Browsing by Author "Leaner, Virna D"

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    A tight balance of Karyopherin β1 expression is required in cervical cancer cells
    (BioMed Central, 2018-11-16) Carden, Sarah; van der Watt, Pauline; Chi, Alicia; Ajayi-Smith, Aderonke; Hadley, Katie; Leaner, Virna D
    Background Karyopherin β1 (Kpnβ1) is the main nuclear import protein involved in the transport of cargoes from the cytoplasm into the cell nucleus. Previous research has found Kpnβ1 to be significantly overexpressed in cervical cancer and other cancer tissues, and further studies showed that inhibition of Kpnβ1 expression by siRNA resulted in cancer cell death, while non-cancer cells were minimally affected. These results suggest that Kpnβ1 has potential as an anticancer therapeutic target, thus warranting further research into the association between Kpnβ1 expression and cancer progression. Here, the biological effects associated with Kpnβ1 overexpression were investigated in order to further elucidate the relationship between Kpnβ1 and the cancer phenotype. Methods To evaluate the effect of Kpnβ1 overexpression on cell biology, cell proliferation, cell cycle, cell morphology and cell adhesion assays were performed. To determine whether Kpnβ1 overexpression influences cell sensitivity to chemotherapeutic agents like Cisplatin, cell viability assays were performed. Expression levels of key proteins were analysed by Western blot analysis. Results Our data revealed that Kpnβ1 overexpression, above that which was already detected in cancer cells, resulted in reduced proliferation of cervical cancer cells. Likewise, normal epithelial cells showed reduced proliferation after Kpnβ1 overxpression. Reduced cancer cell proliferation was associated with a delay in cell cycle progression, as well as changes in the morphology and adhesion properties of cells. Additionally, Kpnβ1 overexpressing HeLa cells exhibited increased sensitivity to cisplatin, as shown by decreased cell viability and increased apoptosis, where p53 and p21 inhibition reduced and enhanced cell sensitivity to Cisplatin, respectively. Conclusions Overall, our results suggest that a tight balance of Kpnβ1 expression is required for cellular function, and that perturbation of this balance results in negative effects associated with a variety of biological processes.
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    Characterising the anticancer effects of a small molecule with potential to inhibit nuclear import via karyopherin beta1
    (2018) Mkwanazi, Nonkululeko; Leaner, Virna D
    The Karyopherin superfamily is a group of soluble transport proteins which are involved in nuclear-cytoplasmic trafficking. Studies have shown the involvement of Karyopherin proteins in nuclear pore assembly, nuclear membrane assembly and DNA replication. Since all these cell regulatory functions are critical for normal cell function, dysregulation of Karyopherin proteins may have an impact on cancer cell survival. Previous research in our laboratory and in that of others has shown that Karyopherin Beta 1 (KPNB1) is elevated in and necessary for the survival of cervical cancer cells as inhibiting its expression with siRNAs interfered with the proliferation of cancer cells. KPNB1 has thus been proposed as an anticancer target. In addition to inhibition by siRNA, an in silico screen for small molecules with potential to bind KPNB1 identified a number of compounds that are currently under investigation for their cancer cell killing effects. In this study, we investigated the ability of a novel small molecule 1-benzyl-4[(4-methoxy-1-naphyl) methylamino]-N-methyl pyrrolidine-2-carboxamide (Compound 53) to kill cancer cells and inhibit the activity of KPNB1 cargo proteins. In addition, the in vitro pharmacokinetic properties and in vivo toxicology of Compound 53 (C53) were investigated. Cervical (HeLa and CaSki) and oesophageal (WHCO6 and Kyse30) cancer cell lines were found to be more sensitive to C53 treatment compared to non-cancer cells (FG₀), with EC₅₀ values of ~20 μM for the cancer cell lines and ~30-40 μM for the non-cancer cells. C53 treatment significantly inhibited proliferation in cancer cell lines. The reduction in proliferation in cancer cells was associated with a block in the G1 phase of the cell cycle and a change in the expression of cell cycle related proteins such as CyclinD1 and CDK4. C53 treatment resulted in cell death via apoptosis as observed using Annexin V staining and PARP cleavage. To assess whether C53 interferes with KPNB1 associated nuclear import, we investigated the effect of C53 on the activity of KPNB1 cargo proteins, NFAT and NF-ĸB as well as investigate its effect on KPNB1 localisation. The results show that C53 has no effect on the localisation of KPNB1 but it does however block the nuclear activity of the KPNB1 cargoes, NFAT and NF-ĸB. In order to predict the behaviour of C53 in a living system, in vitro ADME pharmacokinetic studies showed that C53 has moderate solubility, permeability and protein binding however, rapid clearance was shown by liver microsome assay. In vivo repeated dose toxicology studies showed that C53 is tolerable in nude mice. Taken together, the data presented in this study shows that a novel small molecule, C53 has a negative effect on the proliferation of cancer cells, inhibits the nuclear import of KPNB1 cargoes, displays tolerable in vitro ADME pharmacokinetic properties and showed no toxic side effects in vivo. These results suggest that C53 targets KPNB1 and shows potential as an anticancer molecule.
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    The effect of inhibiting KPNB1-mediated nuclear import on cancer cell biology and inflammatory transcription factor signalling
    (2018) Stelma, Tamara; Leaner, Virna D
    Cancer remains one of the major causes of morbidity and mortality globally. Many novel and innovative approaches have been employed to develop new chemotherapeutic strategies, of which targeted therapies aim to identify a molecular lesion or dysregulated pathway that cancer cells are dependent on. Research in our laboratory and others identified the nuclear import protein, Karyopherin β1 (KPNB1), to be overexpressed in various cancers and that inhibiting its expression blocks the proliferation of cancer cells. However, little is known about the potential role of KPNB1 in other cancer cell phenotypes and inflammatory signalling pathways. The aim of this study was to investigate the anticancer and anti-inflammatory effects of inhibiting nuclear import via KPNB1 and to characterise the in vivo effect of the small molecule inhibitor of nuclear import, INI-43, on tumour formation. Using siRNA and a small molecule inhibitor, INI-43, to inhibit KPNB1 we found that cervical cancer cell migration and invasion was significantly reduced. The reduced motility of cancer cells was associated with a decrease in MMP-2 and -9 expression and an increase in TIMP-1 and -2 expression following INI-43 treatment. This corresponded with a decrease in MMP-9 gelatinase activity in KPNB1-inhibited cervical cancer cells. Extended periods of KPNB1 inhibition lead to decreased proliferation and apoptosis. These changes in cancer cell biology when KPNB1 is inhibited may in part be due to its function as a nuclear transporter of transcription factors associated with cancer cell proliferation, migration and invasion. We therefore investigated the effects of KPNB1 inhibition on the nuclear localisation and transcriptional activity of key transcription factors; NFkB and AP-1, both having been implicated in many of the hallmarks of cancer. Immunofluorescent analysis and nuclear/cytoplasmic fractionation assays showed that KPNB1 inhibition blocked the nuclear localisation of NFkB. Electromobility shift assays confirmed a reduced NFkB binding to an NFkB DNA-binding sequence in the nuclear extract of KPNB1-inhibited cells. Luciferase reporter assays containing NFkB and/or AP-1 consensus binding sites showed reduced transcriptional activity for both transcription factors following KPNB1 inhibition. Associated with these changes in NFkB and AP-1 activity was reduced inflammatory cytokines; IL-6, IL-1β, TNF-α and GM-CSF target gene expression. To further characterise the role of INI-43 as a potential chemotherapeutic, the effects on tumour growth and development were investigated in an ectopic xenograft mouse model. INI-43 treatment significantly reduced tumour growth in mice and associated with the redistribution and reduction in KPNB1 levels. INI-43 treated tumours also showed altered morphological features including; better tissue differentiation and reduced inflammatory stromal infiltration, as well as reduced Ki-67 expression. The expression of extracellular matrix components and the cytoskeletal structure of cancer cells was analysed to further investigate the role of KPNB1 inhibition in tumour development. Inhibition of KPNB1 in cancer cells caused reduced expression of both collagen type IV and MMP-9. The redistribution of B-catenin and F-actin suggested that INI-43 treatment caused a loss of mesenchymal features required for tumour progression. The nuclear transport system has been of particular interest in recent years for the development of targeted anticancer drugs. However, most studies have focused on nuclear export inhibitors with little known on the potential of nuclear import inhibitors as anticancer drugs. This study provides evidence that inhibiting the nuclear import protein, KPNB1, has anti-inflammatory and anticancer effects and shows promise as an anticancer approach requiring further investigation.
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    Inhibition of Kpnβ1 mediated nuclear import enhances cisplatin chemosensitivity in cervical cancer
    (2021-02-02) Chi, Ru-pin A; van der Watt, Pauline; Wei, Wei; Birrer, Michael J; Leaner, Virna D
    Background Inhibition of nuclear import via Karyopherin beta 1 (Kpnβ1) shows potential as an anti-cancer approach. This study investigated the use of nuclear import inhibitor, INI-43, in combination with cisplatin. Methods Cervical cancer cells were pre-treated with INI-43 before treatment with cisplatin, and MTT cell viability and apoptosis assays performed. Activity and localisation of p53 and NFκB was determined after co-treatment of cells. Results Pre-treatment of cervical cancer cells with INI-43 at sublethal concentrations enhanced cisplatin sensitivity, evident through decreased cell viability and enhanced apoptosis. Kpnβ1 knock-down cells similarly displayed increased sensitivity to cisplatin. Combination index determination using the Chou-Talalay method revealed that INI-43 and cisplatin engaged in synergistic interactions. p53 was found to be involved in the cell death response to combination treatment as its inhibition abolished the enhanced cell death observed. INI-43 pre-treatment resulted in moderately stabilized p53 and induced p53 reporter activity, which translated to increased p21 and decreased Mcl-1 upon cisplatin combination treatment. Furthermore, cisplatin treatment led to nuclear import of NFκB, which was diminished upon pre-treatment with INI-43. NFκB reporter activity and expression of NFκB transcriptional targets, cyclin D1, c-Myc and XIAP, showed decreased levels after combination treatment compared to single cisplatin treatment and this associated with enhanced DNA damage. Conclusions Taken together, this study shows that INI-43 pre-treatment significantly enhances cisplatin sensitivity in cervical cancer cells, mediated through stabilization of p53 and decreased nuclear import of NFκB. Hence this study suggests the possible synergistic use of nuclear import inhibition and cisplatin to treat cervical cancer.
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    Inhibition of the transcription factor AP-1 in cervical cancer
    (2007) Maritz, Michelle Frances; Leaner, Virna D
    AP-I is a dimeric transcription factor comprised primarily of Jun and Fos family proteins, that regulates numerous genes involved in cell proliferation, differentiation and oncogenesis. The expression of AP-I is shown to play an important role in many human cancers and plays a key role in the regulation of the E6 and E7 oncoproteins of high-risk Human Papillomaviruses (HPV) that are etiologically associated with cervical cancer. The c-Jun and Jun B components of AP-I were shown to be expressed at higher levels in cervical cancer patients compared to nonnal patient tissue while Jun D levels were largely unchanged. To define the role of AP-I in cervical cancer, the effect of inhibiting AP-I actvity was determined using a dominantnegative deletion mutant T AM67. CaSki cervical cancer cells with a doxycycline inducible T AM67 demonstrated that inhibition of AP-I activity and expression resulted in an altered cell morphology, a significant decrease in cell proliferation and inhibition of colony formation. This was accompanied by a slower progression of T AM67 expressing cells through the cell cycle, with an accompanying increase in G21M phase. An increase in the expression of the cell cycle regulatory protein, p21 CIPI, was observed that appeared independent of p53 expression. siRNA directed at inhibiting individual AP-I components showed that Jun B was an important regulator of CaSki cell proliferation. These results suggest that AP-I is involved in the cell proliferation and tumourigenic phenotype of cervical cancer cells, such as CaSki cells, possibly via a direct repression of cell cycle regulator p21 CIP1
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    Investigating a novel small molecule inhibitor of nuclear import as an anti-cancer approach
    (2016) Chi, Ru-pin Alicia; Leaner, Virna D; Hendricks, Denver T
    The identification of novel cancer-associated biomarkers against which drugs can be developed is anticipated to be beneficial in multiple ways; including their use as monotherapies and in combination with current chemotherapeutic agents for improved anti-cancer treatment outcome. Recently, research in our own laboratory and others have reported elevated expression of the nuclear transporter Kpnβ1 in multiple cancers. Using the cervical cancer model, we showed that its inhibition using small-interfering RNA (siRNA) resulted in cancer cell death via apoptosis while sparing normal cells, suggesting it has potential as a target for anti-cancer therapy. An in silico screen for Kpnβ1 inhibitors identified several small molecules that showed inhibitory effects on nuclear import as well as cancer killing activity. In this study, we aimed to examine the potential of one such small molecule, the Inhibitor of Nuclear Import-43 (INI-43) as a lead compound with anti-cancer activities using multiple cancer models. Through culture-based in vitro assays, we demonstrated that INI-43 inhibited the proliferation of cancer cells grown anchorage-dependently and independently. These effects were similarly observed in Kpnβ1 knock-down cells, and Kpnβ1 over-expression was able to partially reverse these effects, suggesting that the anti-cancer effects of INI-43 is mediated through interference of the Kpnβ1 function. Toxicology studies and liver microsomal assay showed that INI-43 has an acceptable toxicity profile in nude mice and is metabolically stable, allowing its use in in vivo testing. Intraperitoneal administration of INI-43 significantly reduced the growth of subcutaneously xenografted cervical and oesophageal tumour cells in nude mice, supporting its anti-cancer activity in vivo. To examine the potential of using INI-43 in combination therapy, we examined the effects of the combined treatment of INI-43 and Cisplatin (CDDP), a first-line chemotherapeutic agent used in the treatment of many cancers. INI-43 treatment at sub-lethal concentrations enhanced cancer cells' sensitivity to CDDP, which was similarly observed in Kpnβ1 knock-down cells. Using an ovarian cancer model, we demonstrated that CDDP treatment led to elevated expression and nuclear localization of Kpnβ1, suggesting that Kpnβ1 is involved in CDDP-induced stress response. INI-43 treatment impeded the CDDP-induced nuclear accumulation of Kpnβ1 which correlated with increased cell death, suggesting that nuclear localization of Kpnβ1 may be important for ovarian cancer cell survival when challenged with genotoxins such as CDDP. Using the cervical cancer model, we demonstrated that INI-43 enhanced CDDP-induced cell death synergistically, and that the enhanced cell death is mediated through stabilizing p53 protein. This associated with decreased levels of Myeloid Cell Leukemia 1 (Mcl-1), an anti-apoptotic factor negatively regulated by p53. Furthermore, INI-43 treatment reduced the nuclear import of NFκB, a stress-regulated response known to promote cancer cell survival. Decreased levels of various downstream pro-survival and DNA-repair targets of NFκB were observed, including cyclinD1, c-Myc and X-Linked Inhibitor of Apoptosis Protein (XIAP), which correlated with increased DNA damage and apoptosis. Taken together, we show that nuclear import inhibition using small molecules could have therapeutic benefits in the treatment of cancer, and that INI-43 is a promising candidate for further development to be used in anti-cancer monotherapy or combination chemotherapy.
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    Investigating Karyopherin B1: small molecule interactions for cancer therapy
    (2016) Strydom, Erin; Leaner, Virna D
    The advent of gene expression profiling studies has allowed for the identification of genes with potential as disease markers and therapeutic targets. Our laboratory identified the eukaryotic nuclear importer protein Karyopherin B1 (KpnB1), to be up-regulated in different cancer cell lines, including cervical and oesophageal as well as transformed cells. Inhibition of KpnB1 in these cells using small interfering RNA (siRNA) resulted in significant cancer cell death via apoptosis, suggesting KpnB1 is essential for cancer cell survival. Within our laboratory, we established that candidate small molecules targeted against KpnB1 identified using a rational drug design approach. The outcome of this research is for examine inhibitors of KpnB1 for potential as future anti-cancer agents using. Based on the long-term goal of this research, this particular project was aimed at investigating a small molecule inhibitor identified in our laboratory, known as Inhibitor of Nuclear Import-43 (INI-43) for its potential to bind to the nuclear importer, KpnB1. Using conventional assays as well as cutting edged techniques including circular dichrosim (CD) and isothermal titration calorimeter (ITC), an examination of INI-43 and its interactions with KpnB1 was made. In vitro analysis showed that INI-43 exhibits cytotoxic effects on cervical cancer cells with an IC₅₀ of ≈10μM and induces apoptotic cell death. The NFAT dual luciferase assay measured nuclear import of KpnB1 associated proteins, showing that INI-43 inhibits nuclear import/activity of NFAT in a dose dependent manner. Confocal microscopy of exogenous FRFP-KpnB1 as well as endogenous KpnB1 in the presence of INI-43 showed a change in the localisation of KpnB1 upon drug treatment. Both FRFP-KpnB1 and endogenous KpnB1 appear to be prevented from entering the nucleus, and is retained in the peri-nuclear space and the cytoplasm suggesting that INI-43 inhibits KpnB1 movement into the nucleus. To investigate KpnB1-INI-43 interactions, purified KpnB1 was prepared and used in biophysical techniques. Purified KpnB1 protein was prepared using GST-tagged purification methods and the tagged protein confirmed by mass spectrometry. Purified GST-KpnB1 was used in drug binding studies including circular dichrosim (CD) isothermal titration calorimetry (ITC). CD showed a drug concentration dependant shift in the spectra at around 233nm, indicative of drug protein interaction possibly occurring in a region of KpnB1 containing aromatic amino acids. The purified GST-KpnB1 was used in ITC, which confirmed an interaction between KpnB1 and INI-43, a relatively weak interaction. In conclusion, our data shows that the small molecule, INI-43 kills cancer cells, likely by interfering with KpnB1 associated nuclear import pathways. We show that INI-43 interferes with the nuclear localisation of KpnB1 itself and biophysical assays provide evidence for possible KpnB1-INI-34 interactions. Small molecules such as INI-43 present as promising tools to studying the potential of KpnB1 as an anticancer target.
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    Investigating the role of the Renin Angiotensin System in cancer
    (2017) Dunn, Cherise; Leaner, Virna D; Sturrock, Edward D
    It has recently been discovered that cancer shares a link with metabolic diseases, including that of cardiovascular disease, diabetes, amongst others, where common sets of genes show similar gene expression. There is thus interest to investigate current therapies for metabolic diseases as possible anti-cancer agents. The renin-angiotensin system (RAS) regulates blood pressure and cardiovascular homeostasis through Angiotensin Converting Enzyme-1 (ACE-1) and its homolog ACE-2. RAS has also been implicated in the progression of various cancers due to the increased action of the vasoconstrictor, angiotensin II, which requires ACE-1 and specifically the Angiotensin Type 1 Receptor (AT1R) for its function. In this study, we investigated the potential association of the endogenous ACE-1 and ACE-2 enzymes in cervical cancer. Our results showed that ACE-1 and AT1R protein expression was elevated in cervical cancer cell lines compared to normal cells and that this correlated with elevated ACE-1 enzyme activity in cancer cells. Treatment with the ACE-1 inhibitors, Captopril and Lisinopril, reduced this activity. We showed that ACE-1 axis stimulation in cancer cells results in increased calcium signaling preferentially via the AT1R and this associates with cancer cell proliferation. Candesartan, an AT1R blocker significantly reduced these effects. ACE-2 expression and activity were decreased in cancer compared to normal cells. Our data shows that ACE2 activators, the natural peptide angiotensin 1-7 and small molecule Diminazene aceturate (DIZE) have anticancer effects with DIZE inducing a G2/M arrest in cancer cells. We also investigated associations between drugs targeting RAS and current chemotherapeutic agents, Cisplatin (CDDP) and Doxorubicin (DOX). Our data shows that ACE-1 axis inhibitors have an antagonistic effect on CDDP, while the ACE-2 activator DIZE associates synergistically with DOX. Taken together, these results suggest that elevated ACE- 1 expression associates with cervical cancer and that the inhibitors of ACE-1 function or activators of ACE-2 function have potential as anticancer therapies as single agents or in combination treatments with current chemotherapeutics.
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    Lamina-associated polypeptide 2 (LAP2) expression patterns in transformed and cancer cells
    (2009) Ward, Michelle Claire; Leaner, Virna D
    The Lamina-associated polypeptide two (LAP2) proteins comprising three human isoforms, LAP2 +- LAP2β and LAP2 have been shown to provide a structural framework in the nucleus and to facilitate nuclear assembly and disassembly during the cell cycle. Expression profiling studies, using microarrays, identified elevated levels of LAP2α in cervical cancer patient material compared to normal. Altered expression of LAP2 may thus have significance in the development of certain cancers. The aim of this project was thus to independently confirm the up-regulation of LAP2α in cancer material and to determine the effect of inhibiting its expression on the biology of cancer and transformed cells. LAP2α mRNA and protein expression was shown to be elevated in cervical cancer tissue compared to normal cervical tissue by Real-time RT-PCR and immunohistochemical analysis respectively. Interestingly, LAP2 (both the LAP2α and LAP2β isoforms) was shown to be overexpressed in cervical cancer cell lines compared to a normal primary cervical epithelial cell line. Higher LAP2 expression appears to associate with cellular transformation as increased expression was observed in transformed human fibroblast cells compared to normal fibroblasts. LAP2 expression was also elevated in oesophageal cancer cell lines compared to normal suggesting that the overexpression of LAP2 associates with multiple cancer types. In order to determine the role of LAP2 in cancer cell biology, its expression was inhibited using specific siRNA molecules. Inhibition of LAP2 did not have an effect on adherent cell proliferation; however under anchorage-independent growth conditions a significant decrease in cell proliferation and colony formation was observed in LAP2 knockdown cells. This was accompanied by a decrease in cyclin D1 levels and an increase in p16 levels in LAP2 siRNA transfected cells. Our results did not conclusively show xiii that this decrease in proliferation was as a result of an alteration in the cell cycle profile or due to an increase in apoptosis. In addition, inhibition of LAP2 expression resulted in a decrease in Rb protein expression. It is proposed that LAP2 plays a role in stabilizing the Rb protein, as inhibition of LAP2 expression did not affect Rb mRNA levels but substantially reduced the protein half-life. In summary, increased LAP2 expression associates with transformed and cancer cells and suggests potential for use as a cancer biomarker. Its potential as an anti-cancer therapeutic, however requires further investigation.
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    Modulating ADAM-10 activity and expression in cervical and oesophageal cancer cells
    (2016) Wagiet, Mateen; Leaner, Virna D; Hendricks, Denver T
    The ADAMs (A Disintegrin And Metalloproteinase) is a family of transmembrane and secreted proteins essential in cellular fate determination, wound healing, cell migration, proliferation and angiogenesis. Previous studies have linked a range of ADAMs, which include ADAM10 to cancer development and progression. Research in our laboratory found endogenous ADAM10 levels to be higher in both oesophageal and cervical cancer cell lines. Reports in the literature have highlighted a correlation between high levels of ADAM10 expression with that of cancer cell biology; hence ADAM10 shows promise as an anti-cancer target. The aim of this study was to modulate ADAM10 activity in oesophageal and cervical cancer cell lines using the small molecule inhibitor GI254023X as well as previously undescribed two molecules generated en route to synthesizing GI254023X, namely SN-254 and SN-311. A CX₃CL1 ELISA functional assay as an indicator of ADAM10 activity showed a decrease in CX₃CL1 cleavage after treatment with GI254023X, SN-311 and SN-254 suggesting that all three compounds substantially inhibited ADAM10 activity. The effects of these compounds on the cell biology of WHCO5 oesophageal and HeLa cervical cancer cells were monitored. Our data shows that GI254023X, SN-254 and SN-311 inhibit oesophageal and cervical cancer cell proliferation, and cause cell death via apoptosis as observed by PARP cleavage, and elevated Caspase 3/7 activity. Drug treatment also resulted in an increase in cellular adhesion as well as a significant decrease in the invasion and migration of WHC05 and HeLa cells. The effect of ADAM10 inhibition on typical markers of the epithelial to mesenchymal transition state was also examined. An increase in epithelial cell markers (E-Cadherin, B-Catenin) and a decrease in mesenchymal marker expression (Vimentin) post treatment with the compounds tested strongly suggested that ADAM10 plays a role in mesenchymal cell transition. These results suggest that ADAM10 activity is necessary for the biological phenotypes that associate with cervical and oesophageal cancer cells and that targeting ADAM10 with inhibitors have potential as anticancer therapies.
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    Modulating the expression and activity of the nuclear import protein, Karyopherin β1, in cancer cells
    (2017) Carden, Sarah; Leaner, Virna D; Van der Watt, Pauline J
    Cancer is primarily a disease of disordered gene expression; the dysregulation of thousands of different genes has been associated with the progression of many types of cancer. Previous research from our laboratory aimed at identifying genes differentially expressed in cervical cancer compared to normal patient tissue, found Karyopherin β1 (Kpnβ1), the primary nuclear import protein, to be significantly overexpressed in cervical cancer tissue. Further studies showed that inhibition of Kpnβ1 expression by siRNA resulted in cancer cell death, while non-cancer cells were only minimally affected. These results suggest that Kpnβ1 has potential as an anti-cancer therapeutic target, thus warranting further research into the association between Kpnβ1 expression and cancer progression. In this study, we investigated the biological effects associated with Kpnβ1 overexpression in order to further elucidate the relationship between Kpnβ1 and the cancer phenotype. Our data revealed that Kpnβ1 overexpression, above what was already detected in cancer cells, resulted in reduced proliferation and an associated delay in cell cycle progression. Additionally, overexpression of Kpnβ1 caused changes in the morphology and adhesion properties of cells. Co-expression of Ran, an important nuclear transport factor, binding partner and regulator of Kpnβ1, resulted in a further reduction in proliferation (greater than that of overexpression of either Kpnβ1 or Ran alone), suggesting that cells are particularly unable to handle an imbalance in the levels of Kpnβ1 and Ran. Previous work from our laboratory using a newly identified small molecule, Inhibitor of Nuclear Import-43 (INI-43), showed that INI-43 significantly reduced the proliferation of cancer cells of different tissue origins and interfered with the nuclear import function of Kpnβ1. In order to investigate the specificity of INI-43 for Kpnβ1 in this study, we performed experiments to determine whether overexpression of Kpnβ1 could rescue cancer cells from the negative effects associated with INI-43 treatment. Results revealed that stable overexpression of Kpnβ1 was able to rescue cell viability, as well as the inhibitory effects that INI-43 had on the nuclear import of Kpnβ1 cargoes NFκB p65 and NFAT. Kpnβ1 overexpression was also able to rescue cells from an INI-43 induced G2/M cell cycle block. In addition, treatment of cells with INI-43 enhanced the degradation of Kpnβ1, indicating that INI-43 is likely acting by targeting Kpnβ1. In order to determine the effects of Kpnβ1 dysregulation in HeLa cells, live cell time-lapse videomicroscopy was used to study cells as the progressed through the cell cycle. Inducible expression of Kpnβ1-EGFP, as well as the treatment of cells with the small molecule inhibitor INI-43, were used as mechanisms of dysregulation. Results revealed that modulation of Kpnβ1 by either overexpression or inhibition caused a disruption in mitotic progression, with the appearance of distinct mitotic abnormalities. The treatment of cells expressing Kpnβ1-EGFP with INI-43 resulted in a significant reduction in (or rescue of) the negative effects associated with either condition alone. Taken together, this data suggests that a precise balance of Kpnβ1 expression is required for the correct functioning of cancer cells; when the balance is perturbed in either direction (i.e. with overexpression of Kpnβ1 or INI-43-mediated inhibition of Kpnβ1) negative effects associated with a variety of biological processes are observed. In addition, results from rescue experiments conducted using Kpnβ1 overexpression in combination with INI-43 treatment suggest that that INI-43 is acting, at least in part, by targeting Kpnβ1.
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    Overexpression of Kpnβ1 and Kpnα2 Importin Proteins in Cancer Derives from Deregulated E2F Activity
    (Public Library of Science, 2011) van der Watt, Pauline J; Ngarande, Ellen; Leaner, Virna D
    The Karyopherin superfamily comprises nuclear transport proteins, involved in the shuttling of certain cargo proteins into and out of the nucleus. Karyopherin β1 (Kpnβ1) and Karyopherin α2 (Kpnα2) are importin proteins, which work in concert to transport their cargo into the nucleus. We previously identified increased expression of Kpnβ1 and Kpnα2 in cervical tumours compared to normal epithelium and in transformed cells compared to their normal counterparts. This study therefore aimed to identify the transcription regulatory mechanisms associated with high Kpnβ1 and Kpnα2 levels in cancer cells. Kpnβ1 (−2013 to +100) and Kpnα2 (−1900 to +69) promoter fragments were separately cloned into the reporter vector, pGL3-basic, and luciferase assays revealed both as significantly more active in cancer and transformed cells compared to normal. A series of deletion constructs identified the −637 to −271 Kpnβ1 and −180 to −24 Kpnα2 promoter regions as responsible for the differential promoter activity, and a number of highly conserved E2F binding sites were identified within these regions. Mutation analysis confirmed the requirement of E2F sites for promoter activity, and ChIP analysis confirmed E2F2/Dp1 binding to the Kpnβ1 and Kpnα2 promoters in vivo . Dp1 inhibition resulted in decreased levels of the respective proteins, confirming the role of E2F in the overexpression of Kpnβ1 and Kpnα2 proteins in cancer. E2F activity is known to be deregulated in cervical cancer cells due to the inhibition of its repressor, Rb, by HPV E7. The inhibition of E7 using siRNA resulted in decreased Kpnβ1 and Kpnα2 promoter activities, as did the overexpression of Rb. In conclusion, this study is a first to show that elevated Kpnβ1 and Kpnα2 expression in cancer cells correlates with altered transcriptional regulation associated with deregulated E2F/Rb activities.
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    p21-activated kinase 3 (PAK3) is an AP-1 regulated gene contributing to actin organisation and migration of transformed fibroblasts
    (Public Library of Science, 2013) Holderness-Parker, Nina; Donninger, Howard; Birrer, Michael J; Leaner, Virna D
    Activating Protein 1 (AP-1) plays a vital role in cell proliferation, differentiation and apoptosis. While de-regulation of AP-1 has been linked to many cancers, little is known regarding its downstream transcriptional targets that associate with cellular transformation. Previous studies identified 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 investigate 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 expression in cJun/AP-1 over-expressing cells associated with a significant increase in PAK3 promoter activation. This increased promoter activity was lost when a single putative Jun binding site, which can bind AP-1 directly both in vitro and in vivo, was mutated. Further, inhibition of PAK3 using siRNA showed a regression in the cell morphology, migratory potential and actin organisation associated with AP-1 transformed cells. Our study is a first to describe a role for AP-1 in regulating PAK3 expression and suggest that PAK3 is an AP-1 target required for actin organization and migration observed in transformed cells.
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    Phosphoglucomutase 1 (PGM1) expression and regulation in cancer cells
    (2015) Maranyane, Hapiloe 'Mabaruti; Leaner, Virna D
    Cancer cells undergo metabolism that is significantly different to normal cells, with an increased dependence on glucose metabolism as a hallmark of most cancers. Changes in global gene expression patterns are the major driving forces behind cancer progression. These changes trigger events that result in the dysregulation of key enzymes associated with metabolic processes. Gene expression profiling studies done previously in our laboratory identified a group of genes involved in glucose metabolism to be differentially expressed in cervical cancer patient material. Of these, Phosphoglucomutase 1 (PGM1) was identified to have elevated expression in the cancer group. PGM1 is a phosphotransferase that catalyses the reversible conversion of the glycogen breakdown product, glucose-1-phosphate into glucose-6-phosphate, a substrate for glycolysis and the pentose phosphate pathway. This places PGM1 at a critical traffic point of glucose metabolism. In this study we investigated the expression, regulation and biological significance of PGM1 in cancer cells. Our results showed that PGM1 expression was elevated in cervical cancer tissue compared to normal. Its expression was also high in cervical, oesophageal and breast cancer cell lines. Elevated PGM1 expression associated with high promoter activity as well as with E2F and HIF1α activities in cancer cells. PGM1 expression at the level of mRNA, protein and promoter activation was significantly stimulated in hypoxia mimicking conditions. Our data showed that PGM1 expression in cancer cells was required mainly for glycogen accumulation with marginal changes on glycolysis and the pentose phosphate pathway. While PGM1 expression did not appear necessary for cancer cell proliferation in normoxia and nutrient sufficiency, our data shows that it is required for proliferation under conditions of glucose deprivation combined with hypoxia. Together these findings suggest that PGM1 expression is altered in cancer cells, that it is required for aberrant glycogen expression in cancer cells and that it has a role in cancer biology during severe stress conditions.