SCT60103 GTC Group 1 Presentation Strategies are focussed more in cancer treatment

1. Therapeutic strategies that target the
cellular transformation process for cancer
prevention and treatment.
By Chong Jia En, Chong Jia Yii, Lim Yee Hung, Tan Liu Xi and Yeap Tze Huay
SCT60103 Genes and Tissue Culture Technology

2. • The result of a series of events that both depends on and promotes genetic instability.
Transformation
(Freshney 2016, p.495)
(Fearon and Vogelstein 1990).
▪ Infection with virus
▪ Transfection with genes
▪ Ionising radiation
▪ Chemical carcinogens
• Can arise from
• Transformation of cultured cells implied a spontaneous or induced permanent phenotypic
change resulting from a heritable change in DNA and gene expression.
▪ Immortalisation
▪ Aberrant growth control
▪ Malignancy
• Change in gene expression result in
change in gene phenotype

3. Transformation
(Weinberg 2007)
• Genetic alteration of the infected cell leads
to its unlimited proliferative potential. -----------------------------------------
• Performs cellular transformation assay to aid
in development of therapeutic strategies that
can treat and prevent cancer.
• Example of types of therapeutic strategies:
• Due to the limitations and challenges, further
investigation of therapy is needed.
▪ Targeted therapy
▪ Chemotherapy
▪ Precision Medicine

4. WNT Signalling Pathways
WNTs: secreted glycoproteins (Papkoff, Brown and Varmus 1987) which regulate multiple signalling
pathways via both β-catenin (CTNNB1)- dependent and CTNNB1-independent mechanisms
WNT-CTNNB1 signalling (maintenance of tumour-initiating cells) and CTNNB1-independent WNT
signalling pathways can either activate or inhibit tumorigenesis and cancer progression in a
context-dependent manner
Can promote transcriptional changes that can drive epithelial-mesenchymal transition – alteration in protein
expression that results in complex changes in cell behaviours (e.g. cell-cell interaction and enhanced motility)
Changes in WNT signalling pathways and other oncogene and tumour suppressor pathways
cooperate to drive cancer initiation and progression
Targeted cellular pathways/targets:
• WNT signalling pathways
• specific overexpressed WNTs and WNT receptors in tumours (WNT receptors retain the
malignant phenotypes but are not necessary for normal tissue homeostasis)
How does it lead to cellular transformation?

5. ➔Small molecules
◆ lithium chloride- cause CTNNB1 activation by inhibiting GSK3
◆ IWP (inhibitor of WNT production)- a membrane bound acetyltransferase that modifies
WNT ligands for their secretion and signalling activity (Chen et al 2009).
◆ DVL-specific inhibitors – to inhibit DVL function in CTNNB1-dependent and CTNNB1-
independent pathways by protein-protein interaction and structure-based design
algorithms (Fujii et al 2007).
➔Blocking antibodies (target specific overexpressed WNTs and WNT receptors in tumours
that inhibit proliferation and cause apoptosis in different cancers (He et al 2004)).
◆ FZD7-specific antibodies that targets FZD receptors in hepatocellular carcinoma cells
blocks expression of WNT-CTNNB1 gene (Wei, Chua, Gapper and So 2011) and interacts with
DVL to inhibit the growth of HCC cells (Nambotin et al 2011).
● WNT-CTNNB1 signalling is required serial transplantation and self-renewal of both
normal haematopoietic stem cells and subsets of leukemia cells -> inhibition of
pathway may have unwanted side effects on normal adult stem cells
● Current Development by Hudecek et al 2010 and Yang et al 2011: ROR1-targeted
antibody - target ROR1-positive malignant cells for selective killing by the immune
system as ROR1 is only highly expressed in embryonic tissues and blood cancers
but not in normal adult cells (Wu et al 2008).
WNT Signalling Pathways
Therapeutic strategies:

6. Therapeutic strategies:
WNT Signalling Pathways
➔Peptides
◆ FZD7-blocking peptides- block TCF/LEF reporter activity and the expression of WNT-
CTNNB1 target genes in HCC cells; disrupt the growth of HCC cells (did not affect the
viability of normal hepatocytes lacking FDZ7 expression) -> induce apoptosis in colon
and breast cancer cells
◆ Use of peptide ligands that binds to the PDZ domain of FZD2 to disrupt WNT-CTNNB1
signalling
● Useful in targeting CTNNB1 independent signalling
● Activity of many downstream kinases from this signalling pathway are essential
for normal homeostasis and metabolism
➔WNT signalling and combination therapy (sensitize or desensitize cancer cells to
toxic insults)
◆ Cancer cells’ sensitivity to chemotherapeutic agents is enhanced by inhibiting or activating
(overexpression of WNT5A) the WNT-CTNNB1 pathway> cooperative inhibition of tumour
growth
➔Gene therapy
◆ FZD7 knockdown – reduces TCF-dependent transcription and xenograft tumour growth
of triple negative breast cancer
◆ Small interfering RNAs (siRNA) – to reduce ROR1 expression as is decreases the growth
of gastric, lung and breast cancer cells during cell culture and xenografts / induce
apoptosis in CLL, breast carcinoma and cervical carcinoma

7. WNT Signalling Pathways
Challenge
❑ Mutations in APC and AXIN1 hyperactivates the
WNT-CTNNB1 pathway, limiting the potential
molecular targets for pathway modulation. It is
because factors acting upstream of the
destruction complex are not required in
pathway activation.
❑ The WNT pathway cannot be targeted using a
single universal strategy
-------------------
Current development:
 further investigation on the mechanisms of
crosstalk between WNT pathways and related
signalling networks such as oncogene and tumour
suppressor pathways
 combinatorial therapies
 identify the genetic factors and biomarkers that
allow prediction on responses to treatment with
WNT pathway modulators
 identify WNT receptors that are necessary for cell
evasion of senescence and apoptosis
 To identify molecules which affect the interaction
between TCF7L2 and CTNNB1 and hence, inhibit
CTNNB1-dependent transcription.
------------------------------

8. Association between Diabetes Mellitus and Cancer
• First described 81 years ago by Joslin clinician
• Risk increase in diabetes
How a diabetes patient’s cell transform into tumour cell?
• Insulin resistance
• Caused by the increased level of insulin and insulin-like growth factor (I/IGF) which bind to
some receptor of cell
• Activate the downstream (P13K)/Akt and MAPK signaling pathways
• Leads to proliferation of cell
• Results in development of tumour
• Diabetes as inflammatory disease (Donath and Shoaelson 2011)
• produce cytokines and tumour necrosis factor
• activate the transcription of tumour cells
• Hypoimmunity of Diabetic Patients (Geerlings and Hoaepelman 1999)
• more susceptible to opportunistic infection as T2DM patients maybe immunodeficient
Metformin
-------------------------------------------------------------------------

9. Therapeutic Strategy: Metformin
-----------------------------------------------------
Treat Type II Diabetes Mellitus
• inhibiting lipogenesis and mitigates
hyperlipidaemia
• reduce cellular levels of reactive oxygen species
• down regulates proinflammatory cytokines which
constitutes risk factor of cancer
• Obtained from T2DM patients
• Findings
• Reduced risk of cancer
• Improves overall survival
• Suggesting metformin exerts therapeutic effect
• Case control study reviewed clinical records from 923 T2DM patients in UK and found 23% reduction in risk of developing cancer (Evans et al. 2005)
• Reduced risk is found in cancers for liver, colorectal, pancreatic, stomach and esophageal cancers, whereas no consistent results found on cancers from
breast, prostate and lung
Epidemiological Evidence of Anti-tumour Effect of Metformin
--------------------------------------------------------------------------
Function as – antitumor effect
• Depress tumour proliferation (AMPK)
• Induce apoptosis, autophagy, cell cycle arrest
of tumour cells

10. - Metformin systemically ameliorates hyperinsulinemia, hyperglycemia
and hyperlipidemia
- promoting factor of initiation and progression of cancer
- inhibits mTOR pathway
- through AMPK-dependent and independent mechanisms
- activates AMPK
- intermediate downstream effector of tumour suppressor LKBI
- inhibits SREBP-1
- by regulating its expression and phosphorylation
- down-regulation of FASN and ACC
- Phosphorylates and inhibits ACC
Diabetes Associated Cancer
Cellular Level Signalling Pathways
Metformin suppresses de novo syntheses of fatty acids and protein synthesis in cancer cell
(He et al. 2015)
Metformin
How Metformin works?

11. Challenges
DOSE
• Metformin prosecutes anticancer function via systemic but indirect
effect
• Concentration required for direct effect of metformin
• within the range of 5 to 10 mM which is greater than the steady state
levels of plasma when standard dose is given to T2DM patients
• Low concentration of metformin is not sufficient to cause AMPK
activation
• insufficient to inhibit malignant growth of cancer cells
SITE
• According to studies in mice, administration of metformin at
50mg/kg per day results in maximal concentration of 50 to 60 µM
in hepatic portal vein
• Greatest accumulation occurs in the small intestine and secondly in
stomach, colon, kidney and liver
• This suggest that tumour originated from these sites could be the
first target for orally administered metformin
Metformin
-------------------
DOSE
Addition of other activators with different mechanisms may circumvent
the limitation low doses
• E.g.: salicylate binds to a site different from AMP and directly
activates AMPK
• This suggest combined use of metformin and salicylate may
generate synergistic effect which complement the limitation of
Metformin
Solution----------------
Current Development
---------------------------
• Significant contradiction exist among available data
• Longitudinal studies needed to identify treatment efficiency on
different types of cancer
• Clinical trial carried out aiming to determine effect of metformin in
prevention or prognosis of human cancers

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