PROJECT OVERVIEW

Lung Cancer and its Position

Lung cancer is a disease where cells in the Lungs divide uncontrollably. Until date, Lung Cancer continues to be the world's leading cause of cancer-related mortality, with annual deaths of approximately 1,4 million cases worldwide. In Indonesia, Lung cancer is the first contributor of death with 30.065 new cases in 2018. As a lower middle-income country, Indonesia also faces a more complicated solution to overcome this problem. With that being said, as its prevalence keeps increasing and there hasn’t been any effective solution yet, Lung Cancer is inevitably a major national and international problem, and therefore its management, from the diagnostic, preventive, and curative measures, should be improved.

Lung Cancer Epidemiology

The incidence of lung cancer worldwide is increasing and the the rates in men are higher in developed countries and linked with cigarette smoking. 90% of lung cancers attributed to smoking. Lung cancer is the first contributor of death in Indonesia.

Figure 1. Epidemiology of Lung Cancer in Indonesia.

Diagnostic Evaluation of Lung Cancer

The diagnostic evaluation has three simultaneous steps (tissue diagnosis, staging, and functional evaluation), all of which affect treatment planning and determination of prognosis. The tissue diagnosis methods are available in variety that yield cytology samples and small biopsies. The methods consist of Biopsy, Bronchoscopy, or Sputum cytology.

CEAgar: A Rapid, Non Invasive Diagnostic Modality for Lung Cancer

We believe that in order to reduce the prevalence of Lung Cancer, improvements in all aspects related to the management of Lung Cancer are required, from the preventive, diagnostic to curative measures. It is well known that early diagnosis of Lung Cancer improves survival rates and will carry better prognosis. Until date, there are no diagnostic tests to reliably detect this disease with a non-invasive approach. That's why a rapid, practical, and reliable diagnostic method is an utmost importance and a modified tumor seromarker test is undoubtedly an ideal choice. CEAgar, a practical examination of CEA, using CEAker (Taz/ TGFβR1 chimeric receptor of modified E. Coli) will be the solution we are proposing to provide an effective, rapid, and non invasive test to diagnose Lung Cancer.

Why do we choose Carcinoembryonic Antigen (CEA)?

Tumor markers are molecules occurring in blood or tissue that are produced by a tumor associated with a cancer or by the host in response to the cancer. Measurement or identification of tumor markers is useful for clinical diagnosis or patient management. Carcinoembryonic antigen (CEA), the first oncofetal antigens, consists of a large family of cell surface glycoprotein (molecular weight: 150–300 kDa). It is associated with plasma membrane of tumor cells, from which it may be released into the blood. In lung cancer serological tests, one of the most commonly used circulating markers is CEA and CYFRA. But our team chose CEA as our main target to be diagnosed, because CEA is proven to have a high sensitivity and specificity other than tumor markers.

CEAker System:

Multiple biological process such as cell proliferation, differentiation, migration, tumor suppression, metastasis, and apoptosis involve transforming growth factor-β (TGF-β) signaling pathway. TGF-β signaling pathway is one of the most altered cellular signalling in cancers. There are three isoforms of TGF-β such as TGFβ1, TGFβ2, and TGFβ3. Among three isoforms, TGFβ1 is the most abundant and ubiquitously expressed isoform.

Figure 2. CEAker overview: CEAker consists of Taz/ TGFβR1 chimeric receptor.

To initiate TGF-β signaling, TGF-β ligand should bind to type II TGF-β receptors (TβRII). The ligand bind then allows the subsequent incorporation of the TGF-β type I receptor (TβRI), forming a large ligand-receptor complex involving a ligand dimer and four receptor molecules. Binding to the extracellular domains of both types of the receptors by the dimeric ligand induces a close proximity and a productive conformation for the intracellular kinase domains of the receptors, facilitating the phosphorylation and subsequent activation of the type I receptor.

Figure 3. TGFB signalling pathway involves R-SMAD.

Activated TBRI recruits and phosphorylates R-Smads, and enables the resulting complex to bind to Smad4. The activated TBR1 activates R-SMADs (SMAD2 and SMAD3) via phosphorylation then it phosphorylates the R-SMADS, enabling the R-SMADS to bind to SMAD 4 forming the SMAD trimer.The SMAD trimer enters the nucleus to activate gene transcription and promote cell growth and survival[1]. Following binding to Smad4, the complex translocates into nuclear to activate transcription of various target genes.

CEA, especially the CEA B3 domain, directly binds to TBR1 by interacting with the extracellular domain of TGBR1 and inhibits the TGF-β signaling pathway by downregulating the TGF-β signaling pathway.

Figure 4. TGFVR1 extracellular dab intracellular domain.

Tar is one of of the methyl‐accepting chemotaxis proteins (MCPs). MCPs are transmembrane chemoreceptors with periplasmic ligand binding domain and cytoplasmic signaling domain. Tar and ENVZ have the similar architecture. Tar consists of periplasmic receptor domain, transmembranes domain (TM1 and TM2), and cytoplasmic domain. Ligand of the periplasmic receptor is aspartate. The region which connects the TM2 and cytoplasmic domain ins called linker region/HAMP domain (Histidine kinase, Adenylyl cyclases, Methyl‐accepting proteins, and Phosphatases) ENVZ/OmpR is a histidine kinase in E.coli that regulates signal transduction by regulating outer membrane porins protein (OmpF and OmpC) upon medium osmolarity changes. The structure of ENVZ consists of N-terminal cytoplasmic tail (residues 1-15), TM1 (residues 16-34), TM2 (residues 163-179), periplasmic domain (residues 35-162), and cytoplasmic domain (residues 180-450) (Khorchid, Inouye and Ikura, 2005). Cytoplasmic domain consists of linker domain A (residues 180-222) and domain B (residues 290-450) (Zhu et al., 2000). Autophosphorylation of ENVZ occurs in the linker domains which cause the activation of OmpR.

Figure 5. The structure of ENVZ consists of N-terminal cytoplasmic tail (residues 1-15), TM1 (residues 16-34), TM2 (residues 163-179), periplasmic domain (residues 35-162), and cytoplasmic domain (residues 180-450) (Khorchid, Inouye and Ikura, 2005). Cytoplasmic domain consists of linker domain A (residues 180-222) and domain B (residues 290-450).

Taz is Tar-EnvZ chimeric transmembrane receptor in E. Coli by replacing Tar cytoplasmic domain with ENVZ cytoplasmic domain that phosphorylates OmpR to activate OmpC promoter. Om pR‐ P then funct ions as a transcription factor for ompF and ompC genes. In our project we would like to substitute the Aspartate to CEA, the phosphatase activity of Taz will decrease but increase the OmpR‐P level and transcription green fluorescent protein (GFP).

Figure 6. When aspartate binds, the phosphatase activity of TAZ will decrease but increase OMPR-P level; and transcription of GFP cytoplasmic.

Figure 7. CEAker consists of Taz/TGFβR1 chimeric receptor. The chimeric receptor will sense the presence of CEA. The presence of CEA will activate the OMPF/OMPC promoter (BBa_R0083) and initiate the GFP transcription.

Our Project Inspiration

Cancer is one of the most life-threatening diseases that is chronic and causes reduction of the quality of life of the patients. Cancer is indeed a debilitating disease for not only are the physical symptoms and side effects of these diseases difficult and life altering, but also, treating cancer needed huge expenses that is burdening. Lung Cancer is a disease that is characterized by lung cells growing abnormally which is caused my multiple risk factors. One of the main risk factors for Lung Cancer is cigarette smoking and it accounts for almost 90% of lung cancer cases in men and 70 to 80% in women. In today’s world, although there are plentiful treatment options given by science, the mortality of lung cancer is still high in number. According to Global Cancer Observatory (GLOBOCAN) 2018, new cases of Lung Cancer reaches 30.023 in Indonesia and the numbers are estimated to go up each year. In addition, Lung Cancer has succeeded to become the highest contributor of mortality rate among all cancers in Indonesia, being proven by 26.095 cases of death.

As a lower-middle income country with limited budget on the Health Sector, Indonesia continues to struggle in terms of equality of healthcare, with lots of rural areas don’t have sufficient access to Healthcare. In addition, those in rural areas tend to have a low education profile, which by study of epidemiology, they are more susceptible to various risk factors of Lung Cancer, namely smoking from a very early age. A study from the World Health Organization (WHO) shows that 23,4 million from the youth population between 13-17 years old smoke tobacco, and economically productive citizens accounts for 117,3 million that smoke tobacco.

Our team, consisting of members who are passionate in Research, originated from multidiscipline, are aware that it is one of our duty to identify problems around us and to also think for solutions. Therefore, we identified Lung Cancers as one of the devastating problems in our vicinity and we are aware that we must do something about it. We acknowledged that the solutions to reduce the prevalence of Lung Cancer is through comprehensive improvement from the preventive, curative, and diagnostic measures. Through our project, we strive to reduce Lung Cancer prevalence from diagnostic approach.

But we also realized,
That:

“No research without
action, and no action without research” – Kurt Lewin.

This sentence marks our belief and efforts to make change to our society, to contribute to our country’s Healthcare. We believe that through synthetic biology, our well designed CEAgar will provide a non-invasive serological diagnostic tool that could be the answer to early diagnostic modality in the future that could be implemented in all primary healthcare services, both in rural or central areas in our country. The purpose of this project is to create a CEA detecting diagnostic tool through genetically engineered plasmid with Escherichia coli as the vector which can express transforming growth factor-beta receptor type 1 (TGFRBR1), a natural CEA receptor found in human body. Our project also consists of creating campaign about Lung Cancer and its effects, how to prevent it, and most importantly, to socialize about our upcoming project.

REFERENCES

[1] [Internet]. Cancer.org. 2019 [cited 11 October 2019]. Available from: https://www.cancer.org/content /dam/CRC/PDF/Public/8703.00.pdf

[2] Aggarwal A, Lewison G, Idir S, Peters M, Aldige C, Boerckel W et al. The State of Lung Cancer Research: A Global Analysis. Journal of Thoracic Oncology. 2016;11(7):1040-1050.

[3] Zamay T, Zamay G, Kolovskaya O, Zukov R, Petrova M, Gargaun A et al. Current and Prospective Protein Biomarkers of Lung Cancer. Cancers. 2017;9(12):155.

[4] Michalodimitrakis K, Sourjik V, Serrano L. Plasticity in amino acid sensing of the chimeric receptor Taz. Molecular Microbiology. 2005;58(1):257-266.

[5] Chen, J. et al. (2016) ‘Mutational profiles reveal an aberrant TGF-β-CEA regulated pathway in colon adenomas’, PLoS ONE, 11(4), pp. 1–16. doi: 10.1371/journal.pone.0153933.

[6] e Groot, P. M. et al. (2018) ‘The epidemiology of lung cancer’, Translational Lung Cancer Research, 7(3), pp. 220–233. doi: 10.21037/tlcr.2018.05.06.

[7] Khorchid, A., Inouye, M. and Ikura, M. (2005) ‘Structural characterization of Escherichia coli sensor histidine kinase EnvZ: The periplasmic C-terminal core domain is critical for homodimerization’, Biochemical Journal, 385(1), pp. 255–264. doi: 10.1042/BJ20041125.

[8] Li, Y. et al. (2010) ‘Carcinoembryonic antigen interacts with TGF-’, pp. 1–26. Available at: file:///Users/ronaldfaris/Dropbox/Faris master Library (3-8-2014)/Carcinoembryonic antigen interacts with TGF-β receptor and inhibits TGF- β signaling in colorectalcancers.pdf%5Cnpapers3://publication/doi/10.1 158/0008-5472.CAN-10-1073.

[9] Michalodimitrakis, K. M., Sourjik, V. and Serrano, L. (2005) ‘Plasticity in amino acid sensing of the chimeric receptor Taz’, Molecular Microbiology, 58(1), pp. 257–266. doi: 10.1111/j.1365-2958.2005.04821.x.

[10] Vilar, J. M. G., Jansen2, R. and Sander, C. (no date) ‘Signal Processing in the TGF-b Superfamily (PLoS comput., 2006).pdf’.

[11] World Health Organization (2019) ‘Estimated number of cancer cases in Indonesia’, 256, pp. 2018–2019.

[12] Yoshida, T., Phadtare, S. and Inouye, M. (2007) ‘The Design and Development of Tar-EnvZ Chimeric Receptors’, Methods in Enzymology, 423(07), pp. 166–183. doi: 10.1016/S0076-6879(07)23007-1.

[13] Zhu, Y. et al. (2000) ‘Phosphatase activity of histidine kinase EnvZ without kinase catalytic domain’, Proceedings of the National Academy of Sciences of the United States of America, 97(14), pp. 7808–7813. doi: 10.1073/pnas.97.14.7808.

Team:Sriwijaya/Project Overview