Lee Yee Choong

Differential Expression of Novel Tyrosine Kinase Substrates during Breast Cancer Development

To identify novel tyrosine kinase substrates that have never been implicated in cancer, we studied the phosphoproteomic changes in the MCF10AT model of breast cancer progression using a combination of phosphotyrosyl affinity enrichment, iTRAQ™ technology, and LC-MS/MS. Using complementary MALDI- and ESI-based mass spectrometry, 57 unique proteins comprising tyrosine kinases, phosphatases, and other signaling proteins were detected to undergo differential phosphorylation during disease progression. Seven of these proteins (SPAG9, Toll-interacting protein (TOLLIP), WBP2, NSFL1C, SLC4A7, CYFIP1, and RPS2) were validated to be novel tyrosine kinase substrates. SPAG9, TOLLIP, WBP2, and NSFL1C were further proven to be authentic targets of epidermal growth factor signaling and Iressa (gefitinib). A closer examination revealed that the expression of SLC4A7, a bicarbonate transporter, was down-regulated in 64% of the 25 matched normal and tumor clinical samples. The expression of TOLLIP in clinical breast cancers was heterogeneous with 25% showing higher expression in tumor compared with normal tissues and 35% showing the reverse trend. Preliminary studies on SPAG9, on the other hand, did not show differential expression between normal and diseased states. This is the first time SLC4A7 and TOLLIP have been discovered as novel tyrosine kinase substrates that are also associated with human cancer development. Future molecular and functional studies will provide novel insights into the roles of TOLLIP and SLC4A7 in the molecular etiology of breast cancer.

Novel breast cancer metastasis-associated proteins.

With the use of the breast cancer metastatic model, which comprises four isogenic cell lines, iTRAQ-based ESI-LC/MS/MS proteomics was employed to catalog protein expression changes as cancer cells acquire increasing metastatic potential. From more than 1000 proteins detected, 197 proteins, including drug-targetable kinases, phosphatases, proteases and transcription factors, displayed differential expression when cancer cells becomes more metastatic. Overall, the number of protein expression changes was evenly distributed across mildly ( approximately 30%), moderately ( approximately 40%) and aggressively ( approximately 30%) metastatic cancer cells. Some changes were found to be specific to one while others were required for two or more phenotypes. KEGG Orthology suggests major reprogramming in cell metabolism and to smaller extents in genetic and environmental information processing. Ten novel metastasis-associated proteins were identified and the iTRAQ-based expression profiles of 7 proteins were verified to be congruent with antibody-based methods. With the use of tissue microarrays comprising 50 matched cases of invasive and metastatic lesions, the expression profiles of SH3GLB1 and SUB1, SND1, TRIM28 were validated to be down- and up-regulated, respectively, during clinical progression of carcinoma in situ to invasive and metastatic carcinomas. Our study has unraveled proteome-wide molecular aberrations and potentially new players in breast cancer metastasis.

Cathepsin S Mediates Gastric Cancer Cell Migration and Invasion via a Putative Network of Metastasis-Associated Proteins

Cancer progression is governed by multifaceted interactions of cancer cells with their microenvironment and one of these ways is through secreted compounds. Substances released by gastric cancer cells have not being profiled in a proteome-wide manner. ITRAQ-based tandem mass spectrometry was employed to quantify proteins secreted by HFE145 normal, MKN7 well-differentiated, and MKN45 poorly differentiated gastric cancer cell lines. The expression levels of 237 proteins were found to be significantly different between normal and cancer cells. Further examination of 16 gastric cell lines and 115 clinical samples validated the up-regulation of CTSS expression in gastric cancer. Silencing CTSS expression suppressed the migration and invasion of gastric cancer cells in vitro. Subsequent secretomics revealed that CTSS silencing resulted in changes in expression levels of 197 proteins, one-third of which are implicated in cellular movement. Proteome-wide comparative secretomes of normal and gastric cancer cells were produced that constitute a useful resource for gastric cancer research. CTSS was demonstrated to play novel roles in gastric cancer cell migration and invasion, putatively via a network of proteins associated with cell migration, invasion, or metastasis. Cathepsin S is member of a large group of extracellular proteases, which are attractive drug targets. The implicated role of CTSS in gastric cancer metastasis provides an opportunity to test existing compounds against CTSS for adjuvant therapy and/or treatment of metastatic gastric cancers.

Upregulation of plasma C9 protein in gastric cancer patients

Gastric cancer is one of the leading causes of cancer‐related deaths worldwide. Current biomarkers used in the clinic do not have sufficient sensitivity for gastric cancer detection. To discover new and better biomarkers, protein profiling on plasma samples from 25 normal, 15 early‐stage and 21 late‐stage cancer was performed using an iTRAQ‐LC‐MS/MS approach. The level of C9 protein was found to be significantly higher in gastric cancer compared with normal subjects. Immunoblotting data revealed a congruent trend with iTRAQ results. The discriminatory power of C9 between normal and cancer states was not due to inter‐patient variations and was independent from gastritis and Helicobacter pylori status of the patients. C9 overexpression could also be detected in a panel of gastric cancer cell lines and their conditioned media compared with normal cells, implying that higher C9 levels in plasma of cancer patients could be attributed to the presence of gastric tumor. A subsequent blind test study on a total of 119 plasma samples showed that the sensitivity of C9 could be as high as 90% at a specificity of 74%. Hence, C9 is a potentially useful biomarker for gastric cancer detection.

Proteome-Wide Profiling of the MCF10AT Breast Cancer Progression Model

Background Mapping the expression changes during breast cancer development should facilitate basic and translational research that will eventually improve our understanding and clinical management of cancer. However, most studies in this area are challenged by genetic and environmental heterogeneities associated with cancer. Methodology/Principal Findings We conducted proteomics of the MCF10AT breast cancer model, which comprises of 4 isogenic xenograft-derived human cell lines that mimic different stages of breast cancer progression, using iTRAQ-based tandem mass spectrometry. Of more than 1200 proteins detected, 98 proteins representing at least 20 molecular function groups including kinases, proteases, adhesion, calcium binding and cytoskeletal proteins were found to display significant expression changes across the MCF10AT model. The number of proteins that showed different expression levels increased as disease progressed from AT1k pre-neoplastic cells to low grade CA1h cancer cells and high grade cancer cells. Bioinformatics revealed that MCF10AT model of breast cancer progression is associated with a major re-programming in metabolism, one of the first identified biochemical hallmarks of tumor cells (the “Warburg effect”). Aberrant expression of 3 novel breast cancer-associated proteins namely AK1, ATOX1 and HIST1H2BM were subsequently validated via immunoblotting of the MCF10AT model and immunohistochemistry of progressive clinical breast cancer lesions. Conclusion/Significance The information generated by this study should serve as a useful reference for future basic and translational cancer research. Dysregulation of ATOX1, AK1 and HIST1HB2M could be detected as early as the pre-neoplastic stage. The findings have implications on early detection and stratification of patients for adjuvant therapy.

Discovery of SLC3A2 cell membrane protein as a potential gastric cancer biomarker: Implications in molecular imaging

Despite decreasing incidence and mortality, gastric cancer remains the second leading cause of cancer-related deaths in the world. Successful management of gastric cancer is hampered by lack of highly sensitive and specific biomarkers especially for early cancer detection. Cell surface proteins that are aberrantly expressed between normal and cancer cells are potentially useful for cancer imaging and therapy due to easy accessibility of these targets. Combining two-phase partition and isobaric tags for relative and absolute quantification methods, we compared the relative expression levels of membrane proteins between noncancer and gastric cancer cells. About 33% of the data set was found to be plasma membrane and associated proteins using this approach (compared to only 11% in whole cell analysis), several of which have never been previously implicated in gastric cancer. Upregulation of SLC3A2 in gastric cancer cells was validated by immunoblotting of a panel of 13 gastric cancer cell lines and immunohistochemistry on tissue microarrays comprising 85 matched pairs of normal and tumor tissues. Immunofluorescence and immunohistochemistry both confirmed the plasma membrane localization of SLC3A2 in gastric cancer cells. The data supported the notion that SLC3A2 is a potential biomarker that could be exploited for molecular imaging-based detection of gastric cancer.

Progressive loss of epidermal growth factor receptor in a subpopulation of breast cancers: implications in target-directed therapeutics

Understanding the molecular etiology and heterogeneity of disease has a direct effect on cancer therapeutics. To identify novel molecular changes associated with breast cancer progression, we conducted phosphoproteomics of the MCF10AT model comprising isogenic, ErbB2- and ErbB3-positive, xenograft-derived cell lines that mimic different stages of breast cancer. Using in vitro animal model and clinical breast samples, our study revealed a marked reduction of epidermal growth factor receptor (EGFR) expression with breast cancer progression. Such diminution of EGFR expression was associated with increased resistance to Gefitinib/Iressa in vitro. Fluorescence in situ hybridization showed that loss of EGFR gene copy number was one of the key mechanisms behind the low/null expression of EGFR in clinical breast tumors. Statistical analysis on the immunohistochemistry data of EGFR expression from 93 matched normal and breast tumor samples showed that (a) diminished EGFR expression could be detected as early as in the preneoplastic lesion (ductal carcinoma in situ) and this culminated in invasive carcinomas; (b) EGFR expression levels could distinguish between normal tissue versus carcinoma in situ and invasive carcinoma with high statistical significance (P < 0.001, n = 81). However, no significant correlation of EGFR expression with disease-free survival and overall survival was observed. This is the first time EGFR expression has been tracked meaningfully and developmentally from the normal condition through disease progression using in vitro, xenograft, and matched normal and tumor samples. Thus, our study provides a new insight into the role of EGFR in breast cancer development. Although no value of EGFR expression in prognosis was found, our findings are likely to have implications in the design of clinical trials targeting the EGFR family of proteins in breast cancer. [Mol Cancer Ther 2007;6(11):2828–42]

TRPV4 Regulates Breast Cancer Cell Extravasation, Stiffness and Actin Cortex.

Metastasis is a significant health issue. The standard mode of care is combination of chemotherapy and targeted therapeutics but the 5-year survival rate remains low. New/better drug targets that can improve outcomes of patients with metastatic disease are needed. Metastasis is a complex process, with each step conferred by a set of genetic aberrations. Mapping the molecular changes associated with metastasis improves our understanding of the etiology of this disease and contributes to the pipeline of targeted therapeutics. Here, phosphoproteomics of a xenograft-derived in vitro model comprising 4 isogenic cell lines with increasing metastatic potential implicated Transient Receptor Potential Vanilloid subtype 4 in breast cancer metastasis. TRPV4 mRNA levels in breast, gastric and ovarian cancers correlated with poor clinical outcomes, suggesting a wide role of TRPV4 in human epithelial cancers. TRPV4 was shown to be required for breast cancer cell invasion and transendothelial migration but not growth/proliferation. Knockdown of Trpv4 significantly reduced the number of metastatic nodules in mouse xenografts leaving the size unaffected. Overexpression of TRPV4 promoted breast cancer cell softness, blebbing, and actin reorganization. The findings provide new insights into the role of TRPV4 in cancer extravasation putatively by reducing cell rigidity through controlling the cytoskeleton at the cell cortex.

Elevated NRD1 metalloprotease expression plays a role in breast cancer growth and proliferation

Understanding the molecular etiology of cancer and increasing the number of drugs and their targets are critical to cancer management. In our attempt to unravel novel breast‐cancer associated proteins, we previously conducted protein expression profiling of the MCF10AT model, which comprises a series of isogenic cell lines that mimic different stages of breast cancer progression. NRD1 expression was found to increase during breast cancer progression. Here, we attempted to confirm the relevance of NRD1 in clinical breast cancer and understand the functional role and mechanism of NRD1 in breast cancer cells. Immunohistochemistry data show that NRD1 expression was elevated in ductal carcinoma in situ and invasive ductal carcinomas compared with normal tissues in 30% of the 26 matched cases studied. Examination of NRD1 expression in tissue microarray comprising >100 carcinomas and subsequent correlation with clinical data revealed that NRD1 expression was significantly associated with tumor size, grade, and nodal status (P < 0.05). Silencing of NRD1 reduced MCF10CA1h and MDA‐MD‐231 breast‐cancer‐cell proliferation and growth. Probing the oncogenic EGF signaling pathways revealed that NRD1 knock down did not affect overall downstream tyrosine phosphorylation cascades including AKT and MAPK activation. Instead, silencing of NRD1 resulted in a reduction of overall cyclin D1 expression, a reduction of EGF‐induced increase in cyclin D1 expression and an increase in apoptotic cell population compared with control cells.

Biological models of breast cancer: one size does not fit all

Breast cancer is a leading cause of cancerrelated deaths, accounting for 7.4 million deaths or 13% of all deaths worldwide, based on 2004 cancer statistics. Approximately 1 million people are diagnosed with breast cancer each year. This comprises a tenth of all new cancers or 23% of all female cancers [1]. Early detection, radical surgery and adjuvant therapy are cornerstones of breast cancer management. Understanding the molecular etiology of breast cancer through elucidation of signal transduction pathways, discovery of biomarkers and drug targets can contribute to better outcomes for breast cancer patients. Breast cancer is a heterogeneous disease comprising of at least the luminal (ER), Her2/ER and basal or triple-negative (ER/Her2/PR) subtypes with different clinical behaviors and responses to therapy [2]. In addition, the development of breast cancer is influenced by multi faceted interactions between cancer cells and their microenvironment. Technological advances such as mass spectrometry have propelled the concept of systems biology, a very attractive holistic approach towards understanding the complex circuitries within the cells. However, while technological tools are critical, appreciation of the biological intricacies associated with a disease is equally important, if not more so, if we are to avoid running the risk of the ‘junk in–junk out’ reality. Depending on the desired end points, correct choice of biological models is important for the understanding of diseases like breast cancer. Does a holistic biological model exist for breast cancer? A typical breast tumor consists of cancer cells, extra cellular matrix and different cell types that secrete a diverse spectrum of growth factors, cytokines and chemokines that collectively influence processes such as tumor angiogenesis, invasion and metastasis. Although some researchers have attempted to ‘reconstruct’ these cellular and biochemical interactions through coculture experiments, such mimicry is still a far cry from the complete integrative physiology possessed by the tumors. Consequently, clinical tissue is an immensely precious biological resource for investigation as it possesses the entire spectrum of inter active components in the tumor–host system. Moreover, in the case of breast cancer, it is not uncommon to be able to find normal ductal epithelial cells, carcinoma in situ and invasive carcinoma within the resected tumor. This presents an excellent opportunity to study the molecular changes during breast cancer progression. However, there are two caveats. The first is biological – it is conceivable that each type of lesion found within the same tumor was derived from different clonal development of cancerinitiating cells – in which case, the lesions would have undergone different distinct molecular evolutions. Nevertheless, the mere fact that these lesions are isogenic to Lee Yee Choong