SCT 60103 GENE & TISSUE CULTURE TECHNOLOGY GROUP 1 PRESENTATION BY: LEE SHU SHYAN, TAN PEI NI, MISHALNI, POOVIZHI, NOOR SALAH M.

Title of Assignment:

A normal cell can be transformed into a cancerous cell. Discuss the therapeutic strategies that are employed to

target the cellular transformation process for cancer prevention and treatment.

INTRODUCTION• Cancer transformation cause genetic instability • Spontaneous mutation rate is high due to high cell proliferation • Continuous replication of mutant cell cause damaged DNA• Effects of transformation- Growth rate - Mode of growth -Product formation-Life span-Tumorigenicity• Causes of transformation -Virus-Gene transfection-Chemical carcinogen-Ionizing radiation• Change in gene expression result to change in gene phenotype.

Therapeutic strategies to target the cellular transformation

•TGF-β is a multipotent cytokine that involved in many cellular processes, including cell proliferation, cell differentiation, apoptosis, angiogenesis and etc.•It has dual roles during tumorigenesis: i) In normal cell, it functions as inducer of apoptosis at the same time controlling cell proliferation and differentiation. ii) In transformed cell, it inhibits proliferation of transformed cell at the early stage but enhance tumor invasion and metastasis, tumor angiogenesis at the later stage.  

TGF-β Signaling Pathway

(Conolly EC, Freimuth J, Akhurst RJ, 2012)

TGF-beta pathway inhibition can be realized at 3 levels:

i) Ligand level: antisense oligonucleotides delivered directly intravenously or engineered into immune cells to prevent TGF-β synthesis

ii) Ligand-receptor level: ligand-traps and anti-TGFβ-receptor monoclonal antibodies to prevent ligand-receptor interaction

iii) Intracellular level: TGF-β receptor kinase inhibitors to prevent signal transduction

How to inhibit?

(Neuzillet et.al., 2015)

Bacterial TherapyCertain species of non-pathogenic anaerobic bacteria(Clostridium such as M55), thrive and consume oxygen-poor cancerous tissue whereas die when they come in contact with the tumor's oxygenated sides, meaning they would be harmless to the rest of the body. Only spores of anaerobic bacteria that reach an oxygen starved area of a tumor will germinate, multiply and become active. However, bacteria don't consume all parts of the malignant tissue (need to combine with chemotherapeutic treatments).

Ø  Bacteria as vector for gene therapy

- Delivering anticancer agents, cytotoxic peptides, therapeutic proteins or pro-drug converting enzymes to solid tumors.- Strong inhibition of angiogenesis and reduced tumor growth - A cya/crp (genes encoding proteins involved in the regulation of cyclic AMP levels) mutant of S. typhimurium, ×4550, has been engineered to express interleukin-2 for the treatment of liver cancer in preclinical models. Since S. typhimurium, naturally colonizes in liver, it is hypothesized that its attenuated form could be used to deliver cytokines locally to liver, with an effect on hepatic metastases.

Role of bacteria in bacterial therapy

(Patyar et al., 2010)

Ø  Bacterially directed enzyme prodrug therapy- Transformed anaerobic bacteria with an enzyme can convert a non-toxic prodrug into a toxic drug. - With the proliferation of the bacteria in the necrotic and hypoxic areas of the tumor, the enzyme is expressed solely in the tumor.- Thus a systemically applied prodrug is metabolized to the toxic drug only in the tumor.

Ø  Bacterial toxins for cancer treatment- Kill cells / at reduced levels alter cellular processes that control proliferation, apoptosis and differentiation. - Cell-cycle inhibitors, eg: cytolethal distending toxins (CDTs) and the cycle inhibiting factor (Cif), block mitosis of cancer cells.- Protein toxins such as Pseudomonas exotoxin, diphtheria toxin, and ricin are potent cell-killing agents.- Eliminating binding to toxin receptors by conjugating the toxins to cell-binding proteins such as monoclonal antibodies or growth factors can target to specific sites on the surface of cancer cells.

(Patyar et al., 2010)

(China et al., 2014)

- Non-toxic to the host; harmless to normal tissue- Selective for a specific type of tumor;- Has the ability to penetrate deeply into the tumor where ordinary treatment does not reach- Non-immunogenic (does not trigger an immune response immediately but may be cleared by the host)- Able to be manipulated easily;- Has a drug carrier that may be controlled

The ideal criteria for the selection of therapeutic bacteria

• TGF-β has a complex and dichotomous role in cancer  (Smith, Robin and Ford, 2012) , timing of treatment and predictive biomarkers for patient selection  (Neuzillet et al., 2016)

• Systemic inhibition of TGF-β may have deleterious side effects (Smith, Robin and Ford, 2012)

• Delivery ex-vivo - integrating lentiviral vectors may be less desirable because they drive constitutive expression and may result in more off-target activity

• In-vivo  - the relatively small packaging capacity of AAV vectors poses some challenges for nuclease delivery (Turitz Cox, Jeffrey Platt and Zhang, 2016)

Challenges in targeting the TGF-β signaling

Challenges with bacterial therapy

• Incomplete tumor lysis

• Treating small non-necrotic metastases of large primary tumors as metastasis is the major cause of mortality from cancer

• Potential for DNA mutations

• Toxicity at the dose required for therapeutic efficacy

(Patyar et al., 2010)

Future Development   IMMUNOTHERAPY

CHECKPOINT INHIBITORS

Using drugs : Nivolumab and ipilimumab

Function to boost the power of T-cells to destroy the cancer cells.

Works by easing the constraints on the immune system which leads to its better functioning

For Skin, lung and kidney cancer

Chimeric Antigen Receptor Therapy (CAR)

Own T-cells are engineered to destroy cancer cells

T-cells from patients are collected, manipulated and infused back into the patient's bloodstream

For B cell acute lymphoblastic leukemia and some blood cancers

(Kiesler and Begley, 2016)

EPIGENETICS- Using drugs- Transforming cancer cells back to normal rather than destroying them- Target to epigenetic enzymes- Function to regulate cells genetic programming- Set the cells on a path back toward normal growth and development- Drugs such AG-221: For myeloid leukemia (AML)

(Kiesler and Begley, 2016)

CONCLUSIONThere are two therapeutic strategies proposed to target the cancer and it has its respective challenges faced as well• Targeting the TGF beta signaling pathway• Bacterial therapy Future treatments are being designed and tested to overcome and reduce the existing challenges in the therapeutic strategies in treating and preventing cancer• Checkpoint inhibitors• CAR therapy • epigenetics

REFERENCESAmer, M. (2014). Gene therapy for cancer: present status and future perspective. Molecular and Cellular Therapies, 2(1), p.27.

Australia, C. (2016). What is cancer? - Cancer Council Australia. [online] Cancer.org.au. Available at: http://www.cancer.org.au/about-cancer/what-is-cancer/ [Accessed 15 Sep. 2016].

Cancer.org. (2016). Types of Cancer Treatment | American Cancer Society. [online] Available at: http://www.cancer.org/treatment/treatmentsandsideeffects/treatmenttypes/ [Accessed 11 Sep. 2016].

China, W., Stomatology, 610041, S. and China, P.R. (2014) ‘Tumor‑targeting bacterial therapy: A potential treatment for oral cancer (review)’, Oncology Letters, 8(6), pp. 2359–2366.

Choi, K., 2012. Autophagy and cancer. Experimental & Molecular Medicine, [online] 44(2), p.109. Available at: <http://www.nature.com/emm/journal/v44/n2/full/emm201215a.html> [Accessed 12 Sep. 2016].

C. Connolly, E., Freimuth, J. and J. Akhurst, R., 2016. Complexities of TGF-β Targeted Cancer Therapy. [online] Ijbs.com. Available at: <http://www.ijbs.com/v08p0964.htm> [Accessed 17 Sep. 2016].Google Books. (2016). Cancer Cell Culture. [online] Available at: https://books.google.com.my/books?id=TPNzgOqnYcMC&pg=PA210&dq=cancer+cell+spread&hl=en&sa=X&redir_esc=y#v=onepage&q=cancer%20cell%20spread&f=false [Accessed 15 Sep. 2016].

Kiesler, E. and Begley, M., 2016. The Future of Cancer Research: Five Reasons for Optimism | Memorial Sloan Kettering Cancer Center . [online] Mskcc.org. Available at: <https://www.mskcc.org/blog/future-five-reasons-optimism> [Accessed 14 Sep. 2016].

Neuzillet, C., Tijeras-Raballand, A., Cohen, R., Cros, J., Faivre, S., Raymond, E. and de Gramont, A., 2016. Targeting the TGFβ pathway for cancer therapy. Pharmacology & Therapeutics, [online] 147(march 2015), pp.22-31. Available at: <http://dx.doi.org/10.1016/j.pharmthera.2014.11.001> [Accessed 17 Sep. 2016].

Smith, A., Robin, T. and Ford, H. (2012). Molecular Pathways: Targeting the TGF-  Pathway for Cancer Therapy. Clinical Cancer Research, 18(17), pp.4514-4521.

Thundimadathil, J., 2012. Cancer Treatment Using Peptides: Current Therapies and Future Prospects. Journal of Amino Acids, 2012, pp.1-13.

Turitz Cox, D., Jeffrey Platt, R. and Zhang, F., 2016. Therapeutic genome editing: prospects and challenges. nature medicine, [online] 21(2), pp.121-131. Available at: <http://zlab.mit.edu/assets/reprints/Cox_D_Nat_Med_2015.pdf> [Accessed 17 Sep. 2016].

Patyar, S., Joshi, R., Byrav, D.P., Prakash, A., Medhi, B. and Das, B. (2010) ‘Bacteria in cancer therapy: A novel experimental strategy’, Journal of Biomedical Science, 17(1), p. 21. doi: 10.1186/1423-0127-17-21.

Thank you!