ORCID Profile
0009-0001-0659-1222
Current Organisation
University of Newcastle Australia
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Publisher: Springer Science and Business Media LLC
Date: 08-04-2023
Publisher: Springer Science and Business Media LLC
Date: 25-07-2017
DOI: 10.1007/S10549-017-4403-5
Abstract: Protein phosphatase 2A (PP2A) is a family of serine/threonine phosphatases that regulate multiple cellular signalling pathways involved in proliferation, survival and apoptosis. PP2A inhibition occurs in many cancers and is considered a tumour suppressor. Deletion/downregulation of PP2A genes has been observed in breast tumours, but the functional role of PP2A subunit loss in breast cancer has not been investigated. PP2A subunit expression was examined by immunohistochemistry in human breast tumours, and by qPCR and immunoblotting in breast cancer cell lines. PP2A subunits were inhibited by shRNA, and mutant PP2A genes overexpressed, in MCF10A and MCF7 cells, and growth and signalling in standard and three-dimensional cultures were assessed. Expression of PP2A-Aα, PP2A-Bα and PP2A-B'α subunits was significantly lower in primary human breast tumours and lymph node metastases, compared to normal mammary tissue. PP2A-Aα and the regulatory subunits PP2A-Bα, -Bδ and -B'γ were also reduced in breast cancer cell lines compared to normal mammary epithelial cells. Functionally, shRNA-mediated knockdown of PP2A-Bα, -B'α and -B'γ, but not PP2A-Aα, induced hyper-proliferation and large multilobular acini in MCF10A 3D cultures, characterised by activation of ERK. Expression of a breast cancer-associated PP2A-A mutant, PP2A-Aα-E64G, which inhibits binding of regulatory subunits to the PP2A core, induced a similar hyper-proliferative phenotype. Knockdown of PP2A-Bα also induced hyper-proliferation in MCF7 breast cancer cells. These results suggest that loss of specific PP2A regulatory subunits is functionally important in breast tumourigenesis, and support strategies to enhance PP2A activity as a therapeutic approach in breast cancer.
Publisher: Springer Science and Business Media LLC
Date: 16-10-2020
Publisher: Elsevier BV
Date: 04-2014
DOI: 10.1016/J.CELLSIG.2013.12.015
Abstract: Calcium/calmodulin-stimulated protein kinase II (CaMKII) is a multi-functional serine/threonine protein kinase that controls a range of cellular functions, including proliferation. The biological properties of CaMKII are regulated by multi-site phosphorylation and targeting via interactions with specific proteins. To investigate the role specific CaMKII phosphorylation sites play in controlling cell proliferation and cell cycle progression, we examined phosphorylation of CaMKII at two sites (T253 and T286) at various stages of the cell cycle, and also examined the effects of overexpression of wild-type (WT), T286D phosphomimic, T253D phosphomimic and T253V phosphonull forms of CaMKIIα in MDA-MB-231 breast cancer and SHSY5Y neuroblastoma cells on cellular proliferation and cell cycle progression. We demonstrate herein that whilst there is no change in total CaMKII expression or T286 phosphorylation throughout the cell cycle, a marked dephosphorylation of CaMKII at T253 occurs during the G2 and/or M phases. Additionally, we show by molecular inhibition, as well as pharmacological activation, that protein phosphatase 2A (PP2A) is the phosphatase responsible for this dephosphorylation. Furthermore, we show that inducible overexpression of WT, T286D and T253V forms of CaMKIIα in MDA-MB-231 and SHSY5Y cells increases cellular proliferation, with no alteration in cell cycle profiles. By contrast, overexpression of a T253D phosphomimic form of CaMKIIα significantly decreases proliferation, and cells accumulate in mitosis, specifically in metaphase. Taken together, these results strongly suggest that the dephosphorylation of CaMKII at T253 is involved in controlling the cell cycle, specifically the metaphase-anaphase transition.
Publisher: Bioscientifica
Date: 11-2021
DOI: 10.1530/REP-20-0650
Abstract: This study aimed to determine if the (pro)renin receptor (ATP6AP2) changes the cellular profile of choriocarcinomas from cytotrophoblast cells to terminally syncytialised cells and ascertain whether this impacts the invasive potential of choriocarcinoma cells. Additionally, we aimed to confirm that FURIN and/or site 1 protease (MBTPS1) cleave soluble ATP6AP2 (sATP6AP2) in BeWo choriocarcinoma cells and determine whether sATP6AP2 levels reflect the cellular profile of choriocarcinomas. BeWo choriocarcinoma cells were treated with ATP6AP2 siRNA, FURIN siRNA, DEC-RVKR-CMK (to inhibit FURIN activity), or PF 429242 (to inhibit MBTPS1 activity). Cells were also treated with forskolin, to induce syncytialisation, or vehicle and incubated for 48 h before collection of cells and supernatants. Syncytialisation was assessed by measuring hCG secretion (by ELISA) and E-cadherin protein levels (by immunoblot and immunocytochemistry). Cellular invasion was measured using the xCELLigence real-time cell analysis system and secreted sATP6AP2 levels measured by ELISA. Forskolin successfully induced syncytialisation and significantly increased both BeWo choriocarcinoma cell invasion ( P 0.0001) and sATP6AP2 levels ( P = 0.02). Treatment with ATP6AP2 siRNA significantly inhibited syncytialisation (decreased hCG secretion ( P = 0.005), the percent of nuclei in syncytia ( P = 0.05)), forskolin-induced invasion ( P = 0.046), and sATP6AP2 levels ( P 0.0001). FURIN siRNA and DEC-RVKR-CMK significantly decreased sATP6AP2 levels (both P 0.0001). In conclusion, ATP6AP2 is important for syncytialisation of choriocarcinoma cells and thereby limits choriocarcinoma cell invasion. We postulate that sATP6AP2 could be used as a biomarker measuring the invasive potential of choriocarcinomas. Additionally, we confirmed that FURIN, not MBTPS1, cleaves sATP6AP2 in BeWo cells, but other proteases (inhibited by DEC-RVKR-CMK) may also be involved.
Publisher: Springer Science and Business Media LLC
Date: 11-07-2014
Publisher: Springer Science and Business Media LLC
Date: 08-04-2202
Publisher: Impact Journals, LLC
Date: 18-06-2016
Publisher: Springer Science and Business Media LLC
Date: 12-2022
DOI: 10.1186/S12014-022-09385-7
Abstract: Global high-throughput phosphoproteomic profiling is increasingly being applied to cancer specimens to identify the oncogenic signaling cascades responsible for promoting disease initiation and disease progression pathways that are often invisible to genomics analysis. Hence, phosphoproteomic profiling has enormous potential to inform and improve in idualized anti-cancer treatment strategies. However, to achieve the adequate phosphoproteomic depth and coverage necessary to identify the activated, and hence, targetable kinases responsible for driving oncogenic signaling pathways, affinity phosphopeptide enrichment techniques are required and often coupled with offline high-pressure liquid chromatographic (HPLC) separation prior to nanoflow liquid chromatography–tandem mass spectrometry (nLC-MS/MS). These complex and time-consuming procedures, limit the utility of phosphoproteomics for the analysis of in idual cancer patient specimens in real-time, and restrict phosphoproteomics to specialized laboratories often outside of the clinical setting. To address these limitations, here we have optimized a new protocol, p hospho- h eavy-l a beled-spiketide FAIM S st e pped-CV D DA (pHASED), that employs online phosphoproteome deconvolution using high- f ield a symmetric waveform i on m obility s pectrometry (FAIMS) and internal phosphopeptide standards to provide accurate label-free quantitation (LFQ) data in real-time. Compared with traditional single-shot LFQ phosphoproteomics workflows, pHASED provided increased phosphoproteomic depth and coverage (phosphopeptides = 4617 pHASED, 2789 LFQ), whilst eliminating the variability associated with offline prefractionation. pHASED was optimized using tyrosine kinase inhibitor (sorafenib) resistant isogenic FLT3-mutant acute myeloid leukemia (AML) cell line models. Bioinformatic analysis identified differential activation of the serine/threonine protein kinase ataxia-telangiectasia mutated (ATM) pathway, responsible for sensing and repairing DNA damage in sorafenib-resistant AML cell line models, thereby uncovering a potential therapeutic opportunity. Herein, we have optimized a rapid, reproducible, and flexible protocol for the characterization of complex cancer phosphoproteomes in real-time, a step towards the implementation of phosphoproteomics in the clinic to aid in the selection of anti-cancer therapies for patients.
Publisher: Elsevier BV
Date: 03-2023
Publisher: Frontiers Media SA
Date: 05-06-2020
Publisher: Springer Science and Business Media LLC
Date: 30-08-2023
DOI: 10.1007/S43032-023-01333-6
Abstract: Metabolic inactivation of progesterone within uterine myocytes by 20α-hydroxysteroid dehydrogenase (20α-HSD) has been postulated as a mechanism contributing to functional progesterone withdrawal at term. In humans, 20α-HSD is encoded by the gene AKR1C1 . Myometrial AKR1C1 mRNA abundance has been reported to increase significantly during labor at term. In spontaneous preterm labor, however, we previously found no increase in AKR1C1 mRNA level in the myometrium except for preterm labor associated with clinical chorioamnionitis. This suggests that increased 20α-HSD activity is a mechanism through which inflammation drives progesterone withdrawal in preterm labor. In this study, we have determined the effects of various treatments of therapeutic relevance on AKR1C1 expression in pregnant human myometrium in an ex vivo culture system. AKR1C1 expression increased spontaneously during 48 h culture ( p 0.0001), consistent with the myometrium transitioning to a labor-like phenotype ex vivo , as reported previously. Serum supplementation, prostaglandin F 2α , phorbol myristate acetate, and mechanical stretch had no effect on the culture-induced increase, whereas progesterone ( p = 0.0058) and cAMP ( p = 0.0202) further upregulated AKR1C1 expression. In contrast, culture-induced upregulation of AKR1C1 expression was dose-dependently repressed by three histone rotein deacetylase inhibitors: trichostatin A at 5 ( p = 0.0172) and 25 µM ( p = 0.0115) suberoylanilide hydroxamic acid at 0.5 ( p = 0.0070), 1 ( p = 0.0045), 2.5 ( p = 0.0181), 5 ( p = 0.0066) and 25 µM ( p = 0.0014) and suberoyl bis-hydroxamic acid at 5 ( p = 0.0480) and 25 µM ( p = 0.0238). We propose the inhibition of histone rotein deacetylation helps to maintain the anti-inflammatory, pro-quiescence signaling of progesterone in pregnant human myometrium by blocking its metabolic inactivation. Histone deacetylase inhibitors may represent a class of agents that preserve or restore the progesterone sensitivity of the pregnant uterus.
Publisher: MDPI AG
Date: 26-03-2020
Abstract: Breast cancer is the most commonly diagnosed and the second leading cause of cancer-related mortality among women worldwide. miR-518f-5p has been shown to modulate the expression of the metastasis suppressor CD9 in prostate cancer. However, the role of miR-518f-5p and CD9 in breast cancer is unknown. Therefore, this study aimed to elucidate the role of miR-518f-5p and the mechanisms responsible for decreased CD9 expression in breast cancer, as well as the role of CD9 in de novo tumor formation and metastasis. miR-518f-5p function was assessed using migration, adhesion, and proliferation assays. miR-518f-5p was overexpressed in breast cancer cell lines that displayed significantly lower CD9 expression as well as less endogenous CD9 3′UTR activity, as assessed using qPCR and dual luciferase assays. Transfection of miR-518f-5p significantly decreased CD9 protein expression and increased breast cell migration in vitro. Cd9 deletion in the MMTV/PyMT mouse model impaired tumor growth, but had no effect on tumor initiation or metastasis. Therefore, inhibition of miR-518f-5p may restore CD9 expression and aid in the treatment of breast cancer metastasis.
No related grants have been discovered for Richard Kahl.