RFWD3 modulates response to platinum chemotherapy and promotes cancer associated phenotypes in high grade serous ovarian cancer.

Background: DNA damage repair is frequently dysregulated in high grade serous ovarian cancer (HGSOC), which can lead to changes in chemosensitivity and other phenotypic differences in tumours. RFWD3, a key component of multiple DNA repair and maintenance pathways, was investigated to characterise its impact in HGSOC. Methods: RFWD3 expression and association with clinical features was assessed using in silico analysis in the TCGA HGSOC dataset, and in a further cohort of HGSOC tumours stained for RFWD3 using immunohistochemistry. RFWD3 expression was modulated in cell lines using siRNA and CRISPR/cas9 gene editing, and cells were characterised using cytotoxicity and proliferation assays, flow cytometry, and live cell microscopy. Results: Expression of RFWD3 RNA and protein varied in HGSOCs. In cell lines, reduction of RFWD3 expression led to increased sensitivity to interstrand crosslinking (ICL) inducing agents mitomycin C and carboplatin. RFWD3 also demonstrated further functionality outside its role in DNA damage repair, with RFWD3 deficient cells displaying cell cycle dysregulation, reduced cellular proliferation and reduced migration. In tumours, low RFWD3 expression was associated with increased tumour mutational burden, and complete response to platinum chemotherapy. Conclusion: RFWD3 expression varies in HGSOCs, which can lead to functional effects at both the cellular and tumour levels.

Variation in IL6ST cytokine family function and the potential of IL6 trans-signalling in ERα positive breast cancer cells.

High expression of the transmembrane receptor IL6ST (gp130) has been identified as a predictive biomarker of endocrine treatment response in ERα-positive breast cancers. To investigate its function further in this disease, this study evaluated the expression, function and signalling of IL6ST in ERα-positive breast cancer cell lines and investigated crosstalk between ERα and IL6ST. IL6ST was differentially expressed in ERα-positive breast cancer cell lines (low in MCF-7, high in ZR751 and T47D), while multiple soluble isoforms of IL6ST were identified. IL6ST is the common signal transducing receptor component for the IL6ST family of cytokines and the effects of seven IL6ST cytokines on these cell lines were studied. These cytokines caused differential growth and migration effects in these cell lines e.g. MCF-7 cells were growth-stimulated, while ZR751 cells were inhibited by IL6 and OSM.. Activation of the STAT and ERK pathways is associated with these responses. Evidence to support trans-signalling involved in cell growth and migration was obtained in both MCF-7 and ZR751 models. Interaction between cytokines and estrogen on ERα-positive cell lines growth were analysed. High expression of IL6ST (in ZR751) may lead to growth inhibition by interacting cytokines while lower expression (in MCF-7) appears associated with proliferation. High IL6ST expression is consistent with a more beneficial clinical outcome if cytokine action contributes to anti-estrogen action.

Collateral-resistance to estrogen and HER-activated growth is associated with modified AKT, ERα, and cell-cycle signaling in a breast cancer model.

Aim: A model of progressively endocrine-resistant breast cancer was investigated to identify changes that can occur in signaling pathways after endocrine manipulation. Methods: The MCF7 breast cancer model is sensitive to estrogens and anti-estrogens while variant lines previously derived from wild-type MCF7 are either relatively 17ß-estradiol (E2)-insensitive (LCC1) or fully resistant to estrogen and anti-estrogens (LCC9). Results: In LCC1 and LCC9 cell lines, loss of estrogen sensitivity was accompanied by loss of growth response to transforming growth factor alpha (TGFα), heregulin-beta and pertuzumab. LCC1 and LCC9 cells had enhanced AKT phosphorylation relative to MCF7 which was reflected in downstream activation of phospho-mechanistic target of rapamycin (mTOR), phospho-S6, and phospho-estrogen receptor alpha Ser167 [ERα(Ser167)]. Both AKT2 and AKT3 were phosphorylated in the resistant cell lines, but small interfering RNA (siRNA) knockdown suggested that all three AKT isoforms contributed to growth response. ERα(Ser118) phosphorylation was increased by E2 and TGFα in MCF7, by E2 only in LCC1, but by neither in LCC9 cells. Multiple alterations in E2-mediated cell cycle control were identified in the endocrine-resistant cell lines including increased expression of MYC, cyclin A1, cyclin D1, cyclin-dependent kinase 1 (CDK1), CDK2, and hyperphosphorylated retinoblastoma protein (ppRb), whereas p21 and p27 were reduced. Estrogen modulated expression of these regulators in MCF7 and LCC1 cells but not in LCC9 cells. Seliciclib inhibited CDK2 activation in MCF7 cells but not in resistant variants; in all lines, it reduced ppRb, increased p53 associated responses including p21, p53 up-regulated modulator of apoptosis (PUMA), and p53 apoptosis-inducing protein 1 (p53AIP1), inhibited growth, and produced G2/M block and apoptosis. Conclusions: Multiple changes occur with progression of endocrine resistance in this model with AKT activation contributing to E2 insensitivity and loss of ERα(Ser118) phosphorylation being associated with full resistance. Cell cycle regulation is modified in endocrine-resistant breast cancer cells, and seliciclib is effective in both endocrine-sensitive and resistant diseases.

Nuclear factor erythroid 2-related factor 2 modulates HER4 receptor in ovarian cancer cells to influence their sensitivity to tyrosine kinase inhibitors.

Aim: Nuclear factor erythroid 2-related factor 2 (NRF2) is a key component in the cell's response to oxidative and electrophilic stress and is a transcription factor regulating the expression of a collection of anti-oxidative and cytoprotective genes. Human epidermal growth factor receptor 4 (HER4/erbB4) regulates growth and differentiation in many cancer types. Here, NRF2 and HER4 receptor interactions were investigated in a panel of ovarian cancer cell lines. Methods: Pharmacological [tert-butylhydroquinone (tBHQ) and retinoid/rexinoid, bexarotene] and genetic [small interfering RNA (siRNA)] manipulations were used to activate or inhibit NRF2 function in the cell line panel (PE01, OVCAR3, SKOV3). Activity of the HER-targeted tyrosine kinase inhibitors, erlotinib (ERL) and lapatinib (LAP), was evaluated after NRF2 activation. Results: While tBHQ increased the levels of both phosphorylated-NRF2 (pNRF2) and HER4 in PE01, OVCAR3 and SKOV3 cells, bexatorene and NRF2-target siRNA treatment decreased pNRF2 and total HER4 levels. The tBHQ-dependent pharmacological activation of NRF2 attenuated the therapeutic effectiveness of ERL and LAP. Analyses of gene expression data from a HER4 driven reporter system and in vitro or in vivo cancer models, support NRF2 regulation of HER4 expression. Conclusions: These results support the presence of signaling interaction between the NRF2 and HER4 receptor pathways and suggest that intervention modulating this cross-talk could have anticancer therapeutic value.

Estrogen Receptor Signaling in Cancer.

Estrogen receptor signaling plays.

Evaluation of Gene Expression Data From Cybrids and Tumours Highlights Elevated NDRG1-Driven Proliferation in Triple-Negative Breast Cancer.

BACKGROUND: Triple-negative breast cancer is an aggressive type of breast cancer with high risk of recurrence. It is still poorly understood and lacks any targeted therapy, which makes it difficult to treat. Thus, it is important to understand the underlying mechanisms and pathways that are dysregulated in triple-negative breast cancer. METHODS: To investigate the role of mitochondria in triple-negative breast cancer progression, we analysed previously reported gene expression data from triple-negative breast cancer cybrids with SUM-159 as the nuclear donor cell and SUM-159 or A1N4 (c-SUM-159, c-A1N4) as the mitochondrial donor cells and with 143B as the nuclear donor cell and MCF-10A or MDA-MB-231 (c-MCF-10A, c-MDA-MB-231) as the mitochondrial donor cells. The role of potential biomarkers in cell proliferation and migration was examined in SUM-159 and MDA-MB-231 cells using sulforhodamine B and wound healing assays. RESULTS: Rank product analysis of cybrid gene expression data identified 149 genes which were significantly up-regulated in the cybrids with mitochondria from the cancer cell line. Analysis of previously reported breast tumour gene expression datasets confirmed 9 of the 149 genes were amplified, up-regulated, or down-regulated in more than 10% of the patients. The genes included NDRG1, PVT1, and EXT1, which are co-located in cytoband 8q24, which is frequently amplified in breast cancer. NDRG1 showed the largest down-regulation in the cybrids with benign mitochondria and was associated with poor prognosis in a breast cancer clinical dataset. Knockdown of NDRG1 expression significantly decreased proliferation of SUM-159 triple-negative breast cancer cells. CONCLUSIONS: These results indicate that mitochondria-regulated nuclear gene expression helps breast cancer cells survive and proliferate, consistent with previous work focusing on an Src gene signature which is mitochondria regulated and drives malignancy in breast cancer cybrids. This is the first study to show that mitochondria in triple-negative breast cancer mediate significant up-regulation of a number of genes, and silencing of NDRG1 leads to significant reduction in proliferation.

Estrogen Signaling and Its Potential as a Target for Therapy in Ovarian Cancer.

The estrogen receptor (ER) has functionality in selected ovarian cancer subtypes and represents a potential target for therapy. The majority (>80%) of high grade serous, low grade serous and endometrioid carcinomas and many granulosa cell tumors express ER-alpha (ERα), and these tumor types have demonstrated responses to endocrine therapy (tamoxifen and aromatase inhibitors) in multiple clinical studies. Biomarkers of responses to these drugs are actively being sought to help identify responsive cancers. Evidence for both pro-proliferative and pro-migratory roles for ERα has been obtained in model systems. ER-beta (ERß) is generally considered to have a tumor suppressor role in ovarian cancer cells, being associated with the repression of cell growth and invasion. The differential expression of the specific ERß isoforms may determine functionality within ovarian cancer cells. The more recently identified G protein-coupled receptor (GPER1; GPR30) has been shown to mediate both tumor-suppressive and tumor-promoting action in ovarian cancer cells, suggesting a more complex role. This review will summarize recent findings in this field.

Precision Medicine and the Role of Biomarkers of Radiotherapy Response in Breast Cancer.

Radiotherapy remains an important treatment modality in nearly two thirds of all cancers, including the primary curative or palliative treatment of breast cancer. Unfortunately, largely due to tumor heterogeneity, tumor radiotherapy response rates can vary significantly, even between patients diagnosed with the same tumor type. Although in recent years significant technological advances have been made in the way radiation can be precisely delivered to tumors, it is proving more difficult to personalize radiotherapy regimens based on cancer biology. Biomarkers that provide prognostic or predictive information regarding a tumor's intrinsic radiosensitivity or its response to treatment could prove valuable in helping to personalize radiation dosing, enabling clinicians to make decisions between different treatment options whilst avoiding radiation-induced toxicity in patients unlikely to gain therapeutic benefit. Studies have investigated numerous ways in which both patient and tumor radiosensitivities can be assessed. Tumor molecular profiling has been used to develop radiosensitivity gene signatures, while the assessment of specific intracellular or secreted proteins, including circulating tumor cells, exosomes and DNA, has been performed to identify prognostic or predictive biomarkers of radiation response. Finally, the investigation of biomarkers related to radiation-induced toxicity could provide another means by which radiotherapy could become personalized. In this review, we discuss studies that have used these methods to identify or develop prognostic/predictive signatures of radiosensitivity, and how such assays could be used in the future as a means of providing personalized radiotherapy.

The impact of tumour pH on cancer progression: strategies for clinical intervention.

Dysregulation of cellular pH is frequent in solid tumours and provides potential opportunities for therapeutic intervention. The acidic microenvironment within a tumour can promote migration, invasion and metastasis of cancer cells through a variety of mechanisms. Pathways associated with the control of intracellular pH that are under consideration for intervention include carbonic anhydrase IX, the monocarboxylate transporters (MCT, MCT1 and MCT4), the vacuolar-type H+-ATPase proton pump, and the sodium-hydrogen exchanger 1. This review will describe progress in the development of inhibitors to these targets.

Inhibitors of the Fanconi anaemia pathway as potential antitumour agents for ovarian cancer.

The Fanconi anaemia (FA) pathway is an important mechanism for cellular DNA damage repair, which functions to remove toxic DNA interstrand crosslinks. This is particularly relevant in the context of ovarian and other cancers which rely extensively on interstrand cross-link generating platinum chemotherapy as standard of care treatment. These cancers often respond well to initial treatment, but reoccur with resistant disease and upregulation of DNA damage repair pathways. The FA pathway is therefore of great interest as a target for therapies that aim to improve the efficacy of platinum chemotherapies, and reverse tumour resistance to these. In this review, we discuss recent advances in understanding the mechanism of interstrand cross-link repair by the FA pathway, and the potential of the component parts as targets for therapeutic agents. We then focus on the current state of play of inhibitor development, covering both the characterisation of broad spectrum inhibitors and high throughput screening approaches to identify novel small molecule inhibitors. We also consider synthetic lethality between the FA pathway and other DNA damage repair pathways as a therapeutic approach.

Evaluation of the dual mTOR/PI3K inhibitors Gedatolisib (PF-05212384) and PF-04691502 against ovarian cancer xenograft models.

This study investigated the antitumour effects of two dual mTOR/PI3K inhibitors, gedatolisib (WYE-129587/PKI-587/PF-05212384) and PF-04691502 against a panel of six human patient derived ovarian cancer xenograft models. Both dual mTOR/PI3K inhibitors demonstrated antitumour activity against all xenografts tested. The compounds produced tumour stasis during the treatment period and upon cessation of treatment, tumours re-grew. In several models, there was an initial rapid reduction of tumour volume over the first week of treatment before tumour stasis. No toxicity was observed during treatment. Biomarker studies were conducted in two xenograft models; phospho-S6 (Ser235/236) expression (as a readout of mTOR activity) was reduced over the treatment period in the responding xenograft but expression increased to control (no treatment) levels on cessation of treatment. Phospho-AKT (Ser473) expression (as a readout of PI3K) was inhibited by both drugs but less markedly so than phospho-S6 expression. Initial tumour volume reduction on treatment and regrowth rate after treatment cessation was associated with phospho-S6/total S6 expression ratio. Both drugs produced apoptosis but minimally influenced markers of proliferation (Ki67, phospho-histone H3). These results indicate that mTOR/PI3K inhibition can produce broad spectrum tumour growth stasis in ovarian cancer xenograft models during continuous chronic treatment and this is associated with apoptosis.

Carbonic anhydrase inhibitors based on sorafenib scaffold: Design, synthesis, crystallographic investigation and effects on primary breast cancer cells.

Carbonic anhydrase inhibitors (CAIs) of the sulfonamide, sulfamate and coumarin classes bearing the phenylureido tail found in the clinically used drug Sorafenib, a multikinase inhibitor actually used for the management of hepatocellular carcinomas, are reported. All compounds were assayed on human (h) CA isoforms I, II, VII and IX, involved in various pathologies. Among the sulfonamides, several compounds were selective for inhibiting hCA IX, with KI values in the low nanomolar ranges (i.e. 0.7-30.2 nM). We explored the binding modes of such compounds by means of X-ray crystallographic studies on isoform hCA I in adduct with one sulfonamide and a sulfamate inhibitor. Antiproliferative properties of some sulfamates on breast tumor cell lines were also investigated.

Development and characterisation of acquired radioresistant breast cancer cell lines.

BACKGROUND: Radiotherapy plays an important role in the multimodal treatment of breast cancer. The response of a breast tumour to radiation depends not only on its innate radiosensitivity but also on tumour repopulation by cells that have developed radioresistance. Development of effective cancer treatments will require further molecular dissection of the processes that contribute to resistance. METHODS: Radioresistant cell lines were established by exposing MDA-MB-231, MCF-7 and ZR-751 parental cells to increasing weekly doses of radiation. The development of radioresistance was evaluated through proliferation and colony formation assays. Phenotypic characterisation included migration and invasion assays and immunohistochemistry. Transcriptomic data were also generated for preliminary hypothesis generation involving pathway-focused analyses. RESULTS: Proliferation and colony formation assays confirmed radioresistance. Radioresistant cells exhibited enhanced migration and invasion, with evidence of epithelial-to-mesenchymal-transition. Significantly, acquisition of radioresistance in MCF-7 and ZR-751 cell lines resulted in a loss of expression of both ERα and PgR and an increase in EGFR expression; based on transcriptomic data they changed subtype classification from their parental luminal A to HER2-overexpressing (MCF-7 RR) and normal-like (ZR-751 RR) subtypes, indicating the extent of phenotypic changes and cellular plasticity involved in this process. Radioresistant cell lines derived from ER+ cells also showed a shift from ER to EGFR signalling pathways with increased MAPK and PI3K activity. CONCLUSIONS: This is the first study to date that extensively describes the development and characterisation of three novel radioresistant breast cancer cell lines through both genetic and phenotypic analysis. More changes were identified between parental cells and their radioresistant derivatives in the ER+ (MCF-7 and ZR-751) compared with the ER- cell line (MDA-MB-231) model; however, multiple and likely interrelated mechanisms were identified that may contribute to the development of acquired resistance to radiotherapy.

Preclinical Organotypic Models for the Assessment of Novel Cancer Therapeutics and Treatment.

The immense costs in both financial terms and preclinical research effort that occur in the development of anticancer drugs are unfortunately not matched by a substantial increase in improved clinical therapies due to the high rate of failure during clinical trials. This may be due to issues with toxicity or lack of clinical effectiveness when the drug is evaluated in patients. Currently, much cancer research is driven by the need to develop therapies that can exploit cancer cell adaptations to conditions in the tumor microenvironment such as acidosis and hypoxia, the requirement for more-specific, targeted treatments, or the exploitation of 'precision medicine' that can target known genomic changes in patient DNA. The high attrition rate for novel anticancer therapies suggests that the preclinical methods used in screening anticancer drugs need improvement. This chapter considers the advantages and disadvantages of 3D organotypic models in both cancer research and cancer drug screening, particularly in the areas of targeted drugs and the exploitation of genomic changes that can be used for therapeutic advantage in precision medicine.

HER2 regulates HIF-2α and drives an increased hypoxic response in breast cancer.

BACKGROUND: Tumour hypoxia is a driver of breast cancer progression associated with worse prognosis and more aggressive disease. The cellular response to hypoxia is mediated by the hypoxia-inducible transcription factors HIF-1 and HIF-2, whose transcriptional activity is canonically regulated through their oxygen-labile HIF-α subunits. These are constitutively degraded in the presence of oxygen; however, HIF-1α can be stabilised, even at high oxygen concentrations, through the activation of HER receptor signalling. Despite this, there is still limited understanding on how HER receptor signalling interacts with HIF activity to contribute to breast cancer progression in the context of tumour hypoxia. METHODS: 2D and 3D cell line models were used alongside microarray gene expression analysis and meta-analysis of publicly available gene expression datasets to assess the impact of HER2 overexpression on HIF-1α/HIF-2α regulation and to compare the global transcriptomic response to acute and chronic hypoxia in an isogenic cell line model of HER2 overexpression. RESULTS: HER2 overexpression in MCF7 cells leads to an increase in HIF-2α but not HIF-1α expression in normoxia and an increased upregulation of HIF-2α in hypoxia. Global gene expression analysis showed that HER2 overexpression in these cells promotes an exaggerated transcriptional response to both short-term and long-term hypoxia, with increased expression of numerous hypoxia response genes. HIF-2α expression is frequently higher in HER2-overexpressing tumours and is associated with worse disease-specific survival in HER2-positive breast cancer patients. HER2-overexpressing cell lines demonstrate an increased sensitivity to targeted HIF-2α inhibition through either siRNA or the use of a small molecule inhibitor of HIF-2α translation. CONCLUSIONS: This study suggests an important interplay between HER2 expression and HIF-2α in breast cancer and highlights the potential for HER2 to drive the expression of numerous hypoxia response genes in normoxia and hypoxia. Overall, these findings show the importance of understanding the regulation of HIF activity in a variety of breast cancer subtypes and points to the potential of targeting HIF-2α as a therapy for HER2-positive breast cancer.

Emerging role of nuclear factor erythroid 2-related factor 2 in the mechanism of action and resistance to anticancer therapies.

Nuclear factor E2-related factor 2 (NRF2), a transcription factor, is a master regulator of an array of genes related to oxidative and electrophilic stress that promote and maintain redox homeostasis. NRF2 function is well studied in in vitro, animal and general physiology models. However, emerging data has uncovered novel functionality of this transcription factor in human diseases such as cancer, autism, anxiety disorders and diabetes. A key finding in these emerging roles has been its constitutive upregulation in multiple cancers promoting pro-survival phenotypes. The survivability pathways in these studies were mostly explained by classical NRF2 activation involving KEAP-1 relief and transcriptional induction of reactive oxygen species (ROS) neutralizing and cytoprotective drug-metabolizing enzymes (phase I, II, III and 0). Further, NRF2 status and activation is associated with lowered cancer therapeutic efficacy and the eventual emergence of therapeutic resistance. Interestingly, we and others have provided further evidence of direct NRF2 regulation of anticancer drug targets like receptor tyrosine kinases and DNA damage and repair proteins and kinases with implications for therapy outcome. This novel finding demonstrates a renewed role of NRF2 as a key modulatory factor informing anticancer therapeutic outcomes, which extends beyond its described classical role as a ROS regulator. This review will provide a knowledge base for these emerging roles of NRF2 in anticancer therapies involving feedback and feed forward models and will consolidate and present such findings in a systematic manner. This places NRF2 as a key determinant of action, effectiveness and resistance to anticancer therapy.

Biocompatibility of common implantable sensor materials in a tumor xenograft model.

Real-time monitoring of tumor microenvironment parameters using an implanted biosensor could provide valuable information on the dynamic nature of a tumor's biology and its response to treatment. However, following implantation biosensors may lose functionality due to biofouling caused by the foreign body response (FBR). This study developed a novel tumor xenograft model to evaluate the potential of six biomaterials (silicon dioxide, silicon nitride, Parylene-C, Nafion, biocompatible EPOTEK epoxy resin, and platinum) to trigger a FBR when implanted into a solid tumor. Biomaterials were chosen based on their use in the construction of a novel biosensor, designed to measure spatial and temporal changes in intra-tumoral O2 , and pH. None of the biomaterials had any detrimental effect on tumor growth or body weight of the murine host. Immunohistochemistry showed no significant changes in tumor necrosis, hypoxic cell number, proliferation, apoptosis, immune cell infiltration, or collagen deposition. The absence of biofouling supports the use of these materials in biosensors; future investigations in preclinical cancer models are required, with a view to eventual applications in humans. To our knowledge this is the first documented investigation of the effects of modern biomaterials, used in the production of implantable sensors, on tumor tissue after implantation. © 2018 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals, Inc. J Biomed Mater Res Part B, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1620-1633, 2019.

Expression of glycolytic enzymes in ovarian cancers and evaluation of the glycolytic pathway as a strategy for ovarian cancer treatment.

BACKGROUND: Novel therapeutic approaches are required to treat ovarian cancer and dependency on glycolysis may provide new targets for treatment. This study sought to investigate the variation of expression of molecular components (GLUT1, HKII, PKM2, LDHA) of the glycolytic pathway in ovarian cancers and the effectiveness of targeting this pathway in ovarian cancer cell lines with inhibitors. METHODS: Expression of GLUT1, HKII, PKM2, LDHA were analysed by quantitative immunofluorescence in a tissue microarray (TMA) analysis of 380 ovarian cancers and associations with clinicopathological features were sought. The effect of glycolysis pathway inhibitors on the growth of a panel of ovarian cancer cell lines was assessed by use of the SRB proliferation assay. Combination studies were undertaken combining these inhibitors with cytotoxic agents. RESULTS: Mean expression levels of GLUT1 and HKII were higher in high grade serous ovarian cancer (HGSOC), the most frequently occurring subtype, than in non-HGSOC. GLUT1 expression was also significantly higher in advanced stage (III/IV) ovarian cancer than early stage (I/II) disease. Growth dependency of ovarian cancer cells on glucose was demonstrated in a panel of ovarian cancer cell lines. Inhibitors of the glycolytic pathway (STF31, IOM-1190, 3PO and oxamic acid) attenuated cell proliferation in platinum-sensitive and platinum-resistant HGSOC cell line models in a concentration dependent manner. In combination with either cisplatin or paclitaxel, 3PO (a novel PFKFB3 inhibitor) enhanced the cytotoxic effect in both platinum sensitive and platinum resistant ovarian cancer cells. Furthermore, synergy was identified between STF31 (a novel GLUT1 inhibitor) or oxamic acid (an LDH inhibitor) when combined with metformin, an inhibitor of oxidative phosphorylation, resulting in marked inhibition of ovarian cancer cell growth. CONCLUSIONS: The findings of this study provide further support for targeting the glycolytic pathway in ovarian cancer and several useful combinations were identified.

Carbonic Anhydrase IX (CAIX), Cancer, and Radiation Responsiveness.

Carbonic anhydrase IX has been under intensive investigation as a therapeutic target in cancer. Studies demonstrate that this enzyme has a key role in pH regulation in cancer cells, allowing these cells to adapt to the adverse conditions of the tumour microenviroment. Novel CAIX inhibitors have shown efficacy in both in vitro and in vivo pre-clinical cancer models, adversely affecting cell viability, tumour formation, migration, invasion, and metastatic growth when used alone. In co-treatments, CAIX inhibitors may enhance the effects of anti-angiogenic drugs or chemotherapy agents. Research suggests that these inhibitors may also increase the response of tumours to radiotherapy. Although many of the anti-tumour effects of CAIX inhibition may be dependent on its role in pH regulation, recent work has shown that CAIX interacts with several of the signalling pathways involved in the cellular response to radiation, suggesting that pH-independent mechanisms may also be an important basis of its role in tumour progression. Here, we discuss these pH-independent interactions in the context of the ability of CAIX to modulate the responsiveness of cancer to radiation.

Inhibition of pH regulation as a therapeutic strategy in hypoxic human breast cancer cells.

Hypoxic cancer cells exhibit resistance to many therapies. This study compared the therapeutic effect of targeting the pH regulatory proteins (CAIX, NHE1 and V-ATPase) that permit cancer cells to adapt to hypoxic conditions, using both 2D and 3D culture models. Drugs targeting CAIX, NHE1 and V-ATPase exhibited anti-proliferative effects in MCF-7, MDA-MB-231 and HBL-100 breast cancer cell lines in 2D. Protein and gene expression analysis in 2D showed that CAIX was the most hypoxia-inducible protein of the 3 targets. However, the expression of CAIX differed between the 3 cell lines. This difference in CAIX expression in hypoxia was consistent with a varying activity of FIH-1 between the cell lines. 3D expression analysis demonstrated that both CAIX and NHE1 were up-regulated in the hypoxic areas of multicellular tumor spheroids. However, the induction of CAIX expression in hypoxia was again cell line dependent. 3D invasion assays conducted with spheroids showed that CAIX inhibition significantly reduced the invasion of cells. Finally, the capability of both NHE1 and CAIX inhibitors to combine effectively with irradiation was exhibited in clonogenic assays. Proteomic-mass-spectrometric analysis indicated that CAIX inhibition might be combining with irradiation through stimulating apoptotic cell death. Of the three proteins, CAIX represents the target with the most promise for the treatment of breast cancer.