state. Phosphorylation of the protein disrupts that interaction and allows it to refold into its active conformation.

“The structure of the V600E mutant told us that, in cancer, the activating mutations do more or less the same thing as phosphorylation – they mimic it and so disrupt the interaction interface, allowing the active conformation to prevail.”

So the mutations don’t really activate the protein, they destabilize its inactive conformation, locking it into an activated form meaning it: “can’t be dephosphorylated and turned off.”

This data has led to the development of several drugs targeting the inhibition of B-RAF.  Plexxikon for example used the structural data to develop the first of these: vemurafenib. Vemurafenib (now marketed as Zelboraf®) has recently been approved for use in Europe and targets V600E, V600K and a handful of other V600 mutations.

The Marais lab has generated structural data that has led to the development of several drugs targeting the inhibition of B-RAF. (Source: http://info.cancerresearchuk.org)

The use of B-RAF inhibitors

The availability of drugs like vemurafenib not only offers extended survival to melanoma patients with metastatic disease – it has revealed something rather interesting about the use of  inhibitor drugs in cancer treatment.

Of the patients who receive vemurafenib, around a third develop non-melanoma skin lesions in the form of either cutaneous squamous-cell carcinomas (cSCCs) or keratoacanthomas (KAs). The incidence of these lesions is not just random bad luck, neither does it appear that the drug itself is carcinogenic – rather, these lesions appear to be driven by a distinct mechanism in predisposed cells harboring certain pre-existing abnormalities.

Part of the Marais lab current work is to further characterize abnormalities predisposing to these secondary lesions, to improve the use of vemurafenib and inform developers of new B-RAF targeting drugs*. However, the lab already has a pretty firm grasp on why these lesions appear  – knowledge gained from studying those curious mutations resulting in kinase-dead B-RAF…

“The discovery of those rare, loss-of-function mutations begged the question: ‘How do they work?’ After all, the genetics suggested they would be gain-of-function, yet the biology told us they were the opposite. We really wanted to understand that.”

“We’d noticed a small proportion of these kinase-inactive mutants were coincident with RAS mutations in human cancer. Carla Milagre, a PhD student in my lab, recreated these conditions in inducible-expression mice. In this model, we saw a massive increase in the oncogenic activity of RAS in the presence of the kinase-dead B-RAF mutant. We concluded that kinase‑dead B-RAF is not an oncogene by itself, but what it does is it massively potentiates the activity of oncogenic RAS.”

Biochemical studies carried out in parallel by Sonja Heidorn, another PhD student in Marais’ lab, had elucidated a putative mechanism for this. This data was published in Cell.

“Sonja discovered that, paradoxically, B-RAF inhibitors drive activation of the MAPK/ERK pathway in RAS mutant cells, whereas they inhibit the pathway in B-RAF mutant cells.”

The B-RAF paradox

“It seems that B-RAF inhibitors drive the formation of dimer complexes between B-RAF and C-RAF – some of them homodimers, some of them heterodimers – and these drug-bound dimers cause hyperactivation of the pathway. The drug-bound partner constitutively associates itself and its drug-free counterpart with RAS at the cell membrane, leading to uncontrolled propagation of pathway signal.”

What may be happening in the case of patients who receive vemurafenib and go on to develop cSCCs or KAs is that they have latent abnormalities which are activated by the presence of the drug; as Professor Marais explained:

“The patients have these pre-existing indolent lesions – they’ve got RAS-mutant keratinocytes in their skin. These lesions don’t do anything until you administer the drug, but in doing so you activate the drug paradox and drive their development, accelerating their growth.”

A recent paper to come from Marais’ lab proposes the use of a new class of inhibitor drug targeting MEK in tandem with vemurafenib as a possible way to circumvent these lesions. (MEK is a signaling protein downstream of RAS and BRAF, but upstream of their MAPK target).

The use of MEK inhibitors to negate the negative aspects of B-RAF inhibition has already been tested for proof-of-principle, but in a different type of cancer than melanoma.  As a new paradigm in cell signaling, the B-RAF paradox could be relevant to many diseases treated with inhibitor drugs…

“I think the paradox will probably end up having quite a lot of biological and translational importance. The original research papers have been cited nearly 200 times in 2 years; I think that is really telling – people are really interested in this.”

“Are there other paradoxes to be discovered?”

Recently, this translational importance has become apparent in the case of chronic myeloid leukemia (CML), where awareness of the paradox and the use of MEK inhibitors have proven effective:

Proof of principle

“In CML you can have a situation where patients respond to a drug – typically imatinib (Gleevec) – but then they stop responding: it’s not just that the patients become resistant to the drug, it’s actually the case that the drug gives tumor cells a survival advantage.

“Recently, we’ve shown that it is activation of the paradox in drug resistant CML patients that drives survival of the cancer.” Marais’ team was also able to demonstrate that nilotinib – another drug used to treat CML – in combination with MEK inhibitors was able to overcome this resistance and kill CML cells that were no longer responding to a single treatment.

So have the lab found any other drugs in mainstream use that also paradoxically activate B-RAF and cancer survival?

“We’ve looked at a bunch of other drugs, most of them don’t do anything - but you do wonder what else they might be doing…

“Are there other paradoxes to be discovered?”

By @DebGrainger

*Independent research is also being carried out by Levi Garraway’s lab at Harvard Medical School.

Stroma, connective and supportive frameworks of cells that underly the tissues and organs of the body, can have a protective function against cancer – delaying or preventing tumor formation when physiologically normal. However, it is becoming increasingly recognized that in cases of invasive carcinoma stromal changes can create a permissive and supportive environment for tumor growth. In more advanced stages of cancer stroma can even stimulate invasion and metastases, which result inevitably in poor prognosis. Therefore, it is becoming increasingly important for oncology researchers to consider the role of tumor-associated stroma in tumor growth, progression and migration.

Recently, upregulation of the wild-type PALLD gene in stromal cancer-associated fibroblasts (CAFs) has been shown to promote the invasiveness of adjacent tumor cells [1]. The work was carried out by a group of researchers at the University of Washington in Seattle and first-authored by recent interviewee Professor Teresa Brentnall from the Division of Gastroenterology.  Whilst this work was carried out in a pancreatic cancer model, the question of whether PALLD plays a similar role, facilitating pathological interactions between breast cancer cells and their associated fibroblasts, is intriguing.

Immunohistochemistry of a paraffin-embedded ovarian tumor using PALLD palladin antibody 10853-1-AP (viewed under 10x lens.)

Previously, PALLD – encoding the cytoskeletal protein palladin – has been shown to contribute to the invasive motility of human breast cancer cells [2]. A similar study also found that breast cancer cell migration can be regulated by palladin directly [3]. Furthermore, this work reported that the aberrant behavior of wild-type palladin in breast cancer cell migration is linked to the deregulation of the PI3-kinase (PI3K) pathway; this is specifically through enhanced phosphorylation of the palladin protein by Akt2 (PKBβ) [3]. This interplay between faulty PI3K signaling and wild-type palladin emphasizes the idea that PALLD does not have to fall foul of mutations itself to play a role in malignancy – it can be seemingly normal. Moreover, as the Seattle group’s PLoS One paper describes, the function of wild-type palladin can be affected, not only in tumor cells, but in the surrounding stroma – and by another source entirely…

In the pancreatic cancer-model examined by the Brentnall and colleagues, increased, pathological expression of palladin in the stromal CAFs is remotely triggered in a paracrine manner by adjacent ras-mutated epithelial cells. Whilst the paracrine environment of breast tissue is distinctly different from that of the pancreatic environment, the latter presents an interesting scenario – where the function of palladin in the surrounding stroma is modulated by pathological events elsewhere. PALLD studies in breast cancer cells have, so far, focused almost exclusively on the role of the PALLD gene and its protein in the tumor cells, without considering the impact of stromal PALLD; in light of the the findings reported by Brentnall and her fellow researchers, perhaps it is time that changed?

Footnotes

Are you looking into something similar? Well we’d like to hear about it! Drop an email to Europe@ptglab.com and tell us more.

Looking at palladin in your research? All three papers cited above have utilized Proteintech’s anti-palladin antibody – Rabbit polyclonal PALLD, catalog number: 10853-1-AP

Other palladin-related products: Rabbit polyclonal PALLD, 16179-1-AP, Mouse monoclonal PALLD 60167-1-Ig.

You can find out more about these antibodies, as well as thousands of others in our catalog, by visiting www.ptglab.com.

References

[1] Brentnall et al. (2012) Arousal of Cancer Associated Stroma: Overexpression of Palladin Activates Fibroblasts to Promote Tumor Invasion, PLoS One, 7:e30219

[2] Giocoechea et al. (2009) Palladin contributers to invasive motility in human breast cancer cells, Oncogene, 28: 587-598

[3] Chin and Toker (2010) The Actin-Bundling Protein Palladin Is an AKT1-Specific Substrate that Regulates Breast Cancer Cell Migration, Molecular Cell, 38: 333-344

Professor Hans Clevers (source: www.hubrecht.eu/research/clevers)

During the 2012 AACR Annual Meeting at the beginning of the month, I had the opportunity to meet with AACR speaker Professor Hans Clevers, Director of the Hubrecht Institute for Developmental Biology and Stem Cell Research, Utrecht, Netherlands. Catching Clevers’ talk at the meeting, I was keen to ask him about every aspect of his lab’s work up to that point covering the journey from T cells to growing “organoids” in culture. This article covers the first half of our discussion, during which I found myself even questioning the necessity of hypotheses in research…

Professor Hans Clevers is well known for his lab’s work encompassing Lgr5+ stem cells in the gut; work that has now culminated in a technique to grow “organoids” – structures that resemble organs - in culture, independently of any host organism.

The implications of this body of research are potentially numerous and diverse, and the lab’s journey to this present state of scientific capability has been just as varied. It began with Clevers’ origins in immunology and traversed the study of model organisms for a while, before essentially starting again from scratch with the intestine. Since recent times, several more organ tissues have been under the scrutiny of the Clevers lab: pancreas, liver, lung, kidney, the list goes on. Unsurprisingly, I faced a genuine problem upon beginning my discussion with Hans – knowing where to start.

Coincidentally, the subject of deciding where to begin became thematic with my opening question (a rather generic: “What was your experience of the AACR Annual Meeting this year?”) I found my starting point in Professor Clevers’ description of his “Meet the Research Pioneer” session – a forum  organized by the AACR committee to facilitate career discussions between prominent researchers and early-career scientists.

Amongst answering questions in the session such as “How do you make career decisions?” and “Do you fight journal editors over rejections?” Clevers had described to those present his general approach to scientific practice and how he locates starting points in research; I found this fascinating and totally opposed to how I was taught to go about science during my graduate studies….

“I try to discourage people from formulating hypotheses: it is not how science should be learned. This idea that you should first formulate a question – your hypothesis – then test it, often goes wrong I find.” He explained further: “Once you formulate your hypothesis, it’s very natural to then prove that you are right rather than to do the opposite; hypotheses are often formulated in such a way that it takes longer to find out if you are wrong. Another negative aspect of this approach is that you rarely learn much more than one answer to one question – typically, you do not look at other possibilities.”

 “I’m strongly convinced this is how it should work when you really want to discover novel things – you cannot hypothesize how something works.”

So perhaps not having a solid starting question was not such a bad thing for this interview? After all, it is a tactic that seems to have worked for Clevers: “The hypothesis-free approach has worked very well for me, and for others, but…” He admits, “There is a significant hurdle to it: It is very difficult to write grants when you have no hypothesis. We now rely on previous studies to generate grants for new ones.” And there was indeed a time when the Clevers lab found it difficult to secure funding to approach science in this way…

After finishing postdoctoral work at the Dana-Farber institute at Harvard – where he cloned the T cell gene CD3 epsilon – Clevers set up his lab in Utrecht, the Netherlands, primed to chase down a discovery made during his postdoctoral studies. He had found a T cell-specific enhancer back in Boston and, shortly after being established, his lab managed to clone its gene: T cell factor 1 (TCF1). But then a challenge presented itself:

“We cloned TCF1 right away, but then it took 6-7 years to figure out what it did. We found there were three more TCFs in that time too: LEF, TCF3 and TCF4. They all bind DNA beautifully but we were without explanation to their exact function as they couldn’t regulate transcription – so it was not easy to get grants back then.”

The advent of two-hybrid screening technology ushered in an experiment in the lab that was to identify the already famous β-catenin as one of several cofactors binding the Tcf protein: “The moment we pulled out β-catenin from the two-hybrid screen we realized Tcf alone only binds DNA – it requires β-catenin to activate transcription.” But completing the picture of TCF function was not the only conclusion of this work – β-catenin was already famous for its role in the Wnt signaling cascade:

“We essentially found a missing link in the Wnt pathway.”

“Before the two-hybrid screen, nobody knew that β-catenin could control gene expression – we essentially found a missing link in the Wnt pathway.” Something they had not set out to do initially – the hypothesis-free approach was working well.

“With the results in front of us we realized that T cells were not the best model system for furthering our understanding of TCF in the context of Wnt signaling. As such, we decided to work in animal models, and we quite rapidly went on to show that TCF is ubiquitous across the species: frogs, flies and the worm C.elegans.”

So how did the Clevers lab make the transformation from what was – circa 1996 – essentially a developmental biology lab to an organoid growing one, becoming experts in intestinal epithelium renewal and stem cells in between? Why is it that Clevers now frequently appears on the bill of cancer focused forums and conferences when at one time it would make more sense to find him on the speaker list of those dealing with Xenopus development?

Well, the Clevers lab’s involvement in cancer research began with several collaborations with major cancer authority Bert Vogelstein’s lab; inspired by the Vogelstein lab’s work on APC – an upstream regulator of the Wnt pathway – in colon cancer. Together the labs found that colon cancer cells lacking APC have high levels of β-catenin/TCF signaling. Conversely, Clevers then wanted to see what happened if TCF was removed from the equation: “We knocked out TCF4 in mice and we found a phenotype that was essentially the opposite of cancer: we lost the intestinal structures vital to the renewal of gut. This told us that there were two sides to this; one: if you over-activate β-catenin/TCF signaling you get colon cancer; two: this signaling cascade is the normal driver of turnover in the gut.”

“The architecture of the gut…told me this was a great place to begin to locate and study stem cells.”

The intestine then became an intense focus for Clevers’ lab: “So we realized that the gut is incredibly interesting because of this turnover – its epithelium replenishes within 4-5 days. We knew that the signaling program activated by TCF in this tissue caused cancer, but was also responsible for normal cell renewal – we then began to consider that finding whatever genes might be activated by TCF as the secret to finding adult stem cells. Plus, the architecture of the gut – the villi and their crypts – told me this was a great place to begin to locate and study stem cells.”

However, this change in direction was not such a smooth transition for the lab: “It was tough. You cannot just start something new like that – there are no funds available. So it was a slow progression, it took a few years overlapping with the model organism and T cell work. “There was no technology available at the time (around 1998-99) to support a hypothesis-free approach to finding TCF’s target genes; there was no such thing as a microarray just yet. There were all sorts of complicated cloning strategies to begin to ask what genes were controlled by TCF – but we had to take them one at a time, individually.”  Technology however, was catching up…

A GFP knock-in into the Lgr5 locus visualizes the stem cells of the small intestine of mice at the base of crypts, the image features in Barker et al. Nature 2007. (Source: www.hubrecht.eu/research/clevers). 

“We were probably among the first labs to collaborate with Pat Brown – who had just invented DNA microarraying at Stanford. So in 2000 we constructed colon cancer cell lines (where the Wnt pathway is very active) in such a way where we could block their signaling and then do microarrays to assess many genes at a time and see which ones were ‘switched off’. We did an experiment with four microarray chips and found maybe 60-70 genes that were active in colon cancer that were activated by β-catenin/TCF signaling.” And Lgr5, that would become so significant not long after, was one of them.

The next step for the lab was to conduct expression studies on each of these sixty-plus genes – every member of the lab set about working on the list. Nick Barker, a postdoctoral researcher in Clevers lab at the time – who had been a major player in establishing a link between TCF signaling and cancer through the Vogelstein lab collaboration – noticed that one gene in particular had unique expression pattern: “Nick looked at a few genes – expressing them with GFP tags in mice – and realized Lgr5 had this very unusual expression pattern. It basically marked these very small cells in the crypts of the gut that we had never seen before. Immediately upon seeing these cells I knew we had found something. Now when I look at an intestinal crypt I see them immediately – in every crypt you see this cell.”

That cell type came to be known as the Lgr5+ cell, and later studies began to show their proliferative capabilities as well as their other rather unique and interesting traits. It was the identification of these cells that formed foundations for the work that now enables Clevers and his collaborators to grow organ-like structures in a culture dish – completely independently of any complex system or organism. How that leap was made however remains for part two of the interview – which you can catch in an upcoming blog post. A conclusion for the moment though: it seems, despite the difficulties associated with it, there is certainly something to be said for a hypothesis-free approach to conducting science, and Professor Clevers would support that notion: “I’m strongly convinced this is how it should work when you really want to discover novel things – you cannot hypothesize how something works.”

By @DebGrainger.

All evening, the atmosphere in South Branch was abuzz with lively conversation and laughter, and it provided the perfect opportunity for this resident blogger to get snap happy with the camera…

]]> http://proteintechblog.com/2012/04/11/ten-year-anniversary-celebrations-cocktails-in-chicago/feed/ 0 proteintech IMG_3525 IMG_3518 IMG_3519 IMG_3559 IMG_3546 IMG_3590_2 IMG_3523 IMG_3537 IMG_3517 IMG_3545 IMG_3554 IMG_3541 IMG_3535 IMG_3532 IMG_3521 IMG_3585 IMG_3638 IMG_3591 http://proteintechblog.com/2012/04/10/interview-malignant-hyperthermia-research/ http://proteintechblog.com/2012/04/10/interview-malignant-hyperthermia-research/#comments Tue, 10 Apr 2012 15:49:14 +0000 Proteintech Group http://proteintechblog.com/?p=3726 ]]> Last month our resident blogger Deb Grainger paid a visit to the Leeds Institute of Molecular Medicine (LIMM) Postgraduate Symposium, where she met up with malignant hyperthermia researchers Dr. Patrick Booms and PhD student Alan Merritt. During the SciLinks networking session, Deb talked to both Patrick and Alan about their research and learned how they are using molecular biology to help improve knowledge of this rare, genetically inherited condition.

Malignant hyperthermia (or simply MH) describes a rare but serious hyper-metabolic reaction of predisposed individuals to certain general anesthetics, which can result in death if not recognized and treated quickly. During an MH episode the anesthetics trigger a massive release of calcium within the patient’s skeletal muscle cells, leading to an increase in muscle metabolism and contraction. As well as causing a rapid rise in body temperature (hence the hyperthermia nomenclature), this reaction eventually overwhelms the body’s capacity to supply oxygen and damages and breaks down muscle tissue (a process called rhabdomyolysis) – and I’ve already alluded to what could happen if this condition is left untreated.

I was unaware of MH until I came across the work of research fellow Dr. Patrick Booms and graduate student Alan Merritt, who have around a decade or so of experience in MH research between them at Leeds University, UK. I met up with Patrick and Alan at the LIMM Postgraduate Symposium last month, hoping they could educate me a little on this potentially life-threatening drug reaction.

MH is what is classed as a pharmacogenetic disorder; meaning whatever genetics predispose for MH only come into play upon administration of a drug – which in this case is the use of certain anesthetics (the volatile kind). In all other respects the ‘patient’ is often normal and leads a perfectly healthy life; technically, if they never needed an operation they would never discover their susceptibility or encounter an MH episode…

Patrick told me it is actually a little more complicated than the above; an individual doesn’t have to avoid surgery entirely to miss an MH-positive diagnosis: “The problem with MH is you only find out when you are challenged with anesthetics and you can go one, two or even several procedures before you have an MH reaction. You can be challenged once; twice – then the third time it hits.” A few disclaimers were then issued before we continued…

There isn’t much cause for everyone to worry about being MH-susceptible as it is a rare trait – only around 1 in 10,000 of the world’s population is thought to have it. In reality, due to uneventful anesthetic challenges, its occurrence is: “Somewhere between 1 in every 50,000 to 100,000 anesthetizations.” Furthermore, if it turns out you are in fact MH-positive there are safe alternatives to the more commonly used MH-triggering anesthetics; although, these may be more expensive and have an increased number of (but much less serious) side-effects.  Finally, in the event the worst does happen, an MH episode can be treated with quick thinking and the drug Dantrolane (I’m also told that putting the individual on ice helps greatly) – essentially deaths from MH have been significantly reduced since the condition was first identified in the 1960s.

Muscle contraction is triggered via calcium release (as well as being contolled by several other chemicals). In MH-susceptible patients undergoing a reaction to anesthetic, calcium release become deregulated in such a way that large volumes of calcium are released into the muscle cell. This causes rapid and increased muscle contraction and the serious complications that result from this.

Despite the reassurance above, it is vitally important for patients who have a suspected vulnerability to MH – and their blood relatives – to get tested. It is precisely the unpredictability of MH that makes this essential. Family members of individuals with an MH-positive diagnosis need to go for testing as MH susceptibility is, after all, a dominantly inherited trait (and affects both sexes). Like the MH reaction, diagnosing MH is not so straightforward either…

The most definitive way of testing for MH at present – and for the foreseeable future – is via a standard protocol devised by the European MH Group (in North America there is a slightly different protocol). This clinical MH test – only available at Leeds St. James’ Hospital in the UK – involves taking a biopsy of skeletal muscle from an individual and exposing strips of it to certain drugs before measuring for a particular response. In Europe it’s known as an in vitro contracture test (IVCT) – the North American version is a caffeine halothane contracture test (CHCT); Patrick explained a little more about the IVCT:

“It’s quite an invasive test – the biopsy is taken under local anesthetic and, via open surgery, about 2 grams of muscle is removed. The test itself has not really changed since its initial development and involves stimulating the muscle with an electric current (to see if it is still alive) before perfusing it with halothane or caffeine.” I then learned that normal muscle does not contract in the presence of halothane whereas MH muscle will contract; MH-positive muscle is also more sensitive to caffeine and will contract at much lower concentrations of the drug.

The clinical test for MH-susceptibility is the current gold standard and it’s extremely reliable, but it has its downsides – its invasiveness being an obvious one. Currently Patrick and Alan are part of a team working towards a more comprehensive genetic test as an alternative option to the clinical one. Both researchers explained why this is a very good move…

As Alan began: “MH is more frequently detected in early life as children are more likely to be ‘put under’ with anesthetics that trigger MH; for example, the volatile gas inhalation anesthetics.” Patrick then adds, “This is one of the reasons we do our research; we rarely subject children to the invasive testing and ask their parents to be assessed clinically instead. We offer a current genetic test for about thirty known MH-causing mutations to children, it only requires a blood or saliva sample and is a lot less troublesome for them.”

Other benefits of a less invasive test, requiring easily stored and transported samples, are convenience and improved centralization. As Alan pointed out: “At the moment, people come from all over the UK to Leeds to get tested; with a genetic test they could donate samples for us at their local Doctor’s office.”  If it were a matter of testing someone using a saliva swab or a blood sample they could give a sample at their local physicians, removing the need for travel and perhaps increasing the sample size within the UK population and expanding it to other parts of the world.

However, fully fledged genetic testing for MH-susceptibility is still in its very early stages: “At the moment…” Said Patrick, “There are 180 mutations identified in a protein called the ryanodine receptor (RYR1) causing MH susceptibility. Thirty of those fulfill certain criteria as diagnostic mutations and we screen for those thirty in the lab when we test new samples.”

“The main aim of our research is to increase the amount of diagnostic mutations of RYR1 from 30 to 180 to include the whole array of known mutations.” But there’s another hurdle with the development of a fully comprehensive genetic test: “Another problem is that RYR1 mutations are likely to account for only around 50% of MH-causing mutations. For example, our group has identified one of two mutations in a gene encoding the dihydropyridine receptor (DHPR) and these have yet to be functionally characterized… Also there are at least ten other candidate genes encoding protein components of the calcium release unit thought to carry mutations potentially resulting in MH-susceptibility.”

So how are Patrick and Alan helping to developing genetic testing for MH-susceptibility? Well it largely lies in the functional testing of the mutations already identified. As Patrick explains, turning an identified mutation into one of diagnostic value involves a lot of intensive work…

“We’ve selected 10 of the most common mutations [yet to be functionally tested] and are trying to construct DNA vectors based on these that will express their resulting mutated proteins. This expression is done in a cell model – currently we use HEK293 cells for their ease of transfection, but eventually we hope to express in a skeletal muscle-like cell line lacking functional expression of endogenous, wild-type RYR1.”

“We test for the MH-functionality of a given mutant-construct we can successfully express in the HEK cells with calcium (Ca²⁺) live-cell imaging.  Living cells are filmed under a confocal microscope and we use fluorescence dyes to observe the calcium they release in the presence of one of the clinical drugs; halothane (the drug used in the clinic) and caffeine for example, plus 4-CmC and KCl compounds used in the laboratory setting.” The basic biology behind this testing, Patrick explained, is that Ca²⁺is essential for muscle contraction: “It is released in huge amounts from a cell storage compartment called the sarcoplasmic reticulum upon a muscle cell receiving signals to contract. Any MH-causing mutations will elicit this effect (minus the contraction) in the HEK cells upon addition of the relevant drug. This can be captured in real time using the calcium imaging technology.”

Time sequence of caffeine-induced calcium release from the sarcoplasmic reticulum in transduced transgenic differentiated “dyspedic” myotubes carrying a mutation in RYR1. You can see the increase in fluorescence corresponding to increased Ca²⁺ release. Imaging was carried out using a confocal fluorescence microscope.

Alan confirms he has already been successful at expressing three of the 10 commonly mutated versions of the RYR1 protein selected for the study in HEK cells.  One of these is the p.Gly3990Val mutation (they’re still generating more data to submit the other two mutations for publication at present) – and there are more on the way. When you take into consideration the molecular biology involved, having three successfully expressing mutant DNA constructs is definitely good going. As the experts explained:

“The molecular cloning [to make the constructs] is very challenging, because of the size of the RYR1 cDNA (around 15Kb!) everything you do with it is inefficient: gel extraction, sub-cloning, mutagenesis, etc. – they are all inefficient.” As researchers who have experience in such molecular techniques will know: working with a 15,000 base insert and a vector (around 5,000 bases) is tricky business. Alan emphasized this with, “That’s why in 10 years only 30 mutations have been successfully characterized out of 100 plus.”

So apart from the functionality testing, what else is the Leeds MH research group concentrating on? “Our group’s fairly small but we work on [in addition to] expression analysis, microarrays and cell culture models. We also have members of our lab who are dedicated to screening for new mutations. For that project, we also collaborate with Graham Taylor here at Leeds where we have an in-house genomics (next generation sequencing) facility, plus we work with a group in Seattle, WA, on an exome sequencing project. These sequencing projects are searching for other MH-causing mutations in other genes. There is a significant portion of MH cases out there that must be caused by mutations other than those in RYR1.”

The reason MH researchers like Patrick and Alan need to be absolutely sure a given mutation causes MH-susceptibility, and why they are certain there’s much more to the genetics than meets the eye, lies in clues found in certain MH cases. “There must be a modifier affect,” Patrick offered as one example (and one that demonstrates the complexity of MH responders’ genetics extremely well), “Sometimes an individual can have one of the thirty mutations show up in our genetic test, but they don’t have a phenotype.” Alan clarified: “For example, some individuals test positive for a mutation but don’t (IVCT) test positive for MH.” They then tell me the incidence of these cases is quite significant – around 10% of those screened are what Patrick called “discordant” i.e. they really do not fit a ‘single mutation equals MH-positive’ model. “There must be something else out there.” He reiterated, “There must be a modifier.”

So given all this intensive work, when will it be possible to get tested for MH without the need for the current clinical test? Alan fields this one first: “As far as a genetic pre-screening test for MH for the entire population I’d say its reality is distant to perhaps never – mainly as we keep finding different point mutations in MH-positive individuals all the time. And then there’s the fact that people can test positive for MH clinically, but when we screen them genetically we can’t find any of the known mutations. If a mutation can’t be found with the genetic test, you need to undergo clinical assessment as you need to be confirmed either way.”

The future outlook for individuals with anything from undiagnosed, suspected or confirmed MH is good, despite the impracticalities of a nation-wide screen to identify those currently unaware they could fall victim to MH. Patrick concluded: “Technique-wise we are making huge improvements with the genetic test so in the future the need for the clinical test will be lessened, though I don’t see us getting rid of it entirely.” Though he admits: “The frustrating part is that sometimes you have to work for a year [because of the nature of the work involved] before you get a breakthrough, but it will all help out in the end…Maybe come back next year and we’ll have added one or two more diagnostic mutations to the panel?”

I suspect I was not alone in my previous ignorance of MH and its biology, Patrick and Alan agree this is another hurdle: “MH is a rare disease and we only have a small sample of families taking part in our study. Awareness has to be raised in the general population and even amongst peers such as anesthetists…” (And recently their group has even featured in a short film aiming to do just that!) “…It is not like investigating high blood pressure or cancer – it’s an extremely uncommon thing.” Patrick then added thoughtfully: “But in the end, if you can save one life – it is a good thing.”

JP45

Another potential gene candidate causing MH susceptibility has been identified by another member of the MH research group Patrick and Alan belong to  – encoding the JP45 protein. This protein is linked to the function of calsequestrin, a molecule integral to the regulation of intracellular Ca²⁺ homeostasis. The MH-group at Leeds would like to use Proteintech’s anti-JP45 antibody to detect levels of this protein via Western blot and immunohistochemistry (IHC). I ask, briefly, why antibody-use compliments the rest of their work:

“When we designed the Ca²⁺-release imaging experiments, one question that was asked was: ‘how do we control for different protein expression levels? You may see an effect because the mutated proteins are being expressed too highly compared to wild-type controls, rather than because of the mutation itself. So we utilize Western blotting and IHC to make sure any effects we see are down to mutations and not due to expression discrepancies…And that’s what we have planned to do with Proteintech’s JP45 antibody.”

The Annual AACR Meeting was held in the same city as our headquarters this year: Chicago - where there was time before the meeting to enjoy the city's impressive skyline by boat. (Photo Credit: Deb Grainger).

…And it was a good thing our numbers were so plentiful – there were so many of you!

Booth 533 was a busy place for much of the meeting.

We were delighted to be inundated with visitors, but even more delighted at the chance to spend some quality time discussing your research and antibody requirements.

 Even Proteintech CEO and UIC Professor Dr. Jason Li was on hand to talk antibodies.

Talking antibodies with Proteintech CEO Jason Li.

Many delegates also took part in our daily draws for one of two chances to win an iPad.

AACR Annual Meeting delegates sign up for the Proteintech iPad prize draw.

Draw proceedings were always entertaining courtesy of Proteintech staff…

Proteintech's US Marketing Executive Jeff Papp brings a little showmanship to the iPad prize draw.

The luck of the draw: an onlooking delegate is asked to pick a winner.

…And here’s one of the lucky winners!

One of our six iPad winners!

The AACR Meeting this year was also of great significance to Proteintech employees as we were able to celebrate ten years since it first got up and running. Coinciding with the meeting, an anniversary cocktail party was held at the South Branch Tavern in honor of Proteintech’s tenth year (which you can read more about here.)

Thank you for celebrating with us!

When all the excitement and the hustle and bustle of the AACR Annual Meeting exhibition show was over, it was time for our US team to head back to the office and our UK representatives to head home…But not before reflecting on the successes of this year’s meeting and the UK team’s Chicago experience.

Part of the Chicago experience: the sunsets over the city (taken from the 96th floor of the Hancock Tower. Photo credit: Deb Grainger.)

Thanks for visiting us at the 2012 AACR Annual Meeting and we’ll see you again in Washington DC next year!