https://ptsldigital.ukm.my/jspui/handle/123456789/388951 2026-08-03T13:56:29Z https://ptsldigital.ukm.my/jspui/handle/123456789/783851 Title: Targeting tousled-like kinase 1 signalling pathways in glioblastoma multiforme therapy Authors: Kamariah Ibrahim Abstract: Glioblastoma multiforme (GBM) is an aggressive and highly invasive brain tumour that commonly exhibits resistance to conventional chemotherapy and radiotherapy. It is a heterogeneous disease involving complex kinomic alterations causing aberrant dysregulation of signalling pathways. Although many specific kinase inhibitors have been developed, only 30% of patients’ survived due to extreme proliferation, invasion and highly vascularized nature of GBM cells. The aim of this study was to explore novel kinome pathways and to identify potential therapeutic targets for GBM. Meta analysis was performed by filtering significant genes having p-value <0.05 from the Oncomine database using a total of 415 glioblastoma versus normal samples. This analysis identified 113 significantly upregulated kinases. RNAi screening on identified 113 kinases was performed subsequently, using Dharmacon Custom siRNA SMARTpoolTM library on LN18 and U87MG cells. k-Median Absolute Deviation statistical analysis has identified Tousled-like kinase 1 (TLK1) as a potential molecular target. TLK1, a serine-threonine kinase, was selected for an in vitro functional validation as its role in GBM is still unknown. Silencing of TLK1 using 25nM siRNA and shRNA in GBM cells resulted in a significant reduction in cell viability, clonogenicity and proliferation by inducing S-phase cell cycle arrest. In addition, inhibition of TLK1 induced apoptosis signals in GBM cells. Silencing of TLK1 also chemosensitised GBM cells towards sub-lethal dose of temozolomide (250µM). Invasion and migration of GBM cells were also inhibited (p<0.05). Interestingly, in normal human astrocytes, silencing of TLK1 did not cause significant changes in cell viability, apoptosis, invasion as well as migration. The downstream pathways of TLK1 in U87MG cells were interrogated using Illumina HumanHT12-12 v4 BeadChip microarray. WebGestalt pathways analysis on 2,632 significant probes with more than 1.1 fold change difference identified key signalling pathways involved in GBM cancer pathways including DNA replication, cell cycle, focal adhesion, TGF beta, and integrin-mediated cell adhesion signalling pathways. Gene expression analysis using qPCR confirmed the differential expressions of Thrombospondin-2, Paxillin, Ras-related C3 botulinum toxin substrate 2 (RAC2), Collagen type IV α2, FYN proto-oncogene Src family tyrosine kinase and Rho associated coiled-coil containing protein kinase 2 which were involved in the focal adhesion pathway. TLK1 was postulated to regulate invasion and migration of GBM cells through signalling with a small GTP binding protein, RAC2. RAC2 signal was significantly suppressed in both TLK1 knockdown U87MG and LN18 cells using RAC2 activation assay. TLK1 knockdown GBM cells also significantly reduced phosphorylated p70S6 kinase expression which acted downstream of the PI3K/AKT/mTOR signalling pathway. In vivo subcutaneous GBM xenograft of stably transfected U87MG cells with sh-TLK1 in balb/c female nude mice showed significant difference (p<0.05) in tumour growth potential. Subsequent in silico homology modelling of TLK1 and high throughput virtual screening of protein-ligand docking identified two potential compounds that bound to catalytic site of TLK1 modelled protein. These findings suggest that TLK1 and its related pathways are potential molecular targets for GBM therapy. 2017-03-14T00:00:00Z https://ptsldigital.ukm.my/jspui/handle/123456789/783042 Title: Pengenalpastian dan Pencirian Fungsi Varian Genetik Yang Dikaitkan Dengan Rintangan Terhadap Terapi Berasaskan Platinum Dalam Kanser Paru-Paru Bukan Sel Kecil Authors: Hilary Sito Pei-Ching Abstract: Kemoterapi berasaskan platinum (PBC) merupakan rawatan barisan pertama untuk sebahagian besar pesakit kanser paru-paru bukan sel kecil (NSCLC). Namun, hanya 30- 40% pesakit menunjukkan tindak balas berkesan terhadap rawatan ini. Varian genetik telah dikaitkan dengan modulasi tindak balas terhadap PBC, namun penemuan antara kajian adalah tidak konsisten dan terdapat kekurangan pemahaman secara fungsian dan mekanistik. Kajian ini bertujuan untuk mengenal pasti dan mencirikan secara fungsian varian genetik yang dikaitkan dengan rintangan terhadap PBC. Satu analisis meta yang melibatkan 121 kajian dengan 29,478 pesakit NSCLC telah dijalankan untuk menilai hubungan antara 184 varian genetik dengan tindak balas terhadap PBC. Nisbah ganjil terkumpul telah dikira menggunakan lima model genetik, dan kekuatan bukti epidemiologi telah dinilai menggunakan kriteria Venice. Varian yang menunjukkan hubungan signifikan serta kekuatan epidemiologi sederhana atau kuat telah dianalisis menggunakan 33 alat bioinformatik untuk meramal impak kepada fungsi. Varian yang paling memudaratkan telah dipilih untuk pengesahan hiliran dalam sel selanjar NSCLC (NCI-H1299 dan PC9) yang dirawat dengan cisplatin, carboplatin, dan oxaliplatin. Konstruk jenis liar dan varian bagi gen terpilih telah ditransfeksi ke dalam sel selanjar, dan ujian fungsian telah dijalankan untuk menilai ekspresi gen dan protein, kerintangan terhadap ubat platinum, apoptosis, kitaran sel, migrasi, kestabilan protein, dan aktiviti protein hiliran. Daripada analisis meta, 12 varian merentasi 10 gen telah menunjukkan perkaitan yang signifikan. Antaranya, XRCC1 rs1799782 (Arg194Trp) diramalkan mempunyai kesan yang paling memudaratkan. Varian ini meningkatkan ekspresi mRNA dan protein XRCC1. Dalam sel NCI-H1299, varian ini mengurangkan kerintangan terhadap platinum dan meningkatkan perhentian fasa G1 (38% berbanding 33.7%, p<0.001) selepas rawatan carboplatin. Dalam sel PC9, ia mengurangkan apoptosis awal selepas rawatan carboplatin (30.0% berbanding 35.9%, p=0.024) dan oxaliplatin (30.1% berbanding 41.5%, p=0.048), tetapi meningkatkan apoptosis lewat akibat cisplatin (74.9% berbanding 48.6%, p=0.003) dan carboplatin (13.7% berbanding 10.2%, p<0.001). Ia juga meningkatkan penutupan luka dalam sel PC9 selepas rawatan carboplatin (8.1% berbanding 5.5%, p=0.044). Pemblotan Western menunjukkan penurunan PCNA dan peningkatan pemotongan PARP, menandakan kerosakan pada keupayaan pembaikan DNA. Kesimpulannya, XRCC1 rs1799782 mempengaruhi proses selular utama yang berkaitan dengan tindak balas terhadap PBC dalam NSCLC. Hasil ini memberikan pemahaman penting terhadap mekanisme kerintangan terhadap platinum dalam NSCLC. 2026-03-10T00:00:00Z https://ptsldigital.ukm.my/jspui/handle/123456789/782060 Title: Analisis imunogenomik di dalam kanser kolorektal melalui pengenalpastian dan pengesahan kefungsian neoantigen menggunakan kejuruteraan reseptor sel T Authors: Nurul Ainaa' Adilah Rus Bakarurraini Abstract: Kanser kolorektal (CRC) melibatkan kanser usus dan rektum, menyebabkan lebih 500,000 kematian setiap tahun di dunia. Terapi CRC sedia ada kurang berkesan, terutamanya bagi pesakit di tahap lanjut. Oleh itu, penerokaan terapi alternatif bagi pesakit CRC amatlah diperlukan termasuklah terapi imun. Neoantigen, yang berasal daripada mutasi genom pesakit, merupakan antigen yang hanya terdapat di permukaan sel kanser. Dengan menyasarkan neoantigen tertentu, tindak balas sel T boleh diaruhkan untuk membunuh sel kanser. Justeru itu, kajian ini dilakukan untuk mengenalpasti neoantigen dalam pesakit CRC Malaysia dan menilai keberkesanan imunoterapi sel T terhadap neoantigen tersebut. Daripada 50 pesakit CRC, sepuluh pesakit dengan beban mutasi tertinggi dipilih untuk saringan neoantigen. Analisis calon neoantigen, pengekspresan gen, keimunogenan dan kadar keafinan calon terhadap HLA-A*24:02 dilakukan dengan menggunakan pelantar bioinformatik seperti MuPeXi dan sebagainya. Kemudiannya, sel dendritik yang didedahkan dengan calon neoantigen dikultur bersama sel T untuk mengaruh pembentukan reseptor sel T (TCR) spesifik. Jujukan TCR yang dikenalpasti diklonkan ke dalam sel T dari penderma sihat. Asai kefungsian sel T yang direkayasa dilakukan dengan mengukur perembesan sitokin IL2, IFN-γ, grenzim B, dan perforin. Analisis pengenalpastian neoantigen telah mendedahkan 36 calon neoantigen yang berpotensi dengan TMEM97p.R173fs*3 sebagai calon yang paling berpotensi. Pengkulturan sel dendritik dengan sel T menghasilkan 40.4% sel T yang spesifik terhadap TMEM97p.R173fs*3. Keputusan penjujukan TCR mengenalpasti jujukan TCR dominan. Seterusnya, pengklonan TCR menggunakan partikel lentiviral ke dalam sel T penderma sihat menghasilkan 20% populasi sel T dengan gen TCR spesifik terhadap TMEM97p.R173fs*3. Asai kefungsian sel T yang direkayasa menunjukkan peningkatan sitokin IL-2, IFN-γ, grenzim B, dan perforin dalam sampel TMEM97p.R173fs*3 berbanding sampel kawalan (perubahan lipatan > 1.5). Kajian ini membuktikan bahawa sel T yang direkayasa dapat mengenali TMEM97p.R173fs*3 dan menjana tindak balas sitotoksik melalui perembesan sitokin pro-radang. Hal ini membuktikan terapi imun dengan menggunakan kejuruteraan reseptor sel T dari penderma sihat berpotensi menjadi terapi alternatif bagi pesakit CRC. 2025-07-23T00:00:00Z https://ptsldigital.ukm.my/jspui/handle/123456789/781509 Title: Pengkajian sekretom sel kanser payudara sasaran protein AGR2 menggunakan teknologi CRISPR/CAS9 Authors: Syazalina Zahari Abstract: Anterior Gradient-2 (AGR2) is an overexpressed endoplasmic reticulum protein implicated in numerous epithelial-origin cancers, where it plays key roles in promoting tumour growth, metastasis, and cancer drug resistance. Recent studies revealed that AGR2 is secreted into the extracellular microenvironment, where extracellular AGR2 (eAGR2) exhibits pro-oncogenic properties. However, the precise mechanisms through which eAGR2 functions to modulate cancer progression remain poorly understood. Hence, this study aims to elucidate the role of eAGR2 within the cancer secretome—a subset of the proteome comprising extracellular signalling molecules that shape the tumour microenvironment. By expressing AGR2 structural variants, it is revealed that its secretion depends on specific structural elements, including the signal peptide, endoplasmic reticulum retention motif, and dimerization state. Characterization of CRISPR/Cas9-mediated AGR2 knockout (KO) in breast cancer cell lines (MCF-7, T47D, and 1833-BoM) revealed reduction in cell proliferation, migration, and invasion in 2D cultures, along with increased sensitivity to Doxorubicin and Tamoxifen. AGR2 depletion also activated unfolded protein response markers (UPR) IRE1α and ATF6α, underscoring its role in maintaining endoplasmic reticulum proteostasis. In 3D spheroid models, AGR2 KO cells formed significantly smaller and fewer spheroids, highlighting its role in anchorage-independent growth. Secretome analysis via mass spectrometry with data-independent acquisition and neural network (DIA-NN) analysis revealed dynamic shifts in secreted proteins. A total of 1041, 1626, and 1469 proteins were identified in the secretome of MCF-7, T47D, and 1833-BoM cells, respectively, with 40, 401, and 33 proteins differentially expressed upon AGR2 KO. These data reveal distinct, cell line-specific secretome alterations driven by AGR2, underscoring the heterogeneity of its modulation. Protein-protein interaction network analysis identified key AGR2-associated binding partners, with pathways related to cellular growth, metabolism, and immune regulation significantly enriched. Western blot analysis validated the differential expression of PKM, ENO1 and MAPK1 in MCF-7 and T47D, as well as PKM and IL-6 in 1833-BoM secretome. Collectively, this study provides novel insights into the mechanisms of eAGR2 secretion, its modulation of the tumour microenvironment, and its contribution to breast cancer progression. These findings reinforced AGR2 as a critical player in cancer biology and a potential therapeutic target 2025-07-31T00:00:00Z