Browsing by Author "Leaner, Virna"

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    Open Access
    Characterization of ADAM10 in cervical and oesophageal cancers
    (2013) Williams, Cleo Julie-Jean; Leaner, Virna; Hendricks, Denver
    A Disintegrin And Metalloproteinase 10 (ADAM10) is a cell surface molecule that activates proteins such as: adhesion molecules, cytokines, and growth factors. ADAM10 has been associated with the proliferation and metastasis of cancers including, breast, lung, and colon. While ADAM10 has been investigated in these cancers, very little is known of its role in cervical and oesophageal cancer. The aim of this study was to investigate the endogenous expression of ADAM10 in cervical and oesophageal patient material and representative cell lines.
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    Characterizing the role of p21-Activated Kinase 3 (PAK3) in AP-1-induced transformation
    (2014) Holderness-Parker, Nina Alison Victoria; Leaner, Virna
    Previous studies identified p21-Activated Kinase 3 (PAK3), a serine/threonine kinase, as a potential AP-1 target gene. PAK3 has been implicated in a variety of pathological disorders and over-expression of other PAK-family members has been linked to cancer. In this study, we investigated AP-1 regulation of PAK3 expression and the role of PAK3 in cJun/AP-1-associated cellular transformation. Our results showed elevated PAK3 expression at both the mRNA and protein level in cJun-over-expressing Rat1a fibroblasts, as well as in transformed human fibroblasts. Elevated PAK3 protein levels were also seen in cervical, ovarian, oesophageal and breast cancer cells lines, while poor survival tracked with high PAK3 expression in ovarian cancer patient material. Elevated PAK3 levels appear to play no role in the proliferation of transformed or cancerous cells, however appears vital for the transformed morphology and actin distribution. These cytoskeletal changes seem to be the underlying force governing cellular migration, as inhibition of PAK3 significantly reduced the motility of both transformed fibroblasts and cancer cell lines. Our data shows that elevated PAK3 expression in response to AP-1 over-expression is regulated through the transcriptional activation of the PAK3 promoter by AP-1 binding directly to a single site in the promoter. We also show that constitutive activation of PAK3 results in changes in cJun phosphorylation and an increase in AP-1 activity, which can be inhibited by a serine/threonine kinase inhibitor. PAK3 and AP-1 proteins were also shown to directly interact with each other. Our study is a first to describe a role for AP-1 in regulating PAK3 expression, and PAK3 in regulating AP-1 activity, identifying a potential feedback loop in which PAK3 is an AP-1 target required for cytoskeletal reorganization and migration observed in transformed cells.
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    Expression and regulation of the nuclear transport proteins, Crm1 and Kpnß1, in cervical cancer and transformed cells
    (2009) Van der Watt, Pauline Janet; Leaner, Virna
    Includes abstract. Includes bibliographical references (leaves 204-223).
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    Investigating nuclear transport proteins as secreted cancer biomarkers
    (2019) Okpara, Michael Obinna; Leaner, Virna; van der Watt, Pauline
    Previous studies in our laboratory using microarray gene expression analysis identified members of the nuclear transport protein family as significantly upregulated in cervical cancer biopsies compared to normal cervical epithelial tissues. These results were validated at both mRNA and protein levels, and similar upregulation observed in oesophageal cancer. Recent mass spectrometry (MS) analysis of cancer cell secreted proteins identified elevated levels of 13 members of the nuclear transport protein family in the secretomes of transformed, cervical cancer and oesophageal cancer cell lines. The nuclear transport proteins have functions in many cellular processes including proliferation, mitosis, maturation of RNA, activation of the actin cytoskeleton and restructuring of the nuclear envelope. In addition, they are required for the nuclear import and export of numerous cargo proteins such as transcription factors, oncoproteins and kinases, which often display deregulated activity in cancer cells. The aims of this study were to 1) independently validate the MS data showing elevated levels of the nuclear transport proteins in the secretomes of cervical and oesophageal cancer cell lines, 2) investigate the diagnostic potential of members of the nuclear transport protein family using cervical and oesophageal cancer serum samples and 3) identify the potential binding partners of Kpnβ1, a key member of the nuclear transport protein family, in normal and cancer cells. This study investigated the levels of endogenous expression and secretion of 8 members of the nuclear transport protein family; Kpnβ1, IPO5, IPO7, TNPO1, CRM1, CAS, Kpnα2 and Ran in a normal epithelial cell line (hTERT-RPE1) in comparison to transformed (SVWI38 and CT-1), cervical cancer (HeLa and CaSki) and oesophageal cancer (WHCO5 and KYSE 30) cell lines using Western blot analysis. Our data revealed differential endogenous expression in the cell lines. An analysis of the secretomes of the cell lines showed that all 8 proteins assayed were secreted at elevated levels by the transformed, cervical cancer and oesophageal cancer cell lines compared to the normal cell line. These results validate previous MS data generated in our laboratory. To investigate whether members of this protein family can be detected in the serum of cancer patients, ELISA for Kpnβ1, CRM1, Kpnα2 and CAS proteins were performed using commercially available ELISA kits. The results showed significantly elevated levels of Kpnβ1, CRM1 and CAS in the serum of cervical cancer patients compared to the non-cancer controls. Serum levels of Kpnβ1, CRM1, Kpnα2 and CAS were elevated in the oesophageal cancer patients compared to the non-cancer controls. To investigate the diagnostic potential of these proteins, logistics regression analysis was performed. Our results showed that CAS was the best performing individual candidate biomarker in discriminating between cervical cancer cases and non-cancer controls. It had the highest AUC (0.85±0.03) and highest sensitivity (55%) at 95% specificity compared to those of Kpnβ1 (AUC=0.77±0.04 with 35% sensitivity at 95% specificity), CRM1 (AUC=0.64±0.05 with 20% sensitivity at 95% specificity) and Kpnα2 (AUC=0.51±0.05 with <10% sensitivity at 95% specificity). The combination of Kpnβ1, CRM1, Kpnα2 and CAS as a panel of biomarkers had an improved AUC of 0.89 with a sensitivity of 100% at 60% specificity. In discriminating oesophageal cancer cases from the non-cancer controls, CAS (AUC=0.86±0.03 with 56% sensitivity at 95% specificity) similarly performed better compared to Kpnβ1 (AUC=0.62±0.05 with 15% sensitivity at 95% specificity), CRM1 (AUC=0.75±0.04 with 32% sensitivity at 95% specificity) and Kpnα2 (AUC=0.73±0.04 with 21% sensitivity at 95% specificity). The combination of Kpnβ1, CRM1, Kpnα2 and CAS as a panel of biomarkers had the highest diagnostic capacity with an AUC of 0.90 and 84% sensitivity at 86% specificity. These results suggest that individual members of the nuclear transport protein family have potential as diagnostic biomarkers for both cervical and oesophageal cancers, with a combination of Kpnβ1, CRM1, Kpnα2 and CAS being the best predictor. Our investigation aimed at identifying the binding partners of Kpnβ1 in normal, cervical cancer and oesophageal cancer cell lines using immunoprecipitation coupled to mass spectrometry (IP-MS) identified 100 potential Kpnβ1 binding partners in hTERT-RPE1 normal cell extracts, 179 in HeLa cervical cancer cell extracts, 147 in WHCO5 cell extracts and 176 in KYSE30 oesophageal cancer cell extracts. Venn Dis JavaFX-based Venn and Euler diagram software was used to identify common and unique Kpnβ1 binding partners. 38 proteins were identified as common binding partners of Kpnβ1 in normal and cancer cells and 56 common binding partners of Kpnβ1 in the three cancer cell lines. Of these, 18 proteins were found to be unique to the three cancer cell lines and of these, 10 could be linked via protein-protein interaction mapping using STRING bioinformatic analysis. These include nucleoporin 214 (Nup214), Prem RNA 3'-end-processing factor FIP1 (FIP1L1), cell division cycle and apoptosis regulator 1 (CCAR1), cleavage and polyadenylation specific factor 7 (CPSF7), ribosomal protein L7 (RPL7), ribosomal protein L10 (RPL10), ribosomal protein L13A (RPL13A), ribosomal protein S6 (RPS6), ribosomal protein S4, X isoform (RPS4X) and Ras-related nuclear protein (Ran). Among these, FIP1L1, CCAR1 and CPSF7 have not been previously described as binding partners of Kpnβ1. In conclusion, elevated levels of nuclear transport proteins in the extracellular environment of cancer cells and in cancer patient serum samples suggest that they have potential as diagnostic biomarkers for cervical and oesophageal cancers, with a combination of Kpnβ1, CRM1, Kpnα2 and CAS being the best predictor. In addition, this study shows that Kpnβ1 interacts with several different proteins in normal and cancer cells, with some of the interactions unique to cancer cells presenting as novel binding partners for further investigation.
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    Investigating the effect of a small molecule, Inhibitor of Nuclear Import (INI-43) as a pan-cancer treatment
    (2024) Rahman, Shabita; Leaner, Virna
    Karyopherin beta 1 (KPNβ1) is a nuclear import protein that transports cargo molecules from the cytoplasm into the nucleus and plays an important role in other cellular processes such as mitosis and mitotic spindle assembly, DNA replication and cell cycle progression. Previous studies in our laboratory have reported that KPNβ1 is overexpressed in cervical and oesophageal cancer and is important for cancer cell survival and function, suggesting that inhibiting KPNβ1 may be a potential targeted anti-cancer strategy. An in silico screen identified a small molecule inhibitor, Inhibitor of Nuclear Import (INI-43) as an inhibitor of KPNβ1-mediated nuclear import and showed anti-cancer effects against cervical and oesophageal cancer cells using both cell culture and animal models. In this study, we expanded our investigations by determining KPNβ1 expression using bioinformatic analyses and Western blotting, and by investigating the effects of INI-43 in multiple cancer types grown in cell culture to ascertain the potential of KPNβ1 as a pan-cancer therapeutic target. In addition, we determined the ability of INI-43 to interact with KPNβ1 using the biophysical assay - Cellular Thermal Shift Assay (CETSA). Bioinformatic analyses using patient data revealed that KPNβ1 mRNA was significantly overexpressed in multiple cancer tissue compared to non-cancer tissue, including breast cancer, colon cancer, lung cancers and uterine cancers. Interestingly, we also found using bioinformatic approaches, that more than 50% of the cancers, for which data was available to us, show poorer patient survival when KPNβ1 is highly expressed. In vitro analysis using Western blotting on chosen cultured cell lines of different tissue origin revealed that KPNβ1 is overexpressed in multiple cancer types at the proteomic level, including liver cancer, gastric cancer, osteosarcoma and fibrosarcoma. These results suggest that KPNβ1 is a promising diagnostic/prognostic marker for a broad range of cancer types. Cancer cell lines of different tissue origin were found to be more sensitive to INI-43 treatment compared to the non-cancer epithelial cell line (ARPE-19) with EC50 values of ~ 5 - 15 µM for the cancer cell lines and ~ 25 µM for the non-cancer cell line, ARPE-19, which is at least two- RHMSHA007 fold higher than the EC50 values of majority of the cancer cell lines. INI-43 treatment also resulted in cancer cell death by apoptosis as observed by Caspase activity. These results support that INI-43 has potential as a pan-cancer therapeutic agent for multiple cancer types. To investigate KPNβ1:INI-43 interactions in living cells, we analysed thermal melt profiles of treated and untreated KPNβ1 containing protein lysates using the biophysical assay, CETSA. Our study found that treatment with INI-43 has a stabilising effect on KPNβ1, suggesting that INI-43 physically binds to KPNβ1. Furthermore, we obtained data to show that INI-43 does not appear to bind to other nuclear transport proteins that are associated with cancer, including CAS, CRM1, KPNα2, TNPO1 and GAPDH (control). Interestingly, we found that INI43 appears to bind to another nuclear import protein, IPO5. In conclusion, this study is a first to show that KPNβ1 is expressed at high levels in multiple cancer types of different tissue origin and that INI-43, a small molecule inhibitor with antiKPNβ1 activity, has potential as a pan-cancer treatment. The study shows that INI-43 interacts with KPNβ1 in living cells. These results provide evidence that KPNβ1 is a potential anti-cancer therapeutic target for multiple cancer types.
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    Investigating the effect of conventional chemotherapies, Cisplatin and Doxorubicin, in combination with a nuclear transport inhibitor, INI-43, on cerfical cancer cells
    (2023) Wilensky, Gabriella; Leaner, Virna
    Background: Cervical cancer is the most common gynaecological cancer in South Africa, and the burden of disease remains high across the developing world. Like with many other cancers, treatment is limited due to drug resistance and adverse side effects. To overcome these limitations, it has become of interest to inhibit multiple pathways that cancer cells are reliant on, using combination therapies to induce maximal killing of cancer cells. Conventional chemotherapies used in the treatment of cancer include Cisplatin and Doxorubicin, both firstline drugs often used in combination with other treatments. Our laboratory has a focus on identifying novel therapeutic targets and found that Kpnβ1, a member of the nuclear transport protein family is overexpressed and necessary for the survival of cervical cancer cells. Inhibition of Kpnβ1 with a novel Inhibitor of Nuclear Import; INI-43 inhibits Kpnβ1- associated nuclear import pathways and results in cancer cell death. This study investigated the potential of Cisplatin and Doxorubicin when used in combination with INI-43 as an anticancer approach. Methods: Cervical carcinoma cell lines, HeLa, ME-180 and SiHa, were treated with INI-43, Cisplatin and Doxorubicin individually or in combination with each other at fixed ratios. Cell viability was measured by the MTT assay and the Chou-Talalay method was adopted to investigate the nature of the drug interactions in the combination treatments. The Caspase3/7 Glo Assay and Western blot analysis were performed to identify markers of apoptotic death, Caspase-3/7 activation and PARP1 cleavage, respectively, in single and combination treated cells. gH2AX was also investigated via western blot analysis to determine levels of DNA damage in single and combination treated cells. Results: Combination index values were determined to be below 1 at all levels of cell death after treatment with INI-43 and Cisplatin and INI-43 and Doxorubicin combinations, in HeLa, ME-180 and SiHa cell lines, indicating a synergistic effect. Additionally, enhanced levels of PARP1 cleavage were detected in combination treated cells relative to those treated with individual drug doses, indicating that the observed synergism is attributed to both decreased cell viability and elevated levels of apoptotic cell death. Non-cancer ARPE-19 cells were unaffected by single agent treatments and, more importantly, remained unaffected after treatment with drug combinations which resulted in enhanced cell death in the cervical cancer cell lines tested. Conclusion: INI-43, when combined with Cisplatin or Doxorubicin, results in synergistic cytotoxic effects in cervical cancer cells of different histological origins. These results suggest that combining conventional chemotherapies with targeted inhibitors of nuclear import pathways, has potential as an anti-cancer approach that warrants further investigation.
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    Investigating the nuclear import protein KPn߀1 as a cancer therapeutic target
    (2019) Ajayi-Smith, Aderonke Fopesaye; Leaner, Virna
    The dysregulation of numerous genes has been associated with the development and progression of cancer, many of which are being investigated as potential therapeutic targets. Previous work in our laboratory and others reported the elevated expression of the nuclear import protein Karyopherin Beta 1 (Kpnβ1) in various cancers. The inhibition of Kpnβ1 by siRNA silencing inhibited the proliferation of cancer cells and induced cell death via apoptosis while having little effect on non-cancer cells. These findings suggested that Kpnβ1 has potential as an anti-cancer therapeutic target. Using an in silico screening approach to identify small molecule inhibitors of Kpnβ1 with anti-cancer activity, a number of compounds were selected for further investigation in our laboratory. The aim of this study was to investigate a novel small molecule, Compound 60 (9-[(1-methyl-3-piperidinyl)methoxy]-4-[(6-methyl-2-pyridinyl)methyl]-7-(5-methyl-2-thienyl)-2,3,4,5-tetrahydro-1,4-benzoxazepine); by monitoring (i) its effect on cancer cell biology using cervical and oesophageal cancer cell lines, (ii) its effect on nuclear import activities associated with Kpnβ1, (iii) its in vitro ADME pharmacokinetics and in vivo anti-cancer properties and (iv) performing biophysical analysis of Kpnβ1:C60 interactions. Cervical and oesophageal cancer cells were found to be more sensitive to C60 treatment compared to non-cancer epithelial cell. C60 treatment resulted in the inhibition of cancer cell proliferation, colony formation, migration and invasion. G1/S cell cycle arrest and a reduced expression of cell cycle regulatory proteins such as Cyclins D1, B1 and A as well as CDK4 was observed on treatment with C60. C60 induced cell death via apoptosis as observed PARP cleavage. These results suggest that C60 has an inhibitory effect on cancer cell biology. Immunofluorescent analysis and nucleo-cytoplasmic western blot analysis showed that C60 treatment resulted in the cytoplasmic retention of Kpnβ1. Immunofluorescent analysis and luciferase reporter assays showed that C60 inhibited the nuclear entry and transcriptional activity of a Kpnβ1 cargo, NFκB. Similarly, the transcriptional activity of cargo proteins NFAT and AP-1 were also inhibited. This suggests that C60 inhibits the nuclear entry of Kpnβ1 and thus its function as a nuclear importer of cargo proteins. In vitro ADME pharmacokinetics analysis found C60 to have high solubility, permeability and plasma protein binding properties and a short half-life. These findings suggest that C60 may have good oral absorption but rapid clearance in living systems. In vivo toxicology studies showed that C60 is tolerable, allowing for its testing in a xenograft nude mouse model. Intraperitoneal injection of C60 selectively inhibited the growth of oesophageal tumour cells with a significant effect observed on KYSE 30 oesophageal tumours. To investigate Kpnβ1:C60 binding interactions, Kpnβ1 was purified using the GST-tagged purification technique. Purified Kpnβ1:C60 interactions were monitored using the Bio-layer interferometry technique. Our preliminary data suggest an interaction between C60 and Kpnβ1 with Kd values in the micromolar range. We obtained varying Kd values, hence further optimisation is required to arrive at a conclusive Kd value. In conclusion, this study is a first to show that Compound 60 (9-[(1-methyl-3-piperidinyl)methoxy]-4-[(6-methyl-2-pyridinyl)methyl]-7-(5-methyl-2-thienyl)-2,3,4,5-tetrahydro-1,4-benzoxazepine), a small molecule identified to bind Kpnβ1 in an in silico screening approach interferes with the subcellular localisation of Kpnβ1, inhibits the localisation and transcriptional activity of Kpnβ1 cargo proteins and inhibits cancer cell biology in vitro and in vivo. In vitro ADME pharmacokinetics shows that C60 has tolerable drug properties. Biophysical analysis shows that C60 appears to bind Kpnβ1 in the micromolar range. Together, these results provide evidence for C60 as a compound that warrants further investigation as an anti-cancer agent.
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    The regulation of type I collagen gene expression in stromal fibroblast by breast tumour cells
    (2011) Rose, Beverley Ann; Parker, Iqbal; Leaner, Virna
    Recent studies have revealed that interactions between tumour cells and the surrounding stroma play an important role in facilitating tumour growth and invasion. Stromal fibroblasts produce most of the extracellular matrix (ECM) components found in the stroma, including type I collagen. Previous in vivo studies in our laboratory have shown that type I collagen mRNA levels are decreased in stage II and III breast tumour tissue compared to adjacent normal tissue.
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    The role of Karyopherin β1 in the nuclear import of HIV-1 proteins
    (2014) Shaw, Tamlyn Marion; Leaner, Virna; van der Watt, Pauline
    Includes abstract. Includes bibliographical references.
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    Tumour cells down-regulate CCN2 gene expression in co-cultured fibroblasts in a Smad7- and ERK-dependent manner
    (BioMed Central Ltd, 2013) van Rooyen, Beverley; Schafer, Georgia; Leaner, Virna; Parker, M
    BACKGROUND: Recent studies have revealed that interactions between tumour cells and the surrounding stroma play an important role in facilitating tumour growth and invasion. Stromal fibroblasts produce most of the extracellular matrix components found in the stroma. The aim of this study was to investigate mechanisms involved in tumour cell-mediated regulation of extracellular matrix and adhesion molecules in co-cultured fibroblasts. To this end, microarray analysis was performed on CCD-1068SK human fibroblast cells after direct co-culture with MDA-MB-231 human breast tumour cells. RESULTS: We found that the expression of both connective tissue growth factor (CTGF/CCN2) and type I collagen was negatively regulated in CCD-1068SK fibroblast cells under direct co-culture conditions. Further analysis revealed that Smad7, a known negative regulator of the Smad signalling pathway involved in CCN2 promoter regulation, was increased in directly co-cultured fibroblasts. Inhibition of Smad7 expression in CCD-1068SK fibroblasts resulted in increased CCN2 expression, while Smad7 overexpression had the opposite effect. Silencing CCN2 gene expression in fibroblasts led, in turn, to a decrease in type I collagen mRNA and protein levels. ERK signalling was also shown to be impaired in CCD-1068SK fibroblasts after direct co-culture with MDA-MB-231 tumour cells, with Smad7 overexpression in fibroblasts leading to a similar decrease in ERK activity. These effects were not, however, seen in fibroblasts that were indirectly co-cultured with tumour cells. CONCLUSION: We therefore conclude that breast cancer cells require close contact with fibroblasts in order to upregulate Smad7 which, in turn, leads to decreased ERK signalling resulting in diminished expression of the stromal proteins CCN2 and type I collagen.