Protein Tyrosine Phosphatases (PTP) are a family of genes that regulate many important cellular processes involved in the cell cycle, including those central to cancer development. This study aimed to identify mutated PTP genes across fifteen solid tumour cancers and to determine biological pathways affected by the oncogenic PTP that was most frequently mutated. Once the oncogenic PTP was identified, chemical inhibitors for the oncogenic PTP were found for potential use in cancer treatment. PTPN6 (protein tyrosine phosphatase non-receptor type 6) was the most upregulated and mutated PTP (in 11 out of 15 tumours). Cellular pathways disrupted by PTPN6 mutations were the JAK-STAT, VEGF, MAP-Kinase and PI3K pathways. CHEMBL509443 and CHEMBL449613 inhibitors were active against PTPN6 and had limited cross-reactivity. The upregulation of PTPN6 in tumour samples indicates that it may be an oncogene. The pathways affected by PTPN6 mutations have been linked to cancer growth and metastasis. The inhibitors identified, if developed into clinical drugs, could potentially be used to treat solid tumours with PTPN6 mutations, which accounts for 38% of solid tumours on cBioportal.

Introduction 

This study is a bioinformatics analysis of some members of the Protein Tyrosine Phosphatase (PTP) gene family which looks at the potential development of new therapeutics for cancer treatment, using these PTP genes as targets. The PTP gene family consists of 125 members which play an essential role in maintaining a healthy cellular environment.1 In normal human physiology, PTPs work in coordination with Protein Tyrosine Kinases (PTKs) to regulate the process of tyrosine phosphorylation.2 The PTPs remove phosphate groups from tyrosine residues on proteins while PTKs add phosphate groups to the same.2 Since the PTKs catalyse phosphorylation and activation of many growth-factor mediated pathways, the PTPs were intuitively considered to be negative regulators of these pathways and were thought to be tumour-suppressive and unspecific in nature.1 Due to this erroneous assumption, PTPs were considered to be less attractive than PTKs in terms of therapeutic targets for cancer treatment.1 However, increasing evidence suggests that the role of PTPs is far more complex than initially anticipated.1

Recent research has found that some PTPs act as oncogenes, increasing the activity of certain pathways which are important in the development of neoplastic disease.1 Others exhibit both tumour suppressive and oncogenic characteristics depending on the cellular environment.1 Dysregulation of PTP-PTK activity can give rise to a number of diseases and PTPs have been linked with metabolic and autoimmune diseases as well as neurodegeneration.3 Due to the significant role that PTPs play in the development and progression of human disease, some members of this gene family are considered to be attractive targets for new therapeutics for not only cancer treatment but other applications such as diabetes mellitus type 2 management.4

The primary aim of this study was to further elucidate the role of potentially oncogenic PTPs specifically in solid tumours. Secondly, the study aimed to identify chemical inhibitors that have the potential to be developed into therapeutics for the treatment of certain cancers in which there is PTPN6 upregulation. Some research has been carried out on the role of PTPN6 (SHP-1) in haematological malignancies and a selective number of solid tumours namely breast ductal carcinoma and nasopharyngeal carcinoma.1 However, there is still limited literature on the role of PTPN6 in solid tumours. This study analysed datasets of 15 solid tumours using several bioinformatics tools (Table 1) for upregulation of and mutations in PTPN6. Lung adenocarcinoma, stomach adenocarcinoma, breast invasive carcinoma, colorectal adenocarcinoma and prostate adenocarcinoma, were chosen to be used in this study. These cancers were primarily carcinomas, which were chosen since carcinomas are the most common types of cancer diagnosed in the UK.5 The three most common types of cancers causing cancer deaths globally in 2018 were found to be lung cancer (1.76 million deaths), colorectal cancer (862,000 deaths) and stomach cancer (783,000 deaths).6

materials and methods

identification of ptp gene mutants

The cBioPortal Database was used to identify members of the PTP gene family that were mutated in solid tumours.7 After logging on to the cBioPortal for Cancer Genomics website, the 'DATA SETS' setting was enabled. On the 'DATA SETS' homepage, a list of cancers is visible. From this list, a cancer dataset was chosen; for example:

Cancer Study of Colorectal Adenocarcinoma (TCGA, Provisional) - 631 samples.

Once the dataset has been chosen, information such as the survival rate of patients, the mutations and copy number variations of genes etc. is available to view. The ‘STUDY SUMMARY’ option was selected. The ‘MUTATED GENES’ refinement setting was chosen from the ‘STUDY SUMMARY’ page, and the PTPs in this dataset were noted, along with their frequency.

This was repeated for 14 other datasets.  

Analysis of copy number variants

To establish if these PTPs could be potential oncogenes or tumour suppressor genes, the 'COPY NUMBER VARIATION' filter in the cBioPortal database was enabled and the PTP genes that were amplified in the tumour sample were noted.7 This was done by selecting the ‘COPY NUMBER ALTERATIONS’ refinement setting instead of the ‘MUTATED GENES’ setting.

Determining ptp function

UniProt was used to obtain information about the function of PTPs that were found to be mutated.8 The names of each mutated PTP was entered into the search box eg: PTPN11. As there were results available for different organisms, the 'HOMO SAPIENS' filter was applied. Information regarding the functional role was then analysed from the database (this is outlined in the results section).

Determining signalling pathways affected by ptps

KEGG Pathway Analysis was used to determine what signalling pathways were affected by the activity of the chosen PTP.9 On the KEGG website, the KEGG Pathway option was chosen. The name of the chosen organism and protein, which is Homo sapiens and PTPN6, was entered into the search engine. The pathways in which PTPN6 is involved (shown in Figures 1,2,3) were displayed on the computer screen. The pathways affecting cell proliferation were analysed as well as the location of PTPN6 in the pathways.

searching for inhibitors

PubChem BioAssay from the NCBI website was used to search for inhibitors of the chosen PTP and to check for cross reactivity.10 The ‘GENE’ option was selected on the NCBI website. The protein name was entered into the PubChem BioAssay search engine and the Homo sapiens option was selected. The first option on the list for PTPN6 was chosen. After scrolling down to the ‘RELATIVE INFORMATION’ section, the ‘BIOASSAY BY TARGET (SUMMARY)’ tab was selected. The 'BIOACTIVITY DATA' for gene PTPN6 was displayed. All the known inhibitors of PTPN6 were recorded, which amounted to 84 inhibitors. The cross-reactivity of these chemicals was then checked by going onto the PubChem Project website and entering the chemical’s name into the compound search engine. The ‘BIOLOGICAL TEST RESULTS’ section on compound summary page was then selected. The ‘REFINE/ANALYSE BIOASSAY RESULTS’ option was enabled and the ‘BIOACTIVITY ANALYSIS TOOL’ was chosen. A list of the total bioassays was brought up and active proteins that were inhibited excluding PTPN6 were noted. This method was used for each of the chemical inhibitors of PTPN6.

results

cBioPortal Results - Identifying mutated PTPs in 15 solid tumours

After analysing the datasets of different solid tumours, it was found that at least 1 PTP was mutated in 87% of all solid tumours on the database. PTPN11 and PTPN6 were mutated most frequently (in 11 out of the 15 cancers investigated). PTPN11 was mutated in 34% of all solid tumours on the database. PTPN6 was mutated in 38% of all solid tumours on the database. PTPN11 was upregulated in 47% of solid tumours on the database. PTPN6 was upregulated in 73% of solid tumours on the database. This upregulation strongly suggested that they may be oncogenes, hence it was decided to further investigate PTPN11 and PTPN6.

UniProt Results - obtaining data on the functional role of ptpn11 and ptpn6

From UniProt, it was noted that PTPN11 mutations have been associated with several pathologies such as Noonan syndrome, LEOPARD syndrome (type 1) and metachondromatosis, all of which are genetic disorders. In relation to cancer, mutations in PTPN11 can cause juvenile myelomonocytic leukaemia, which is a rare childhood cancer of the blood. PTPN11 is widely expressed with the highest levels found in the heart, brain and skeletal muscle. Since PTPN11 has been extensively researched and is a well-known oncogene, PTPN6 became the main focus of the study from this point.

For PTPN6, there were no associated pathologies listed on the UniProt database. In terms of expression, isoform 1 is expressed in haematopoietic cells while isoform 2 is expressed in non-haematopoietic cells.

The JAK-STAT pathway represents a signalling cascade which regulates cell proliferation and haematopoiesis. A physiologically normal PTPN6 (SHP-1) protein would be expected to dephosphorylate the JAK protein. If the JAK protein was phosphorylated, it would cause the MAP (mitogen-activated protein) kinase pathway to be activated leading to proliferation and differentiation. It would furthermore activate the PI3K (Phosphoinositide-3-kinase) pathway, which promotes cell survival. Inhibition of a normal PTPN6 gene would allow these pathways to be activated more frequently. However, if PTPN6 was mutated, it could dysregulate both MAP kinase and PI3K pathways, which could potentially initiate a cell’s route to becoming cancerous.

The T-cell Receptor pathway involves the activation of T-cells, key components of the immune system that regulate many immune responses.2 PTPN6 normally dephosphorylates ZAP70 (ZetaChain (TCR) Associated Protein Kinase 70kDa) which phosphorylates p38 and hence, NFAT (Nuclear factor of activated T-cells) protein. The activation of NFAT results in proliferation, differentiation and immune response or in some cases anergy (absence of the normal immune response to a particular antigen or allergen). A mutated PTPN6 gene would again cause dysregulation in this pathway, which could result in uncontrolled proliferation along with as imbalance in immune responses and differentiation. This particular pathway, however, is more relevant to development of haematological malignancies as opposed to solid tumours.

The vascular endothelial growth factor (VEGF) pathway is responsible for angiogenesis.2 Angiogenesis is a crucial process needed for tumour growth. It allows the formation of new blood vessels which give the tumour its own blood and oxygen supply. Non-mutated PTPN6 dephosphorylates VEGFR2, the protein responsible for cell migration, proliferation and survival.2 This inactivation of VEGFR2 would result in inhibition of migratory, proliferative and survival processes, however, a mutated PTPN6 genetic function would be altered and override inactivated VEGFR2.

NCBI PubChem BioAssay Results - Identifying inhibitors for PTPN6

This tool was used to search for potential inhibitors of PTPN6. Furthermore, this tool allowed analysis of cross-reactivity between the compounds with inhibitory effects on PTPN6 and other proteins. 84 compounds were found to inhibit the action of PTPN6. Only 2 of these chemicals targeted PTPN6 without being active against other proteins. These were:

CHEMBL509443 and CHEMBL449613

Discussion

Nearly 50% of all PTP genes in the human genome have been linked with a human disease.4 Thus, inhibitors or activators of PTPs could be efficient as drugs in the treatment of several pathological conditions where physiological PTP function is perturbed. Targeting PTKs, the biological counterparts of PTPs, has yielded very positive results in terms of cancer treatment. Cohen et al. (2013) outline that over the past fifteen years, protein kinases have become one of the most important class of drug targets in cancer.11 Approximately twenty drugs that target kinases have been approved for clinical use over the past decade, and hundreds more are undergoing clinical trials.11

In terms of PTPs, there are currently no drug targets that are in clinical use.1 Sodium stibogluconate, a PTPN6 inhibitor that has been studied in phase I clinical trials, showed poor outcomes in malignant melanoma.1 No clinical trials have been carried out to examine the effect of PTPN6 inhibitors in solid tumour cancers yet, although there are currently ongoing trials for PTPN6 inhibitors in inflammatory diseases.1 No phase II studies of smallmolecule PTPN6 - inhibition have been conducted so far.1

A more thorough understanding of the role of PTPN6 in various cellular pathways is essential in developing novel therapeutic targets for solid tumours. This study highlighted the effects and position of PTPN6 in several pathways that are linked to cancer development. Two chemical inhibitors which exhibit PTPN6–specificity were also identified. These inhibitors have not been used in clinical trials previously. With further research and investigation, there is potential for these two chemicals to be developed into therapeutic targets for the treatment of solid tumours in which there is PTPN6 upregulation.

Acknowledgements

Dr. Niamh McNally, Loreto Secondary School, Balbriggan; Mr. Chris Garvey, Loreto Secondary School, Balbriggan; Dr. Alex Eustace, Royal College of Surgeons, Ireland; Professor Cormac Taylor, University College Dublin; Mr. Edward Fynes; Ms. Rachel Gallen; Ms. Siobhán Murray.

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02. Julien SG, Dubé N, Hardy S et al, ‘Inside the human cancer tyrosine phosphatome’. Nat Rev Cancer. 2011 Jan; 11(1):35-49. [Available at: https://www.ncbi.nlm.nih. gov/pubmed/21179176]

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04. He Rong-jun, Yu Zhi-hong, Zhang Ruo-yu et al. “Protein Tyrosine Phosphatases as Potential Therapeutic Targets.” Acta Pharmacologica Sinica , 2014 Sep 15, 35(10); 1227 -1246 [Available at: https://www.ncbi.nlm.nih. gov/pmc/articles/PMC4186993/]

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06. Cancer, World Health Organisation, September 2018 [available at: http://www.who.int/newsroom/fact-sheets/detail/cancer]

07. Cbioportal Database for Cancer Genomics, Memorial Sloan Kettering Cancer Centre. [Available at: http://www.cbioportal. org/]

08. The UniProt Consortium, UniProt: the universal protein knowledgebase, Nucleic Acids Res. 45: D158-D169 (2017)

09. Kanehisa, Furumichi, M., Tanabe et al.; KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Res. 2017; 45, D353-D361 . [Available at: https://www.kegg.jp/]

10. Kim S, Thiessen PA, Bolton EE, Chen J et al, PubChem Substance and Compound databases. Nucleic Acids Res. 2016 Jan 4; 44(D1) [Available at: https://www.ncbi.nlm.nih. gov/pubmed/26400175]

11. Cohen P, Alessi DR. ‘Kinase Drug Discovery – What’s Next in the Field?’ ACS chemical biology. 2013 Jan 18 ;8(1):96-104. [Available at: https://www.ncbi.nlm.nih. gov/pubmed/23276252]