List of Thesis Advisors & Research Projects

Project Title 1:
Targeting Cysteine Metabolism in Ovarian Clear Cell Carcinoma

Research Theme:
Tissue Specific Carcinogenesis

Name of Thesis Advisor(s)/ Department:
Main thesis advisor: A/Prof Deng Lih Wen (Dept of Biochemistry)
Co-thesis advisor: A/P Tam Wai Leong (CSI)

Brief Description:
Cisplatin is the key chemotherapeutic drug in the clinics for the treatment of patients with various cancers, and >50% of cancer patients are treated with cisplatin. However, drug resistance is a major clinical setback and underscores the urgent need for novel approaches to overcome cisplatin resistance. Intracellular cisplatin accumulation is one of the main modes of cisplatin-induced toxicity, and resistant cells have adapted their cellular pathways to negate this. Increased efflux of cisplatin by conjugation with glutathione (GSH) and extrusion of this platinum-GSH conjugate by cellular transporters is a major mode for the acquisition of cisplatin resistance. Cysteine is a rate-limiting precursor for intracellular GSH synthesis, which is vital for compound detoxification and maintaining redox balance. Our pilot study supports the notion that depletion of the extracellular cysteine/cystine pool can re-sensitize cisplatin-resistant cells to the cytotoxic effects of cisplatin. We aim to study the utility of engineered cysteine-degrading enzymes as a mode to systematically deplete extracellular cysteine for therapeutic applications as a cisplatin sensitizer, and to investigate the potential applicability of using novel delivery vehicle to deliver the engineered enzymes to tumour sites for cysteine deprivation locally using in vitro cellular and 3D organoid models as well as in vivo xenograft mouse models. Ovarian cancer model is used in this proof-of-concept study as ovarian carcinoma is the most lethal gynecological cancer with a majority of patients eventually becoming platinum-resistant with subsequent relapses. The project is part of a multidiscipline research program with basic scientists, translational researchers and clinical scientists.

Project Title 2:
Identify molecular signatures of early prediction of therapy resistance and recurrence to develop surveillance strategies for personalized treatment

Research Theme:
Tissue Specific Carcinogenesis

Name of Supervisor(s)/ Department:
Main thesis advisor: A/Prof Deng Lih Wen (Dept of Biochemistry)
Co-thesis advisor: Dr Cheong Jit Kong (Dept of Biochemistry)

Brief Description:
Current treatment for cervical cancer and head and neck cancer includes concurrent chemo-radiotherapy followed by brachytherapy. However, treatment failure characterized by recurrent/metastatic tumours is not uncommon. These tumours are often aggressive and resistant to current therapeutic strategies leaving these patients with little therapeutic options. In a pilot study, 43 first-biopsy samples from patients who were either disease-free (n=23) or recurrent/metastatic (n=20) after standard treatment, were subjected to an unbiased molecular screen and a recurrent/metastatic specific molecular signature was identified. Several candidates in this signature have been suggested to have roles in maintaining the cancer stem cell (CSC) phenotype, which has been associated with disease recurrence/metastasis. However, their specific molecular functions remain to be elucidated and understanding how this signature affects the CSC population will enable the development of molecularly-targeted therapies for this subset of cells. Identification of our molecular signature from samples obtained at initial diagnosis suggests the exciting possibility of using this signature to predict possible relapse/metastatic disease at point of diagnosis. Together with molecular studies, the candidate is expected to apply this molecular signature to develop a liquid biopsy-based non-invasive molecular surveillance strategy. The long-term goal of this work aims to enable clinicians to predict recurrence/metastasis at initial diagnosis to better-tailor therapeutic regimes and prescribe them in a timelier manner to improve patient outcome. The project is part of a multidiscipline research program with basic scientists, translational researchers and clinical scientists.

Project Title 3:
Investigate the impact of electroacupuncture on tumor growth and tumor microenvironment

Research Theme:
Tissue Specific Carcinogenesis

Name of Thesis Advisor(s)/ Department:
Main thesis advisor: A/Prof Deng Lih Wen (Dept of Biochemistry)

Brief Description:
Acupuncture, an ancient Chinese therapeutic strategy, is widely used in the clinical treatment of various acute and chronic diseases, including cancer. Recent studies suggest that electroacupuncture, an alternative form of acupuncture, can inhibit or delay tumor growth in breast cancer and osteosarcoma in mice. It also affects the distribution of paclitaxel in lung cancer mice, promoting its accumulation by modifying the tumor microvasculature and microenvironment. Emerging evidence suggests that electroacupuncture has the ability to regulate tumor immunoactivity by increasing NK cell activity and peripheral IFN-γ levels, leading to reduced tumor sizes in patients with cervical squamous carcinoma. However, the mechanism underlying NK cell activation by electroacupuncture remains unclear. Some studies suggest that electroacupuncture induces beta-endorphin release, a possible mediator of NK cell induction, although this theory has been challenged by the suppressive effects of morphine administration on NK cell activity. Additionally, the endocannabinoid receptor can activate NK cell activity. Previous research has shown that electroacupuncture can activate the endocannabinoid system, and further exploration of its role in electroacupuncture-induced NK cell activity is warranted. The effects of electroacupuncture on cytotoxic CD8+ T cell activation, which play a crucial role in cancer immunotherapy, remain undefined. Therefore, this study aims to evaluate the impact of electroacupuncture on tumor immunogenicity using the murine ID8 ovarian cancer cell model. The study will focus on examining the effect of electroacupuncture on immune cell recruitment in ovarian cancer growth, studying differential gene expression crucial for tumor reduction upon electroacupuncture, and characterizing the possible roles of catecholamines in electroacupuncture-induced tumor regression.

Project Title:
The Immunomodulatory Role of Magnetically Induced Muscle Secretome in Cancer

Research Theme:
Precision Cancer Medicine

Name of Thesis Advisor(s)/ Department:
Main Thesis Advisor: A/Prof Alfredo Franco-Obregon (Dept of Surgery)
Co-Thesis Advisor: Dr Tai Yee Kit, Alex (Dept of Surgery), A/Prof Lim Hsiu Kim, Lina (Dept of Physiology)

Brief Description:

Skeletal muscle has emerged as a major secretory organ that produces various factors that are transported in the extracellular bodily fluids [1-3]. The muscle secretome is commonly activated by physical exercise; more precisely the activation of mitochondrial respiration and downstream mitohormetic adaptation [4-6]. These factors are secreted either in soluble form or encapsulated within extracellular vesicles (EVs) that could attune neighboring or distal recipient cells, functionally modulating physiological and pathological phenotypes such as muscle conditioning, adiposity, and tumorigenesis [7, 8]. Given the importance of physical activity that impinges on the benefits of circulating myokines including EVs, it is no wonder that exercise is now prescribed as medicine for the management of metabolic diseased states including cancer [1, 9]. Indeed, exercise has been associated with improved physical functioning of cancer patients, and consequently better treatment outcomes [10]. The importance of exercise in cancer management is supported by the role of secreted myokines and EVs during physical activity which show promising anticancer properties. These anticancer myokines include irisin, myostatin, decorin, and IL-6 [11, 12]. EVs from activated muscles also contain miRNA, lipids, and small peptides that may have a modulatory role in cancer [7].

Yet, the prescription of exercise to cancer patients presents many challenges due to factors such as the severity of the disease, the physical condition of the patient before and during cancer treatment, and the intensity of the cancer treatment [13]. Significant physical fatigue may arise from cancer treatment such as chemotherapy, radiation therapy, or surgery; making it difficult for patients to engage in physical activity [14]. Moreover, these treatments may result in physical impairments and treatment-related side effects such as neuropathy, muscular pains, joint stiffness, nausea, and a weakened immune system. Consequently, these side effects may be challenging for cancer patients to initiate, adhere to, and sustain regular exercise.

The unmet need is therefore an exercise mimetic modality that could activate the skeletal muscle secretome, which possesses anticancer properties, without the need for physical exertion or pharmacological intervention. Our laboratory has developed a patented pulsed electromagnetic field (PEMF) signature that can invoke the secretome responses from muscle cells. The single brief exposure of muscle cells to these non-invasive low-energy magnetic fields efficiently replicates the beneficial pathways like exercise, in particular, eliciting the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) pathway that plays a crucial role in mitochondrial biogenesis, improves oxidative capacity and efficient energy utilization of cells [4-6]. Moreover, the activation of muscular PGC-1α promotes the mobilization of the muscle secretome [15], and like exercise, PEMF is also effective to elicit muscle secretome responses [4, 5].

 Proposed study:

We have found that PEMF-induced muscle secretome in vitro, also known as PEMF-conditioned media (pCM), inhibits the growth, migration, and invasiveness of breast cancer cells. We also observed a pCM-associated reduction in tumor volume and vascularity in breast cancer (MCF-7) microtumors implanted onto the chorioallantoic membrane model (CAM). Serum from PEMF-exposed (10 min/wk) mice also inhibited the invasiveness and migration of breast cancer cells more than sera from exercised (25 min/twice weekly) mice for the same period of intervention (8 weeks). These results suggest that pCM or sera from PEMF-exposed mice exhibit robust anticancer effects on breast cancer cells. In this proposal, we want to explore the impact of magnetically-induced muscle secretome on the tumor microenvironment (TME). The TME plays a crucial role in the development and progression of cancer, including the overall responses to cancer treatment. Besides cancer cells, the TME is infiltrated with various immune cells such as tumor-infiltrating lymphocytes (TILs), macrophages, and dendritic cells which are under the influence of secretions from cancer cells [16]. Cancer cells often take advantage of the phenotypic plasticity of immune cells by hijacking their cellular pathways to promote tumor growth. Among the major subtypes of immune cells, tumor-associated macrophages (TAMs) and regulatory T cells (T_Regs) cells are two major pro-tumoral contributors. TAMs and T_Regs contribute to multiple facets of cancer progression including proliferation, invasion, and metastasis [17, 18].  To counter this, promoting the recruitment and infiltration of anti-tumoral immune cells in the TME is an attractive treatment strategy in cancer. Studies have shown that exercise promotes the infiltration of anti-tumoral immune cells within the TME while concurrently reducing the infiltration of pro-tumoral immune cells [19, 20]. Exercise-stimulated muscle secretions of cytokines and chemokines like IL-15, CXCL9 and CXCXL11 can have anti-tumoral effects through the recruitment of CD8+ T-cells [21, 22]. Exercise also promotes the infiltration of natural killer (NK) cells while reducing the infiltration of TAMs and T_Regs [19, 23]. Moreover, muscle PGC-1α activation, commonly induced by exercise, has been associated with the production and secretion of B-type natriuretic peptide (BNP) that activates tissue-resident macrophages [24]. Given the profound interaction between the muscle secretome and the TME, we propose to study the (1) modulatory role of magnetically-induced muscle secretome on immune cells and (2) to investigate how muscle PEMF exposure can attenuate breast cancer progression in transgenic mice of spontaneous mammary tumors.

We hypothesize that magnetically-induced muscle secretome can modulate the responses of immune cells and ultimately create an anti-tumoral environment to halt cancer growth. We aim to characterize the phenotypes and key factors secreted by immune cells (macrophages and T-cells) in vitro after the provision of magnetically-induced muscle secretome. We will also utilize the MMTV-PyMT transgenic mouse model that recapitulates the multiple stages of breast cancer progression in humans. The expression of tumors of the PyMT model is highly invasive with a high frequency of pulmonary metastasis [25] which is well suited as a cancer model for our study. Moreover, exercise intervention has been shown to reduce the development of early breast cancer in the PyMT model [25, 26] and is likely mediated by exercise-mediated muscle secretion. The efficacy of PEMF therapy (10 min/wk) to delay cancer development and metastasis will be compared to exercise (50 min/wk) using this model. Anticancer synergism between PEMF and chemotherapy will be studied in the PyMT model. Finally, magnetically-induced muscle secretome and blood plasma from the PyMT mice will be analyzed by microarray and ELISA analyses to monitor changes in inflammatory cytokines and chemokines. Isolated muscle secretome-derived exosomes and blood plasma will also be analyzed using a miRNA cancer panel to unravel potential miRNAs important for the modulation of the immune system. Our proposal will seek to establish the utility of muscle PEMF therapy to modulate the responses of immune cells to combat cancer and provide an understanding of how to best exploit the immunomodulatory potential of magnetic fields as an adjuvant to chemotherapeutics.

References
1. Chow LS, Gerszten RE, Taylor JM, Pedersen BK, van Praag H, Trappe S, Febbraio MA, Galis ZS, Gao Y, Haus JM et al: Exerkines in health, resilience and disease. Nat Rev Endocrinol 2022, 18(5):273-289.
2. Severinsen MCK, Pedersen BK: Muscle-Organ Crosstalk: The Emerging Roles of Myokines. Endocr Rev 2020, 41(4).
3. Louzada RA, Bouviere J, Matta LP, Werneck-de-Castro JP, Dupuy C, Carvalho DP, Fortunato RS: Redox Signaling in Widespread Health Benefits of Exercise. Antioxid Redox Signal 2020.
4. Wong CJK, Tai YK, Yap JLY, Fong CHH, Loo LSW, Kukumberg M, Frohlich J, Zhang S, Li JZ, Wang JW et al: Brief exposure to directionally-specific pulsed electromagnetic fields stimulates extracellular vesicle release and is antagonized by streptomycin: A potential regenerative medicine and food industry paradigm. Biomaterials 2022, 287:121658.
5. Tai YK, Ng C, Purnamawati K, Yap JLY, Yin JN, Wong C, Patel BK, Soong PL, Pelczar P, Frohlich J et al: Magnetic fields modulate metabolism and gut microbiome in correlation with Pgc-1alpha expression: Follow-up to an in vitro magnetic mitohormetic study. FASEB J 2020, 34(8):11143-11167.
6. Yap JLY, Tai YK, Frohlich J, Fong CHH, Yin JN, Foo ZL, Ramanan S, Beyer C, Toh SJ, Casarosa M et al: Ambient and supplemental magnetic fields promote myogenesis via a TRPC1-mitochondrial axis: evidence of a magnetic mitohormetic mechanism. FASEB J 2019, 33(11):12853-12872.
7. Papadopetraki A, Maridaki M, Zagouri F, Dimopoulos MA, Koutsilieris M, Philippou A: Physical Exercise Restrains Cancer Progression through Muscle-Derived Factors. Cancers (Basel) 2022, 14(8).
8. Darragh IAJ, O’Driscoll L, Egan B: Exercise Training and Circulating Small Extracellular Vesicles: Appraisal of Methodological Approaches and Current Knowledge. Front Physiol 2021, 12:738333.
9. Sabaratnam R, Wojtaszewski JFP, Hojlund K: Factors mediating exercise-induced organ crosstalk. Acta Physiol (Oxf) 2022, 234(2):e13766.
10. Park JH, Lee DH, Kim SI, Kim NK, Jeon JY: Moderate to vigorous physical activity participation associated with better quality of life among breast and colorectal cancer survivors in Korea. BMC Cancer 2020, 20(1):365.
11. Karstoft K, Pedersen BK: Skeletal muscle as a gene regulatory endocrine organ. Curr Opin Clin Nutr Metab Care 2016, 19(4):270-275.
12. Turkel I, Ozerklig B, Atakan MM, Aktitiz S, Kosar SN, Yazgan B: Exercise and Metabolic Health: The Emerging Roles of Novel Exerkines. Curr Protein Pept Sci 2022, 23(7):437-455.
13. Andersen HH, Mikkelsen MK, Obarzanek CE, Paludan C, Nielsen D: Reasons for declining participation in an exercise-based trial among older women with breast cancer receiving systemic anti-cancer treatment – a qualitative interview study. Physiother Theory Pract 2023:1-11.
14. Dean A: The holistic management of fatigue within palliative care. Int J Palliat Nurs 2019, 25(8):368-376.
15. Zunner BEM, Wachsmuth NB, Eckstein ML, Scherl L, Schierbauer JR, Haupt S, Stumpf C, Reusch L, Moser O: Myokines and Resistance Training: A Narrative Review. Int J Mol Sci 2022, 23(7).
16. Malfitano AM, Pisanti S, Napolitano F, Di Somma S, Martinelli R, Portella G: Tumor-Associated Macrophage Status in Cancer Treatment. Cancers (Basel) 2020, 12(7).
17. Kazakova A, Sudarskikh T, Kovalev O, Kzhyshkowska J, Larionova I: Interaction of tumor‑associated macrophages with stromal and immune components in solid tumors: Research progress (Review). Int J Oncol 2023, 62(2).
18. Li YR, Wilson M, Yang L: Target tumor microenvironment by innate T cells. Front Immunol 2022, 13:999549.
19. Esteves M, Silva C, Bovolini A, Pereira SS, Morais T, Moreira A, Costa MM, Monteiro MP, Duarte JA: Regular Voluntary Running is Associated with Increased Tumor Vascularization and Immune Cell Infiltration and Decreased Tumor Growth in Mice. Int J Sports Med 2023.
20. Djurhuus SS, Simonsen C, Toft BG, Thomsen SN, Wielsoe S, Roder MA, Hasselager T, Ostergren PB, Jakobsen H, Pedersen BK et al: Exercise training to increase tumour natural killer-cell infiltration in men with localised prostate cancer: a randomised controlled trial. BJU Int 2023, 131(1):116-124.
21. Batatinha H, Diak DM, Niemiro GM, Baker FL, Smith KA, Zuniga TM, Mylabathula PL, Seckeler MD, Lau B, LaVoy EC et al: Human lymphocytes mobilized with exercise have an anti-tumor transcriptomic profile and exert enhanced graft-versus-leukemia effects in xenogeneic mice. Front Immunol 2023, 14:1067369.
22. Kurz E, Hirsch CA, Dalton T, Shadaloey SA, Khodadadi-Jamayran A, Miller G, Pareek S, Rajaei H, Mohindroo C, Baydogan S et al: Exercise-induced engagement of the IL-15/IL-15Ralpha axis promotes anti-tumor immunity in pancreatic cancer. Cancer Cell 2022, 40(7):720-737 e725.
23. Gomes-Santos IL, Amoozgar Z, Kumar AS, Ho WW, Roh K, Talele NP, Curtis H, Kawaguchi K, Jain RK, Fukumura D: Exercise Training Improves Tumor Control by Increasing CD8(+) T-cell Infiltration via CXCR3 Signaling and Sensitizes Breast Cancer to Immune Checkpoint Blockade. Cancer Immunol Res 2021, 9(7):765-778.
24. Furrer R, Eisele PS, Schmidt A, Beer M, Handschin C: Paracrine cross-talk between skeletal muscle and macrophages in exercise by PGC-1alpha-controlled BNP. Sci Rep 2017, 7:40789.
25. Goh J, Tsai J, Bammler TK, Farin FM, Endicott E, Ladiges WC: Exercise training in transgenic mice is associated with attenuation of early breast cancer growth in a dose-dependent manner. PLoS One 2013, 8(11):e80123.
26. Mader T, Chaillou T, Alves ES, Jude B, Cheng AJ, Kenne E, Mijwel S, Kurzejamska E, Vincent CT, Rundqvist H et al: Exercise reduces intramuscular stress and counteracts muscle weakness in mice with breast cancer. J Cachexia Sarcopenia Muscle 2022, 13(2):1151-1163.

Project Title:
Single centre prospective evaluation of 68Gallium-FAPI PET/MRI in Hepatocellular Carcinoma

Research Theme:
Precision Cancer Medicine

Name of Thesis Advisor(s)/ Department:
Main Thesis Advisor: Prof Khong Pek-Lan (Dept of Diagnostic Radiology)
Co-Thesis Advisor: Dr Chee Cheng Ean (Dept of Medicine)

Brief Description:

To evaluate the clinical utility of a novel radiotracer, 68Ga-FAPI (Fibroblast activation protein-inhibitor) for PET imaging of Hepatocellular carcinoma (HCC); specifically, in its additional benefit to MRI alone, which is conventionally used as the imaging modality of choice for HCC, and as a combined hybrid imaging modality of PET/MRI.

We aim to study the added value of 68Ga-FAPI PET, combining it with MRI in a single hybrid modality, PET/MRI, in the context of the current diagnostic pathway in patients with or suspected to have HCC. Specifically, we aim to evaluate its role in (i) lesion detection and characterisation prior to surgery (ii) systemic staging after radiological or histological confirmation of HCC (iii) characterization of indeterminate lesions on conventional MRI and (iv) work-up for the detection of HCC in patients with unexplained elevations of serum alpha-fetoprotein (AFP) concentration.

Project Title: 
LYN Kinase in Cytoskeletal Rearrangements and its Effect on Tumor Immune Escape in Breast Cancer

Research Theme: 
Precision Cancer Medicine

Name of Thesis Adivisor(s)/Department:
Main thesis advisor: Dr Alan Prem Kumar (Dept of Pharmacology)
Co-thesis advisor: A/Prof Celestial Yap (Department of Physiology)

Brief Description:

Emerging evidence indicates that cytoskeletal alterations in breast cancer cells derived from aggressive tumour subtypes render them resistant to killing by cytotoxic immune cells found in the tumour microenvironment. Moreover, these breast cancer cells commonly upregulate expression of the SRC-family kinase, LYN, an important signalling intermediary modulating proliferation, invasion, migration, and immune regulation. Recent studies have shown that LYN kinase promotes formation of actin-rich structures associated with increased migration and facilitates epithelial-to-mesenchymal transition in response to increased stiffness of the extracellular tumour microenvironment. However, mechanisms by which LYN kinase promotes cancer cell cytoskeletal rearrangements and its roles in immune evasion are not explored.

The aim of this project is to examine the mechanisms by which LYN kinase signalling influences alterations in cell shape, cytoskeletal architecture and interactions with cytotoxic immune cells. Analysis will be carried out by assessing the composition of actin fibres, cell-extracellular matrix adhesions, actin-rich migratory structures as well as cytotoxic properties of the relevant immune cells using immunofluorescent imaging, time-lapse microscopy, FACS and LYN-specific inhibition or genetic knockdown. Findings from these studies will form the basis for development of novel therapeutic modalities for difficult to treat aggressive breast cancer.

Project Title 1:
Investigating DDIT3 proteoform-specific role in regulating the mesenchymal state of GBM stem cells

Research Theme:
Tissue Specific Carcinogenesis

Name of Thesis Advisor(s)/Department
Main Thesis Advisor: Dr Derrick Ong Sek Tong (Dept of Physiology)

Brief Description:

Glioblastoma (GBM) is the most common and malignant form of adult astrocytoma, with no effective treatment to date. The consensus in GBM research is that a subpopulation of tumor cells, so called glioma stem cells, drive GBM pathogenesis and recurrence. Recent single cell transcriptomic analysis of primary and recurrent GBM tissues has revealed a proneural to mesenchymal state shift upon tumor recurrence, which is linked to the AP-1 transcription factor, potentially explaining for the increased invasiveness of GBM cells upon tumor recurrence. Notably, ongoing work in our lab has identified DDIT3 as another mesenchymal state-associated factor, and DDIT3 and AP-1 belong to the same family of C/EBP transcription factors, supporting the idea that DDIT3 may also play a crucial role in maintaining the GBM mesenchymal state. More interestingly, members of the C/EBP family can generate protein isoforms due to differential initiation of translation (so called proteoforms), and these proteoforms have different function in gene regulation and cell state. This project explores the exciting idea that the DDIT3 proteoforms may exert different function in gene regulation and cell state of glioma stem cells.

Specific Aims

(1) Determine the expression of DDIT3 proteoforms in patient-derived glioma stem cells.

There are two DDIT3 protein isoforms that are encoded by the same RNA transcript as indicated in Uniprot, but whether the DDIT3 proteoforms are expressed remains unknown as most DDIT3 studies have only focused on the shorter gene product. Our preliminary data show that two DDIT3 bands can be detected by using SDS-PAGE analysis of GBM cell lysates. In this aim, we propose to examine the expression of DDIT3 proteoforms more broadly by using a panel of mesenchymal and proneural state-associated glioma stem cells. Next, we will address if the two DDIT3 protein bands are indeed proteoforms or the same protein with different post-translational modification (e.g. phosphorylation). Last, we will use CRISPR editing tools to alter the translation initiation sites to assess if the DDIT3 proteoforms may be generated by alternative translational initiation.

(2) Investigate the role of the DDIT3 proteoforms in regulating GBM proliferation, mesenchymal state and tumorigenicity.

Our preliminary data indicate a differential role of the DDIT3 proteoforms in regulating glioma stem cell proliferation and invasiveness. In this aim, we will create glioma stem cells that harbour CAS9, an inducible DDIT3 sgRNA (to elicit inducible gene knockout), along with the genetic complementation of short or long DDIT3 proteoforms. At least two patient-derived glioma stem cells of the mesenchymal subtype and a IKK-constitutive active overexpressing glioma stem cell line will be used for the abovementioned endeavour. The resulting cells will be employed for a variety of in vitro assays to evaluate glioma stem cell proliferation, stemness and invasiveness, as well as tumorigenicity in an intracranial GBM model using NSG mice. (NB: IKK-constitutive active overexpression activates the NFkB pathway that transforms proneural state-associated glioma stem cells to the mesenchymal state. This allows us to have a more homogenous system to study the mesenchymal state as compared to patient-derived mesenchymal subtype glioma stem cells which may be heterogeneous.)

(3) Elucidate the mechanisms through which the DDIT3 proteoforms exert their effects on glioma stem cells.

To investigate the underlying mechanisms of the DDIT3 proteoforms, we will employ a multi-omic approach, including (i) proximity-labeling based mass spectrometry to identify DDIT3 proteoform-specific protein interactors; (ii) DDIT3 proteoform-specific ChIP-Seq analyses to examine their genome-wide occupancy; (ii) DDIT3 proteoform-specific transcriptomic regulation to identify genes/ pathways that may be differentially regulated by the DDIT3 proteoforms. The integration of these multi-layered datasets would allow us to characterize the transcriptional changes induced by the DDIT3 proteoforms, providing insights into their differential gene regulatory roles and function in glioma stem cells.

To address if our mechanistic insights hold relevance to glioma patients, we will perform correlative analyses of the identified DDIT3 proteoform-specific genes/ pathways with glioma grade and survival, as well as between primary versus recurrent tumors using publicly available glioma datasets, including TCGA, NCI REMBRANDT, Gravendeel and CGGA. We will complement such RNA-based analysis with protein analysis through immunohistochemical analysis of glioma tumor microarrays.

 References

1          Yoon et al. A chemical biology approach reveals a dependency of glioblastoma on biotin distribution. Sci Adv. 7(36):eabf6033. doi: 10.1126/sciadv.abf6033 (2021).

2          Yoon et al. E2F and STAT3 provide transcriptional synergy for histone variant H2AZ activation to sustain glioblastoma chromatin accessibility and tumorigenicity. Cell Death Differ. 29(7):1379-1394. doi: 10.1038/s41418-021-00926-5 (2022). 

Project Title 2:
Developing a HDAC7 targeting PROTAC degrader as an anti-cancer therapy

Research Theme:
Tissue Specific Carcinogenesis

Name of Thesis Advisor(s)/Department
Main Thesis Advisor: Dr Derrick Ong Sek Tong (Dept of Physiology)

Brief Description:

Pan-histone deacetylase (HDAC) inhibitors effectively suppress tumor growth in pre-clinical models, but show limited clinical utility due to their overt toxicity. Thus, the ability to selectively target a given HDAC isoform, which has strong clinical relevance in carcinogenesis, may allow the clinical translation of HDAC-centric therapeutic agents. In this proposal, we focus on developing HDAC7 targeting PROTAC degrader to deplete HDAC7, which has a well-established pro-tumorigenic role in diverse cancer types, including glioblastoma (GBM), breast and ovarian cancers. The rationale to degrade HDAC7 protein, instead of inhibiting it, stems from the lack of mechanistic clarity on how HDAC7 promotes tumorigenesis, as well as the advantage PROTACs offer in being capable of degrading its target along with the associated proteins (hence eliminating both canonical and non-canonical functions of the target). We propose to uncover compounds that bind to HDAC7 (i.e. binders) by using a two-step in silico small molecule screen. Following, the top five candidate binders will be synthesized, and the binding affinity to HDAC7 and anti-GBM activity of the binders will be evaluated. The top two HDAC7 binders will be further assessed for their binding specificity to HDAC7 by performing streptavidin pulldown-mass spectrometry analysis of GSC lysates that were treated with the biotin-modified binders. The best binder will be used for the development of the HDAC7 targeting PROTAC degrader, which will be used for the evaluation of their anti-GSC properties via a variety of in vitro assays, followed by mechanistic investigation of how the HDAC7 targeting PROTAC degrader acts to elicit its anti-GSC activity. We envisage that the HDAC7 targeting PROTAC degrader will have wide clinical utility in cancer treatment.

References

1. Yoon et al. A chemical biology approach reveals a dependency of glioblastoma on biotin distribution. Sci Adv. 7(36):eabf6033. doi: 10.1126/sciadv.abf6033 (2021).
2. Ang et al. Deciphering nanoparticle trafficking into glioblastomas uncovers an augmented antitumor effect of metronomic chemotherapy. Advanced Materials, 2022, 34(3):e2106194.
3. Yoon et al. E2F and STAT3 provide transcriptional synergy for histone variant H2AZ activation to sustain glioblastoma chromatin accessibility and tumorigenicity. Cell Death Differ. 29(7):1379-1394. doi: 10.1038/s41418-021-00926-5 (2022). 
4. Chuah et al. CAMK2D serves as a molecular scaffold for RNF8-MAD2 complex to induce mitotic checkpoint in glioma. Cell Death Diff, 2023. 30(8):1973-1987. 

Project Title:
Cytoskeletal Rearrangements and its Effect on Tumour Immune Escape in Ovarian Cancer

Research Theme:
Tissue-Specific Carcinogenesis

Name of Thesis Advisor(s)/ Department:
Main Thesis Advisor: A/Prof Celestial Yap (Dept of Physiology)
Co-Thesis Advisor: Dr Alan Prem Kumar (Dept of Pharmacology)

Brief Description:

Ovarian cancers account for a significant proportion of gynaecological cancers worldwide, with a majority of patients presenting at advanced stages. Although initial response to treatment may be promising, relapse rates are Ovarian cancers account for a significant proportion of gynaecological cancers worldwide, with a majority of patients presenting at advanced stages. Although initial response to treatment may be promising, relapse rates are high and 5-year survival is often poor.

Migration and invasion of cancer cells constitute fundamental processes in tumour progression and metastasis and are governed by alterations of various families of cytoskeletal proteins, such as microtubules, actin, and intermediate filaments. These cytoskeletal proteins act in a coordinated manner and mediate numerous cellular and biochemical processes that influence cell shape, structure, division, motility as well as interactions of cells with their extracellular environment. Emerging evidence indicates that cytoskeletal alterations in cancer cells derived from aggressive tumour subtypes render them resistant to killing by cytotoxic immune cells found in the tumour microenvironment. Recent studies have shown that actin-rich structures are associated with increased migration and facilitates epithelial-to-mesenchymal transition in response to increased stiffness of the extracellular tumour microenvironment. However, mechanisms by which cancer cell cytoskeletal rearrangements and its roles in immune evasion are not fully understood.

The aim of this project is to examine the cytoskeletal mechanisms influencing ovarian tumour cell behaviour, such as alterations in cell shape, cytoskeletal architecture and how these affect the interactions with the cells in the tumour microenvironment, such as immune cells. Analysis will be carried out by assessing the composition of actin fibres, cell-extracellular matrix adhesions, actin-rich migratory structures as well as cytotoxic properties of the relevant immune cells using immunofluorescent imaging, time-lapse microscopy, FACS, modulation of genetic expression of key cytoskeletal proteins, including gelsolin and other actin-binding proteins.

Our group has previously established the impact of the cytoskeleton on the reorganisation of the extracellular matrix, invasion and oncogenic signalling.

The project will examine the cytoskeletal mechanisms that contribute to the aggressive behaviour of ovarian cancer. These include the cytoskeletal alterations underlying the interactions between cancer cells and the tumour microenvironment, including immune evasion by cancer cells.

Collaborations for this project include working with clinical oncology and immunology. Findings from these studies will form the basis for development of novel therapeutic modalities for difficult to treat aggressive ovarian cancer.