67 research outputs found
Molecular detection and significance of circulating colorectal cancer cells / Jennifer E. Hardingham.
No full textBibliography: leaves 214-236.xviii, 238 leaves : ill. (some col.) ; 30 cm.Thesis (Ph.D.)--University of Adelaide, Dept. of Physiology, 199
Stem cell marker olfactomedin 4: critical appraisal of its characteristics and role in tumorigenesis
No full textOlfactomedin 4 (OLFM4), a member of the olfactomedin domain-containing proteins, is a glycoprotein with molecular weight of approximately 64 kDa. The protein is a "robust marker" of Lgr5+ stem cells and has been localised to mitochondria, nuclei and cell membranes. The bulk of OLFM4 exists in a polymeric form which is held together by disulfide bonds and carbohydrate interactions. Earlier studies revealed that the protein binds to lectins and cadherins, and facilitates cell-cell adhesion. Recent data demonstrated that the protein possesses several hallmarks of carcinogenesis. OLFM4 has also been purported to be an inducible resistance factor to apoptotic stimuli such as radiation and anticancer drugs. Here, we review its synonyms and classification, gene structure, protein structure, intracellular and tissue distribution, adhesive and antiapoptotic; mitotic; migratory and cell cycle regulatory characteristics. We also critically evaluate recent advances in understanding of the transcriptional regulation of OLFM4 and its upstream signalling pathways with special emphasis on carcinogenesis and outline future perspectives in the field.Phulwinder K. Grover, Jennifer E. Hardingham, Adrian G. Cummin
Differential inhibition of water and ion channel activities of mammalian aquaporin-1 by two structurally related bacopaside compounds derived from the medicinal plant bacopa monnieri
No full textAquaporin-1 (AQP1) is a major intrinsic protein that facilitates flux of water and other small solutes across cell membranes. In addition to its function as a water channel in maintaining fluid homeostasis, AQP1 also acts as a nonselective cation channel gated by cGMP, a property shown previously to facilitate rapid cell migration in a AQP1-expressing colon cancer cell line. Here we report two new modulators of AQP1 channels, bacopaside I and bacopaside II, isolated from the medicinal plant Bacopa monnieri Screening was conducted in the Xenopus oocyte expression system, using quantitative swelling and two-electrode voltage clamp techniques. Results showed bacopaside I blocked both the water (IC50 117 μM) and ion channel activities of AQP1 but did not alter AQP4 activity, whereas bacopaside II selectively blocked the AQP1 water channel (IC50 18 μM) without impairing the ionic conductance. These results fit with predictions from in silico molecular modeling. Both bacopasides were tested in migration assays using HT29 and SW480 colon cancer cell lines, with high and low levels of AQP1 expression, respectively. Bacopaside I (IC50 48 μM) and bacopaside II (IC50 14 μM) impaired migration of HT29 cells but had minimal effect on SW480 cell migration. Our results are the first to identify differential AQP1 modulators isolated from a medicinal plant. Bacopasides could serve as novel lead compounds for pharmaceutic development of selective aquaporin modulators.Jinxin V. Pei, Mohamad Kourghi, Michael L. De Ieso, Ewan M. Campbell, Hilary S. Dorward, Jennifer E. Hardingham and Andrea J. Yoo
Biology and therapeutic implications of VEGF-A splice isoforms and single-nucleotide polymorphisms in colorectal cancer
No full textTumor growth, dissemination and metastasis are dependent on angiogenesis. The predominant vascular endothelial growth factor (VEGF) isoform that plays a major role in angiogenesis is VEGF-A. Indeed, VEGF-A is implicated in promoting angiogenesis of numerous solid malignancies, including colorectal cancer (CRC). A large body of preclinical and clinical evidence indicates that the expression of specific VEGF-A isoforms represents a predominant pro-angiogenic factor, which is associated with formation of metastases and poor prognosis in CRC patients. Different isoforms of human VEGF-A have been identified, all of which arise from alternative splicing of the primary transcript of a single gene. Notably, it has been recently demonstrated that expression of type 3 isoform pattern is significantly correlated with venous involvement in CRC as well as in progression to metastatic colorectal cancer (mCRC), although it remains unclear what proportion of CRC tumors express these isoforms. This review highlights the importance of investigating the genetic and the epigenetic variations in VEGF-A pathways in CRC, the functions of different VEGF-A isoforms and their potential application as prognostic markers and/or therapeutic targets. Better understanding of the mechanisms controlling angiogenesis in liver metastases is necessary to address the limitations of current anti-angiogenic therapies.Miriam Canavese, Doan T.M. Ngo, Guy J. Maddern, Jennifer E. Hardingham, Timothy J. Price and Ehud Haube
Survey of germline variants in cancer‐associated genes in young adults with colorectal cancer
No full textCan we accurately report PTEN status in advanced colorectal cancer?
No full textBACKGROUND: Loss of phosphatase and tensin homologue (PTEN) function evaluated by loss of PTEN protein expression on immunohistochemistry (IHC) has been reported as both prognostic in metastatic colorectal cancer and predictive of response to anti-EGFR monoclonal antibodies although results remain uncertain. Difficulties in the methodological assessment of PTEN are likely to be a major contributor to recent conflicting results. METHODS: We assessed loss of PTEN function in 51 colorectal cancer specimens using Taqman® copy number variation (CNV) and IHC. Two blinded pathologists performed independent IHC assessment on each specimen and inter-observer variability of IHC assessment and concordance of IHC versus Taqman® CNV was assessed. RESULTS: Concordance between pathologists (PTEN loss vs no loss) on IHC assessment was 37/51 (73%). In specimens with concordant IHC assessment, concordance between IHC and Taqman® copy number in PTEN loss assessment was 25/37 (68%). CONCLUSION: Assessment PTEN loss in colorectal cancer is limited by the inter-observer variability of IHC, and discordance of CNV with loss of protein expression. An understanding of the genetic mechanisms of PTEN loss and implementation of improved and standardized methodologies of PTEN assessment are required to clarify the role of PTEN as a biomarker in colorectal cancer.Christopher Hocking, Jennifer E Hardingham, Vy Broadbridge, Joe Wrin, Amanda R Townsend, Niall Tebbutt, John Cooper, Andrew Ruszkiewicz, Chee Lee, and Timothy J Pric
Identification of early-stage colorectal cancer patients at risk of relapse post-resection by immunobead reverse transcription-PCR analysis of peritoneal lavage fluid for malignant cells
No full text© 2006 American Association for Cancer ResearchPurpose: Colorectal cancer patients diagnosed with stage I or II disease are not routinely offered adjuvant chemotherapy following resection of the primary tumor. However, up to 10% of stage I and 30% of stage II patients relapse within 5 years of surgery from recurrent or metastatic disease. The aim of this study was to determine if tumor-associated markers could detect disseminated malignant cells and so identify a subgroup of patients with early-stage colorectal cancer that were at risk of relapse. Experimental Design: We recruited consecutive patients undergoing curative resection for early-stage colorectal cancer. Immunobead reverse transcription-PCR of five tumor-associated markers (carcinoembryonic antigen, laminin 2, ephrin B4, matrilysin, and cytokeratin 20) was used to detect the presence of colon tumor cells in peripheral blood and within the peritoneal cavity of colon cancer patients perioperatively. Clinicopathologic variables were tested for their effect on survival outcomes in univariate analyses using the Kaplan-Meier method. A multivariate Cox proportional hazards regression analysis was done to determine whether detection of tumor cells was an independent prognostic marker for disease relapse. Results: Overall, 41 of 125 (32.8%) early-stage patients were positive for disseminated tumor cells. Patients who were marker positive for disseminated cells in post-resection lavage samples showed a significantly poorer prognosis (hazard ratio, 6.2; 95% confidence interval, 1.9-19.6; P = 0.002), and this was independent of other risk factors. Conclusion: The markers used in this study identified a subgroup of early-stage patients at increased risk of relapse post-resection for primary colorectal cancer. This method may be considered as a new diagnostic tool to improve the staging and management of colorectal cancer.Julia M. Lloyd, Cassandra M. McIver, Sally-Anne Stephenson, Peter J. Hewett, Nicholas Rieger and Jennifer E. Hardingha
Biomarkers of resistance to anti-EGFR in wild type KRAS/BRAF colorectal cancer cell lines
No full textColorectal cancer (CRC) is a leading cause of cancer death worldwide and despite significant improvement the median survival remains relatively poor. The use of targeted therapies like cetuximab and panitumumab inhibiting the epidermal growth factor receptor (EGFR) offer promise in improving patient outcomes. However, a high proportion of CRC patients show resistance to anti-EGFR therapy. Biomarkers such as mutant KRAS or BRAF predict resistance to anti-EGFR therapy in only a subset of patients and we hypothesise that other biomarkers for resistance to EGFR targeted therapies exist. The studies presented in this thesis aimed to determine other biomarkers of resistance to anti-EGFR therapy in wild type KRAS and BRAF CRC cell lines. Following RT-Profiler Array analysis, the 3 most significantly upregulated genes amongst the 3 anti-EGFR resistant CRC cell lines (SNU-C1, SW48 and COLO-320DM) were chosen as candidate biomarkers of resistance: HBEGF (heparin-binding epidermal growth factor-like growth factor), EGR1 (early growth response protein 1) and AKT3 (protein kinase B gamma) were validated using qRT-PCR. HBEGF is a member of EGF-like growth factor family is a potent inducer of tumour growth, angiogenesis, and implicated in metastasis. EGR1 is a transcription factor implicated in cell growth, survival, transformation, tumour progression. AKT3 is a serine/threonine kinase and a downstream mediator of PI3K-AKT-mTOR pathway resulting in cell proliferation, cell survival and angiogenesis. HBEGF was knocked down by 79.4% in SNUC1, EGR1 was knocked down by 85.6% in SW48 and AKT3 was knocked down by 95.3% in COLO-320DM, as validated by qRT-PCR and western blot. Following knockdown, these cell lines were treated with anti-EGFR, and SNU-C1 had proliferation rate of 49.1% (83.8% before knockdown), SW48 yielded proliferation rate of 46.9% (70% before knockdown) and COLO-320DM had proliferation rate of 64.1% (68.3% before knockdown). This suggests that the resistant phenotype of these cell lines was reversed. The expression of these markers was also elucidated using immunohistochemistry on mCRC primary tumour tissues from 10 patients that had undergone cetuximab monotherapy. Some 50% of these patients had overexpression of two or more of these markers, and these patients did not respond to cetuximab, suggesting that these overexpressed biomarkers might be involved in circumventing cetuximab to confer resistance. One of the studies presented in this thesis also explored the KRAS G13D phenomenon and the effect of cetuximab and panitumumab on cell lines harbouring different mutational status. Previous clinical studies have demonstrated that a proportion of KRAS G13D harbouring tumour patients respond to the anti-EGFR therapies, and a large proportion of KRAS WT patients do not respond. After treatment with cetuximab or panitumumab, the KRAS G13D mutant cell lines showed intermediate sensitivity to both treatments, between the resistant KRAS G12V mutant cell line and the sensitive WT KRAS cell line. One of the G13D cell lines was significantly more sensitive to panitumumab than to cetuximab. This study demonstrated that specific KRAS mutation determines the responsiveness to anti-EGFR monoclonal antibody treatment, corresponding to previously reported clinical observations. In conclusion, the studies presented in this thesis have demonstrated that components of EGFR signalling cascade have emerged as important biomarkers of resistance for anti-EGFR targeted therapies. Further assessment of the molecular mechanisms that dictate this resistance and identification of other specific biomarkers for these agents will provide valuable information to identify the most effective therapy for primary and mCRC patients.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Medical Sciences, 2015
Control of anti-apoptotic and antioxidant pathways in neural cells
No full textOxidative stress is a feature of many chronic neurodegenerative diseases as well as a
contributing factor in acute disorders including stroke. Fork head class of
transcription factors (Foxos) play a key role in promoting oxidative stress-induced
apoptosis in neurons through the upregulation of a number of pro-apoptotic genes.
Here I demonstrate that synaptic NMDA receptor activity not only promotes Foxos
nuclear exclusion but also suppresses the expression of Foxo1 in a PI3K-dependent
fashion. I also found that Foxo1 is in fact, a Foxo target gene and that it is subject to
a feed-forward inhibition by synaptic activity, which is thought to result in longerterm
suppression of Foxo downstream gene expression than previously thought. The
nuclear factor (erythroid 2-related) factor 2 (Nrf2) is another transcription factor
involved in oxidative stress and the key regulator of many genes, whose products
form important intrinsic antioxidant systems. In the CNS, artificial activation of Nrf2
in astrocytes has been shown to protect nearby neurons from oxidative insults.
However, the extent to which Nrf2 in astrocytes could respond to endogenous signals
such as mild oxidative stress is less clear. The data presented herein, demonstrate for
the first time that endogenous Nrf2 could be activated by mild oxidative stress and
that this activation is restricted to astrocytes. Contrary to the established dogma, I
found that mild oxidative stress induces the astrocytic Nrf2 pathway in a manner
distinct from the classical Keap1 antagonism employed by prototypical Nrf2
inducers. The mechanism was found to involve direct regulation of Nrf2's
transactivation properties. Overall these results advance our knowledge of the
molecular mechanism(s) associated with the control of endogenous antioxidant
defences by physiological signals
Nanostructured polystyrene well plates allow unbiased high-throughput characterization of circulating tumor cells
No full textRapid, reliable and unbiased circulating tumor cell (CTC) isolation and molecular characterization methods are urgently required for implementation in routine clinical diagnostic and prognostic procedures. We report on the development of a novel unbiased CTC detection approach that combines high-throughput automated microscopy with a simple yet efficient approach for achieving a high level of tumor cell binding in standard tissue culture polystyrene (PS) well plates. A single 5 min high-power oxygen plasma treatment was used to create homogeneous nanoscale roughness on standard PS tissue culture plates and, in turn, drastically enhance the binding of a range of tumor cells. After physical adsorption of an adlayer of poly-l-lysine, binding yields above 97% were obtained at 2 h for all tumor cell lines used in the study. Morphological analysis of the cells confirmed strong adherence to the nanorough PS substrates. Clinically relevant concentrations of a highly metastatic breast cancer cell line, used as model for CTCs, could be reliably detected among blood cells on the nanorough polystyrene plates using an automated microscopy system. The approach was then successfully used to detect CTCs in the blood of a stage IIIc colorectal cancer patient. By combining the high binding abilities of nanorough PS well plates with the high-throughput nature of high-content analysis systems, this methodology has great potential toward enabling unbiased routine clinical analysis of CTCs. It could be applied, once clinically validated, in any clinical center equipped with an automated microscopy facility at a fraction of the cost of current CTC isolation technologies.Yuan Wan, Marnie Winter, Bahman Delalat, Jennifer E. Hardingham, Phulwinder K. Grover, Joseph Wrin, Nicolas H. Voelcker, Timothy J. Price, and Benjamin Thierr
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