Team:ESBS-Strasbourg/proteolux/application/cancer

• Alzeimer disease
• Occurrence and costs
• Genetics
• Pathology
• The Tau protein
• Calpains
• Caspases
• Utilization of the ProteoLux® Pro system

Proteomics technology is an approach of science to understand the expression of the whole set of proteins and its function at the cellular level. Its role in cancer research becomes more significant nowadays. Consulting Medline reveals that, since 1997, when the word proteomics first appeared as an entry, approx. 0.12% of all articles on cancer also contain the word proteomics. Comparing this frequency with the occurrence of the search string ‘genomics’ in 0.17% of the cancer literature, it becomes obvious that proteomics has attracted major scientific attention in the field of cancer research. Vice versa, a search for ‘cancer AND proteomics’ shows that 16% of the proteomics literature deals with cancer, testifying that the attraction is mutual.

Indeed, proteomics technology enables the researchers to look into the proteins level of the cancer cells, proteins being the physiological and pathological indispensible players. Proteins represent the majority of drug targets. However, currently only around 500 out of the estimated >3000 proteins that are predicted to be druggable are exploited.

Proteomics has the great potential to be one of the most powerful tools for cancer research. The ProteoLux® Pro system offers a new proteomic tool for cancer research. It can help in therapy designing by characterizing the potency of drug targets. The multiple targets of the system and its advantages will be exemplified in the case of the pancreatic ductal adenocarcinoma (PDAC), a major unsolved health problem.

Pancreas is a gland organ found in vertebrates. The pancreas is comprised of separate functional units that regulate two major physiological processes: digestion and glucose metabolism. It presents endocrine and exocrine functions. In fact this gland permits to produce hormones like insulin and to secrete digestive enzymes contained in a pancreatic juice which are inserted into the small intestine to help for assimilation of nutrients. The exocrine pancreas consists of acinar and duct cells. The Figure 12 describes in detail the pancreas anatomy.

Figure 1 | Anatomy of the pancreas.
The pancreas is comprised of separate functional units that regulate two major physiological processes: digestion and glucose metabolism. The exocrine pancreas consists of acinar and duct cells. The acinar cells produce digestive enzymes and constitute the bulk of the pancreatic tissue. They are organized into grape-like clusters that are at the smallest termini of the branching duct system. The ducts, which add mucous and bicarbonate to the enzyme mixture, form a network of increasing size, culminating in main and accessory pancreatic ducts that empty into the duodenum. The endocrine pancreas, consisting of four specialized cell types that are organized into compact islets embedded within acinar tissue, secretes hormones into the bloodstream. The α- and β-cells regulate the usage of glucose through the production of glucagon and insulin, respectively. Pancreatic polypeptide and somatostatin that are produced in the PP and δ-cells modulate the secretory properties of the other pancreatic cell types. a | Gross anatomy of the pancreas. b | The exocrine pancreas. c | A single acinus. d | A pancreatic islet embedded in exocrine tissue.
Bardeesy N and DePinho R A. (2002) ”PANCREATIC CANCER BIOLOGY AND GENETICS” Nature Reviews Cancer VOL.2 pp897-909

Pancreatic Cancer is a cancer of the digestive system. Different kinds of pancreatic cancer exist : Adenocarcinomas, which represents 95% of cases, adenosquamous carcinomas, singet ring cell carcinomas, hepatoid carcinomas, colloid carcinomas, undifferentiated carcinomas and undifferentiated carcinomas with osteoclast-like giant cells which represent the remaining 5%.

Figure 2 | Gross photograph of an infiltrating adenocarcinoma
Note the dramatic narrowing of the pancreatic duct associated with the poorly defined white neoplasm.
Maitra A, 1,2 and Ralph H. Hruban R H. (2008) “Pancreatic cancer” Annu. Rev. Pathol. Mech. Dis. 3:157–88

Pancreatic ductal adenocarcinoma is a malignant tumor of epithelia originating in glandular tissue. It grossly produces a firm, highly sclerotic mass (Figure 2). Aggressive and devastating disease, it is one of the most common causes of cancer related death. It is characterized by invasiveness, rapid progression and profound resistance to treatment.

Figure 3 | Mortality of the pancreas cancer per year
http://commons.wikimedia.org/wiki/File:Pancreas_cancer_world_map_-_Death_-_WHO2004.svg

Pancreatic cancer is predominantly a disease of the elderly. Besides, men are more concerned than women. Age and gender are so risk factors. Other established risk factors include diets high in meats and fat, low serum folate levels, obesity, long-standing diabetes mellitus, and chronic pancreatitis. Pancreatic cancer may be genetic. This is the case for 5 to 10% of patient but the gene responsible of this disease is not yet identified. Furthermore for the moment there are no real guidelines for preventing from pancreatic cancer.

Developed countries are the most affected by this disease as shown in Figure 3. This cancer causes the death of thousands people in the world each year of whom 7000 French and 36 800 American.

This cancer is very hard to detect. Indeed, there is often no symptom attached to early pancreatic cancer. Furthermore the later symptoms are non specific and varied. That is why this disease is also called “silent killer”. Pain in the abdomen, loss of appetite and so loss of weight, painless jaundice, diabetes mellitus or elevated blood sugar level, trousseau sign or clinical depression are common symptoms of pancreatic cancer.

The prognosis of the pancreatic cancer is poor. The 5- year survival rate of all patients is below 5%, and the median survival time after diagnosis is 6 months. Surgery offered the only possibility of cure. But surgery may only be used if the tumor is not too advanced and if the cancer does not present metastasis. This surgery is not easy because of the high amount of veins surrounding the pancreas and this operation is possible only in 20% of cases. Furthermore a relapse is common (70 to 80% of people). Thus, only 20% of the patients who have undergone surgery survive longer than 5 years. For those who cannot undergo this surgery their median survey is approximately of 6 months. Radiotherapy may be used in case of evolved cancer and is established after surgery. Then chemotherapy may be used when the cancer presents metastasis. Gemcitabine, Fluorouracil, cisplatine and oxaliplatine are used. Nevertheless, PDAC is insensitive to most therapies including chemotherapy, radiotherapy and immunotherapy.

Figure 4 |Progression model of pancreatic adenocarcinoma
http://www.nature.com/modpathol/journal/v15/n4/images/3880544f1.jpg + medscape.com

The progression from histologically normal epithelium to low-grade pancreatic intraepithelial neoplasia (PanINs are microscopic lesions in the smaller (<5 mm) pancreatic ducts), to high-grade PanIN, to invasive carcinoma (left to right in Figure 4) is associated with the accumulation of specific genetic alterations. This signature molecular profile consists notably in mutations in the oncogene K-RAS and the tumor suppressors CDKN2, TP53, SMAD4/DPC4 and BRCA2. Other recognized precursor lesions of adenocarcinoma (intraductal papillary mucinous neoplasm and mucinous cystic neoplasm) likely harbor a distinct compendium of genetic alterations in their path to invasive cancer.

The Figure 5 presents a schematic diagram of intracellular signalling pathways in pancreatic adenocarcinoma.

Figure 5 | Schematic diagram of intracellular signalling pathways in pancreatic adenocarcinoma
O'Reilly E and Epstein A (2010) “Recent Findings in Pancreatic Cancer: Illuminating Emerging Treatment Strategies” MedscapeCME Oncology

EGFR = epidermal growth factor receptor; ERK = extracellular signal-regulated kinases; FGF-R = fibroblast growth factor receptor; GDP = guanosine diphosphate; GTP = guanosine triphosphate; HER2 = human EGFR 2; IGF = insulin growth factor; MEK = mitogen-activated protein kinase; MEKK = mitogen-activated protein kinase kinase; MMP = matrix metalloproteinase; mTOR = mammalian target of rapamycin; NF-kappaB = nuclear factor kappa-light-chain-enhancer of activated B cells; VEGF = vascular endothelial growth factor receptor.

The majority (90%) of pancreatic adenocarcinomas have a mutated K-Ras oncogene. KRAS encodes a member of the RAS family of guanosine triphosphate (GTP)-binding proteins that mediate a range of cellular functions, including proliferation, cell survival, cytoskeletal remodeling, and motility, among others. Activating mutations impair the intrinsic GTPase activity of the KRAS gene product, resulting in a protein that is constitutively active in intracellular signal transduction. In addition to its role in pancreatic cancer initiation, constitutive RAS signaling appears to be required for pancreatic cancer maintenance as well.

For many years targeting, Ras has been a holy grail in the treatment of pancreatic cancer. Either single-agent or combination therapies with farnesyl transferase inhibitors (SWOG 9924 study) that target Ras translocation have shown limited activity. Ras still represents an attractive and ubiquitous target.

Activated Kras engages a number of downstream effector pathways, including RAF–mitogen-activated protein kinase (RAFMAPK), phosphoinositide-3-kinase (PI3K), and RalGDS pathways (as shown in Figure 6) producing thus a remarkable array of cellular effects, including induction of proliferation, survival and invasion through the stimulation of several effector pathways.

Figure 6 | Some pathways in which KRAS is involved in PDAC
Lauth M, Toftgard R and al (2010) “DYRK1B-dependent autocrine-to-paracrine shift of Hedgehog signaling by mutant RAS” Nat. Struct. Mol. Biol. 17, 718 - 725

Mirk/Dyrk1B as potential target in pancreas cancer

DYRK1B/Mirk is a RAS effector kinase (Figure 6), which is activated by phosphorylation by its upstream activator, the MAP kinase kinase MKK3, suggesting that Mirk, like p38, is activated by certain environmental stress agents.

Mirk/Dyrk1B is a member of the Minibrain/dyrk family of kinases, more precisely an arginine-directed serine/threonine protein kinase. It is expressed at low levels in most normal tissues but at elevated levels in quiescent pancreatic cancer cells. Mirk is expressed in about 90% of pancreatic cancers and is amplified in a subset. Mirk appears not to be an essential gene for normal cells from embryonic knockout studies in mice and RNA interference studies on cultured cells. Mirk/Dyrk1B mediates the prolonged survival of cancer cells through increasing expression of a cohort of antioxidant genes. These unusual characteristics suggest that Mirk may be a selective target for therapeutic intervention [70].

Indeed, one major issue in pancreas cancer is the insensitivity to treatment due to the activation of defense systems, notably those producing antioxydants in which Mirk acts. By inhibiting Mirk, the protection of cancer cells will be inhibited, making them more sensitive to actual treatment (chemotherapy, radiotherapy…).

Advantages of the ProteoLux® Pro system in cancer research

The ProteoLux® Pro system enable to degrade specifically one or more proteins expressed in vivo at a given time and in a given cell type.

The ProteoLux® Pro system also enables you to control the concentration of a protein according to the lighting alternation between far red light and red light. The ProteoLux® Pro system also enables you to degrade specifically muted protein (not the normal one) by using specific intrabodies.

The ProteoLux® Pro system : an amazing tool to study every overexpressed protein, notably oncoproteins.

Indeed, oncogenes which code for oncoproteins are mutated proto-oncogenes, very important genes involved in the regulation of cell cycle, cell growth and cell proliferation. Mutations in proto-oncogenes can lead:

• either to a conformational change of the oncoprotein, thus to an increase of its activity or a loss of its regulation
• or to an increase of its activity, an increase of its stability, a duplication of the oncogene, a retroviral insertion, all this leading to an increase of the concentration of the oncoprotein
• or to a chromosomic translocation (expression in the wrong cell type, at the wrong time or constitutive expression of the oncogene).

The ProteoLux® Pro system : a rapid access to the potency of therapeutic targets

The potency of Mirk as a therapeutic target can easily and relatively fastly be studied with the ProteoLux® system.

Figure 7 | Principle of the assessment of the potency as therapeutic target by the ProteoLux® Pro system

Besides, by using several intrabodies it is possible to target several proteins at the same time. Therefore, the ProteoLux® Pro system permits an easy assessment of combinatory targeting.

The ProteoLux® Pro system : solution to the specific difficulty of clinical management in cancer

As shown in Figure 4, tumors possess cytogenetically different clones corresponding to different stages of progression of the disease. This heterogeneity contributes to differences in clinical behavior and responses to treatment of tumors of the same diagnostic type. With the ProteoLux® system you can target several muted proteins by intrabodies. Thus, you will only target muted proteins but all the muted proteins. Therefore you will target all the stages of progression of the disease.

The discovery and understanding of the altered genes causing cancer (oncogenes, tumor-suppressor genes, and microRNA genes) and of the pathways involved permit the development of cancer therapies. Unfortunately, research goes slowly. The ProteoLux® Pro system gives research a boost.

The ProteoLux® system can interest big pharmaceutical firms like Roche or Novartis which are already focusing on pancreatic cancer. Indeed, Roche has developed a molecule for chemotherapy, Traceva and Novartis has created a new product Afinitor, which permit also to increase life expectancy of people affect by pancreatic endocrine tumor. Our system permits these companies to broaden their scope against pancreatic cancer by studying easily the potency of a new target. Notably, they will be able to develop cancer stage-specific drugs basing on the ProteoLux® system.

One possible purchaser will be the Cancer Research Technology Limited (CRT). It is the cancer-focused technology development and commercialisation arm of Cancer Research UK, the world’s largest cancer charity. Their aim is providing best in class technology transfer services to cancer researchers. The ProteoLux®system will complete their platform technology and research tools.

This list is far from being exhaustive.