Unlocking the paracrine crosstalk: adipocyte-derived factors affect carbonic anhydrase IX expression in colon and breast cancer cells.

Obesity is a major public health concern because it increases the risk of several diseases, including cancer. Crosstalk between obesity and cancer seems to be very complex, and the interaction between adipocytes and cancer cells leads to changes in adipocytes' function and their paracrine signaling, promoting a microenvironment that supports tumor growth. Carbonic anhydrase IX (CA IX) is a tumor-associated enzyme that not only participates in pH regulation but also facilitates metabolic reprogramming and supports the migration, invasion, and metastasis of cancer cells. In addition, CA IX expression, predominantly regulated via hypoxia-inducible factor (HIF-1), serves as a surrogate marker of hypoxia. In this study, we investigated the impact of adipocytes and adipocyte-derived factors on the expression of CA IX in colon and breast cancer cells. We observed increased expression of CA9 mRNA as well as CA IX protein in the presence of adipocytes and adipocyte-derived conditioned medium. Moreover, we confirmed that adipocytes affect the hypoxia signaling pathway and that the increased CA IX expression results from adipocyte-mediated induction of HIF-1α. Furthermore, we demonstrated that adipocyte-mediated upregulation of CA IX leads to increased migration and decreased adhesion of colon cancer cells. Finally, we brought experimental evidence that adipocytes, and more specifically leptin, upregulate CA IX expression in cancer cells and consequently promote tumor progression.

Carbonic Anhydrase IX in Tumor Tissue and Plasma of Breast Cancer Patients: Reliable Biomarker of Hypoxia and Prognosis.

Carbonic anhydrase IX (CA IX) is recognized as an excellent marker of hypoxia and an adverse prognostic factor in solid tumors, including breast cancer (BC). Clinical studies confirm that soluble CA IX (sCA IX), shed into body fluids, predicts the response to some therapeutics. However, CA IX is not included in clinical practice guidelines, possibly due to a lack of validated diagnostic tools. Here, we present two novel diagnostic tools-a monoclonal antibody for CA IX detection by immunohistochemistry and an ELISA kit for the detection of sCA IX in the plasma-validated on a cohort of 100 patients with early BC. We confirm that tissue CA IX positivity (24%) correlates with tumor grading, necrosis, negative hormone receptor status, and the TNBC molecular subtype. We show that antibody IV/18 can specifically detect all subcellular forms of CA IX. Our ELISA test provides 70% sensitivity and 90% specificity. Although we showed that this test could detect exosomes in addition to shed CA IX ectodomain, we could not demonstrate a clear association of sCA IX with prognosis. Our results indicate that the amount of sCA IX depends on subcellular CA IX localization, but more strictly on the molecular composition of individual molecular subtypes of BC, particularly on metalloproteinases inhibitor expression.

ADAM10 mediates shedding of carbonic anhydrase IX ectodomain non­redundantly to ADAM17.

Carbonic anhydrase IX (CA IX) is a transmembrane enzyme participating in adaptive responses of tumors to hypoxia and acidosis. CA IX regulates pH, facilitates metabolic reprogramming, and supports migration, invasion and metastasis of cancer cells. Extracellular domain (ECD) of CA IX can be shed to medium and body fluids by a disintegrin and metalloproteinase (ADAM) 17. Here we show for the first time that CA IX ECD shedding can be also executed by ADAM10, a close relative of ADAM17, via an overlapping cleavage site in the stalk region of CA IX connecting its exofacial catalytic site with the transmembrane region. This finding is supported by biochemical evidence using recombinant human ADAM10 protein, colocalization of ADAM10 with CA IX, ectopic expression of a dominant­negative mutant of ADAM10 and RNA interference­mediated suppression of ADAM10. Induction of the CA IX ECD cleavage with ADAM17 and/or ADAM10 activators revealed their additive effect. Similarly, additive effect was observed with an ADAM17­inhibiting antibody and an ADAM10­preferential inhibitor GI254023X. These data indicated that ADAM10 is a CA IX sheddase acting on CA IX non­redundantly to ADAM17.

Comparative Study of Classical and Alternative Therapy in Dogs with Allergies.

Allergy is a malfunction of the immune system that causes an inappropriate reaction to normally harmless substances known as allergens, such as food components, pollen, parasites, mites, medication, etc. It is very important to make a correct diagnosis, to identify and to eliminate the offending allergen from the body, and provide control and long-term management to achieve a comfortable life for the animal. In the case of highly intensive pruritus, drugs such as glucocorticoids, antihistamines, and Janus kinase inhibitors are generally administered. Unfortunately, common drugs are not always able to resolve the problem. This comparative clinical-outcomes study focused on the application of alternatives, where a combination of acupuncture with phytotherapy and nutrition was applied. These traditional methods do not affect the body only symptomatologically; instead, they treat the patient as a whole. In this clinical study, the therapeutic effects and partial or complete stabilization of the allergic condition of fourteen dogs divided into two groups were observed, compared, and evaluated.

Any Future for Faecal Microbiota Transplantation as a Novel Strategy for Gut Microbiota Modulation in Human and Veterinary Medicine?

Alterations in the composition of the intestinal microbiome, also known as dysbiosis, are the result of many factors such as diet, antibiotics, stress, diseases, etc. There are currently several ways to modulate intestinal microbiome such as dietary modulation, the use of antimicrobials, prebiotics, probiotics, postbiotics, and synbiotics. Faecal microbiota transplantation (FMT) represents one new method of gut microbiota modulation in humans with the aim of reconstructing the intestinal microbiome of the recipient. In human medicine, this form of bacteriotherapy is successfully used in cases of recurrent Clostridium difficile infection (CDI). FMT has been known in large animal medicine for several years. In small animal medicine, the use of FMT is not part of normal practice.

Novel humanized monoclonal antibodies for targeting hypoxic human tumors via two distinct extracellular domains of carbonic anhydrase IX.

BACKGROUND: Hypoxia in the tumor microenvironment (TME) is often the main factor in the cancer progression. Moreover, low levels of oxygen in tumor tissue may signal that the first- or second-line therapy will not be successful. This knowledge triggers the inevitable search for different kinds of treatment that will successfully cure aggressive tumors. Due to its exclusive expression on cancer cells, carbonic anhydrase IX belongs to the group of the most precise targets in hypoxic tumors. CA IX possesses several exceptional qualities that predetermine its crucial role in targeted therapy. Its expression on the cell membrane makes it an easily accessible target, while its absence in healthy corresponding tissues makes the treatment practically harmless. The presence of CA IX in solid tumors causes an acidic environment that may lead to the failure of standard therapy. METHODS: Parental mouse hybridomas (IV/18 and VII/20) were humanized to antibodies which were subsequently named CA9hu-1 and CA9hu-2. From each hybridoma, we obtained 25 clones. Each clone was tested for antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activity, affinity, extracellular pH measurement, multicellular aggregation analysis, and real-time monitoring of invasion with the xCELLigence system. RESULTS: Based on the results from in vivo experiments, we have selected mouse monoclonal antibodies VII/20 and IV/18. The first one is directed at the conformational epitope of the catalytic domain, internalizes after binding to the antigen, and halts tumor growth while blocking extracellular acidification. The second targets the sequential epitope of the proteo-glycan domain, does not internalize, and is able to block the attachment of cancer cells to the matrix preventing metastasis formation. In vitro experiments prove that humanized versions of the parental murine antibodies, CA9hu-1 and CA9hu-2, have preserved these characteristics. They can reverse the failure of standard therapy as a result of an acidic environment by modulating the TME, and both are able to induce an immune response and have high affinity, as well as ADCC and CDC activity. CONCLUSION: CA9hu-1 and CA9hu-2 are the very first humanized antibodies against CA IX that are likely to become suitable therapies for hypoxic tumors. These antibodies can be applied in the treatment therapy of primary tumors and suppression of metastases formation.

The effect of surface modification of TiMg composite on the in-vitro degradation response, cell survival, adhesion, and proliferation.

This study is aimed to evaluate the influence of mechanical surface treatment on the degradation response, cell survival, adhesion, and proliferation of a TiMg composite material. Two sets of the TiMg samples with different surface characteristics were studied: i) as-machined samples (TiMg-T) and ii) samples with a mechanically modified surface (TiMg-P). Surface roughness was determined using a confocal microscope. Degradation rates (DR) were evaluated in artificial Plasma, HBSS, and NaCl 0.9%. The cell viability was evaluated using an MTT assay. The initial cell adhesion and spreading were investigated using the direct contact assay. An xCELLigence system was employed to provide real-time cell proliferation. The focal adhesion and cell morphological changes were also examined. The DR of TiMg-P decreased by ⁓5 times compared with that of TiMg-T. Surface of the TiMg-P specimens after 72 h exposure to either HBSS or Plasma was passivated by a layer enriched with bioactive Ca/P species. The cell viability of L929 and Saos-2 after 72 h incubation for TiMg-P was 94.6% and 94.8% compared with 73.8% and 74.3% obtained for TiMg-T, respectively. The direct contact assay showed that the initial adhesion and spreading of the L929 cells incubated with TiMg-P was more pronounced compared with that of TiMg-T. The proliferation rate of Saos-2 cells incubated with TiMg-P was higher when compared with that of TiMg-T, and was almost comparable to that of the DMEM-blank between the 24 and 72 h interval. TiMg-P had a pronounced difference in the number and area of Focal Adhesions (FA) compared with that of TiMg-T. The morphology of cells incubated with TiMg-P was not altered. The results confirmed that the smooth and less strained surface of the TiMg-P samples effectively improved the in-vitro degradation response, cell survival, adhesion, and proliferation.

Effect of Hydrolyzed Yeast Administration on Faecal Microbiota, Haematology, Serum Biochemistry and Cellular Immunity in Healthy Dogs.

Fungal microorganisms are regularly found in the gastrointestinal tract of healthy and diseased dogs especially from the phyla Ascomycota and Basidiomycota; however, it is necessary to better understand their role in host health. One of the most commonly studied yeast species in humans or animals is Saccharomyces cerevisiae especially in its live cell form. Scarce knowledge on its hydrolysate product effects in dogs forced us to test diet supplemented with hydrolyzed brewery S. cerevisiae (at a dose 0.3% of the diet) for 14 days to healthy adult dogs. Twenty German Shepherds were randomly divided into 2 groups: control and experimental, ten dogs in each. The experiment lasted 42 days (blood and faeces sample collection at days 0, 14, 28 and 42). The results of this straighforward experiment showed significant increase in the abundance of bifidobacteria (day 14), lactic acid bacteria (day 42) and clostridia (day 42). The faecal pH was significantly increased at day 28. In blood serum, the concentration of triglyceride and cholesterol decreased (day 42) while activities of alanine aminotransferase (at day 14) and aspartate aminotransferase significantly increased (at days 28 and 42). Activities of these enzymes were above reference range top in 7 dogs (ALT) and 4 dogs (AST). Haematological paramaters and activity of phagocytes as well as on percentage of lymphocyte subsets CD4+, CD8+, CD4+CD8+ and CD21+ were not changed during the experiment. The important point of these results is their onset mostly in the post-supplementation period. The observation of some unexpected effects emphasizes the need for reassessment to use yeasts products for dogs but further studies using different doses are necessary.

Understanding metabolic alterations and heterogeneity in cancer progression through validated immunodetection of key molecular components: a case of carbonic anhydrase IX.

Cancer metabolic heterogeneity develops in response to both intrinsic factors (mutations leading to activation of oncogenic pathways) and extrinsic factors (physiological and molecular signals from the extracellular milieu). Here we review causes and consequences of metabolic alterations in cancer cells with focus on hypoxia and acidosis, and with particular attention to carbonic anhydrase IX (CA IX). CA IX is a cancer-associated enzyme induced and activated by hypoxia in a broad range of tumor types, where it participates in pH regulation as well as in molecular mechanisms supporting cancer cells' invasion and metastasis. CA IX catalyzes reversible conversion of carbon dioxide to bicarbonate ion plus proton and cooperates with a spectrum of molecules transporting ions or metabolites across the plasma membrane. Thereby CA IX contributes to extracellular acidosis as well as to buffering intracellular pH, which is essential for cell survival, metabolic performance, and proliferation of cancer cells. Since CA IX expression pattern reflects gradients of oxygen, pH, and other intratumoral factors, we use it as a paradigm to discuss an impact of antibody quality and research material on investigating metabolic reprogramming of tumor tissue. Based on the validation, we propose the most reliable CA IX-specific antibodies and suggest conditions for faithful immunohistochemical analysis of molecules contributing to heterogeneity in cancer progression.

Clinical and Pre-Clinical Evidence of Carbonic Anhydrase IX in Pancreatic Cancer and Its High Expression in Pre-Cancerous Lesions.

Hypoxia is a common phenomenon that occurs in most solid tumors. Regardless of tumor origin, the evolution of a hypoxia-adapted phenotype is critical for invasive cancer development. Pancreatic ductal adenocarcinoma is also characterized by hypoxia, desmoplasia, and the presence of necrosis, predicting poor outcome. Carbonic anhydrase IX (CAIX) is one of the most strict hypoxia regulated genes which plays a key role in the adaptation of cancer cells to hypoxia and acidosis. Here, we summarize clinical data showing that CAIX expression is associated with tumor necrosis, vascularization, expression of Frizzled-1, mucins, or proteins involved in glycolysis, and inevitably, poor prognosis of pancreatic cancer patients. We also describe the transcriptional regulation of CAIX in relation to signaling pathways activated in pancreatic cancers. A large part deals with the preclinical evidence supporting the relevance of CAIX in processes leading to the aggressive behavior of pancreatic tumors. Furthermore, we focus on CAIX occurrence in pre-cancerous lesions, and for the first time, we describe CAIX expression within intraductal papillary mucinous neoplasia. Our review concludes with a detailed account of clinical trials implicating that treatment consisting of conventionally used therapies combined with CAIX targeting could result in an improved anti-cancer response in pancreatic cancer patients.

CAIX-Mediated Control of LIN28/let-7 Axis Contributes to Metabolic Adaptation of Breast Cancer Cells to Hypoxia.

Solid tumors, including breast cancer, are characterized by the hypoxic microenvironment, extracellular acidosis, and chemoresistance. Hypoxia marker, carbonic anhydrase IX (CAIX), is a pH regulator providing a selective survival advantage to cancer cells through intracellular neutralization while facilitating tumor invasion by extracellular acidification. The expression of CAIX in breast cancer patients is associated with poor prognosis and metastases. Importantly, CAIX-positive hypoxic tumor regions are enriched in cancer stem cells (CSCs). Here we investigated the biological effects of CA9-silencing in breast cancer cell lines. We found that CAIX-downregulation in hypoxia led to increased levels of let-7 (lethal-7) family members. Simultaneously with the increase of let-7 miRNAs in CAIX-suppressed cells, LIN28 protein levels decreased, along with downstream metabolic pathways: pyruvate dehydrogenase kinase 1 (PDK1) and phosphorylation of its substrate, pyruvate dehydrogenase (PDH) at Ser-232, causing attenuation of glycolysis. In addition to perturbed glycolysis, CAIX-knockouts, in correlation with decreased LIN28 (as CSC reprogramming factor), also exhibit reduction of the further CSC-associated markers NANOG (Homeobox protein NANOG) and ALDH1 (Aldehyde dehydrogenase isoform 1). Oppositely, overexpression of CAIX leads to the enhancement of LIN28, ALDH1, and NANOG. In conclusion, CAIX-driven regulation of the LIN28/let-7 axis augments glycolytic metabolism and enhances stem cell markers expression during CAIX-mediated adaptation to hypoxia and acidosis in carcinogenesis.

Carbonic Anhydrase IX-Mouse versus Human.

In contrast to human carbonic anhydrase IX (hCA IX) that has been extensively studied with respect to its molecular and functional properties as well as regulation and expression, the mouse ortholog has been investigated primarily in relation to tissue distribution and characterization of CA IX-deficient mice. Thus, no data describing transcriptional regulation and functional properties of the mouse CA IX (mCA IX) have been published so far, despite its evident potential as a biomarker/target in pre-clinical animal models of tumor hypoxia. Here, we investigated for the first time, the transcriptional regulation of the Car9 gene with a detailed description of its promoter. Moreover, we performed a functional analysis of the mCA IX protein focused on pH regulation, cell-cell adhesion, and migration. Finally, we revealed an absence of a soluble extracellular form of mCA IX and provided the first experimental evidence of mCA IX presence in exosomes. In conclusion, though the protein characteristics of hCA IX and mCA IX are highly similar, and the transcription of both genes is predominantly governed by hypoxia, some attributes of transcriptional regulation are specific for either human or mouse and as such, could result in different tissue expression and data interpretation.

Prognostic value of intratumoral carbonic anhydrase IX expression in testicular germ cell tumors.

Testicular germ cell tumors (TGCTs) represent a highly curable malignancy, however a small proportion of patients fails to be cured with cisplatin-based chemotherapy. Carbonic anhydrase IX (CA IX) is upregulated by hypoxia in several cancer types and correlates with a poor prognosis. The present translational study evaluated expression and prognostic value of CA IX in TGCTs. Surgical specimens from 228 patients with TGCTs were processed by the tissue microarray method and subjected to immunohistochemistry with the M75 monoclonal antibody. CA IX expression was evaluated in tumors vs. adjacent normal testicular tissues and correlated with clinicopathological characteristics and clinical outcome. CA IX expression was detected in 62 (30.2%) of TGCTs compared to 0 (0%) of normal tissue adjacent to testicular tumor (P<0.001). The highest frequency of the CA IX expression was detected in teratoma (39.0%), followed by seminoma (22.7%), yolk sac tumor (22.2%), embryonal carcinoma (11.9%) and choriocarcinoma (7.7%). None of germ cell neoplasias in situ (GCNIS) exhibited CA IX expression. Patients without the CA IX tumor expression showed significantly better progression-free survival, but not overall survival, compared to patients with the CA IX expression [hazard ratio (HR), 0.57; 95% CI, 0.32-1.02; P=0.037 and HR, 0.58; 95% CI, 0.29-1.16; P=0.088, respectively]. There was no significant correlation between the CA IX expression and clinicopathological variables. The intratumoral CA IX expression can serve as a prognostic marker in the TGCT patients. These results suggest that activation of the hypoxia-induced pathways may be important in the treatment failure in TGCTs patients.

Prognostic value of serum carbonic anhydrase IX in testicular germ cell tumor patients.

Despite the fact that testicular germ cell tumors (TGCTs) are one of the most chemosensitive solid tumors, a small proportion of patients fail to be cured following cisplatin-based first line chemotherapy. Upregulation of carbonic anhydrase IX (CA IX) in various solid tumors is associated with poor outcome. The current prospective study investigated the prognostic value of serum CA IX level in TGCTs. In total, 83 patients (16 non-metastatic following orchiectomy with no evidence of disease, 57 metastatic chemotherapy-naïve and 10 metastatic relapsed chemotherapy-pretreated) starting adjuvant and/or new line of chemotherapy and 35 healthy controls were enrolled in the study. Serum CA IX values were determined using an enzyme-linked immunosorbent assay, and intratumoral CA IX was analyzed by immunohistochemistry. Metastatic chemotherapy-naïve patients had significantly higher mean CA IX serum levels than healthy controls (490.6 vs. 249.6 pg/ml, P=0.005), while there was no difference in serum CA IX levels in non-metastatic or relapsed TGCT patients compared with healthy controls. There was no significant difference in the mean serum CA IX levels between different groups of patients and between the first and second cycle of chemotherapy, nor association with patients/tumor characteristics. Serum CA IX was not prognostic for progression-free survival [hazard ratio (HR)=0.81, P=0.730] or overall survival (HR=0.64, P=0.480). However, there was a significant association between intratumoral CA IX expression and serum CA IX concentration (rho=0.51, P=0.040). These results suggest that serum CA IX level correlates with tumor CA IX expression in TGCT patients, but fails to exhibit either a prognostic value or an association with patients/tumor characteristics.

Dexamethasone downregulates expression of carbonic anhydrase IX via HIF-1α and NF-κB-dependent mechanisms.

Dexamethasone is a synthetic glucocorticoid frequently used to suppress side-effects of anticancer chemotherapy. In the present study, we showed that dexamethasone treatment leads to concentration-dependent downregulation of cancer-associated marker, carbonic anhydrase IX (CA IX), at the level of promoter activity, mRNA and protein expression in 2D and 3D cancer cell models. The effect of dexamethasone on CA IX expression under hypoxic conditions is predominantly mediated by impaired transcriptional activity and decreased protein level of the main hypoxic transcription factor HIF-1α. In addition, CA9 downregulation can be caused by protein-protein interactions between activated glucocorticoid receptors, major effectors of glucocorticoid action, and transcription factors that trigger CA9 transcription (e.g. AP-1). Moreover, we identified a potential NF-κB binding site in the CA9 promoter and propose the involvement of NF-κB in the dexamethasone-mediated inhibition of CA9 transcription. As high level of CA IX is often linked to aggressive tumor behavior, poor prognosis and chemo- and radiotherapy resistance, uncovering its reduction after dexamethasone treatment and implication of additional regulatory mechanisms can be relevant for the CA IX-related clinical applications.

Encapsulation of anti-carbonic anhydrase IX antibody in hydrogel microspheres for tumor targeting.

Encapsulation is a well-established method of biomaterial protection, controlled release, and efficient delivery. Here we evaluated encapsulation of monoclonal antibody M75 directed to tumor biomarker carbonic anhydrase IX (CA IX) into alginate microbeads (SA-beads) or microcapsules made of sodium alginate, cellulose sulfate, and poly(methylene-co-guanidine) (PMCG). M75 antibody release was quantified using ELISA and its binding properties were assessed by immunodetection methods. SA-beads showed rapid M75 antibody release in the first hour, followed by steady release during the whole experiment of 7 days. In contrast, the M75 release from PMCG capsules was gradual, reaching the maximum concentration on the 7th day. The release was more efficient at pH 6.8 compared to pH 7.4. The released antibody could recognize CA IX, and target the CA IX-positive cells in 3D spheroids. In conclusion, SA-beads and PMCG microcapsules can be considered as promising antibody reservoirs for targeting of cancer cells.

Hypoxia increases the heterogeneity of melanoma cell populations and affects the response to vemurafenib.

A hypoxic microenvironment is one of the predominant reasons for incomplete response to melanoma treatment. Vemurafenib, which targets the mutated BRAF-V600 kinase, improves melanoma patient survival, however, resistance invariably develops. The present study evaluated the effect of hypoxia on three BRAF-V600E mutant melanoma cell lines, M14, A375 and 518A2, treated with vemurafenib. Compared with the other two cell lines, hypoxic vemurafenib-treated A375 cells exhibited an enhanced cell proliferation rate and migratory capacity compared with normoxic vemurafenib-treated A375 cells. Immunoblotting analyses revealed that the expression levels of hypoxia inducible factor (HIF)1α and carbonic anhydrase IX were reduced in vemurafenib­treated M14 and 518A2 cells, however, not in A375 cells. The expression levels of the mitogen­activated protein kinase, Janus kinase-signal transducer and activator of transcription, and phosphatidylinositol-4,5-bisphosphate 3­kinase signaling pathway proteins revealed a cell­type specific response to vemurafenib and hypoxia. Knockdown experiments of HIF1α performed in hypoxic A375 cells decreased the expression of phosphorylated (p­)protein kinase B, which was restored following vemurafenib treatment, and increased the expression of p­extracellular­signal­regulated kinases. Therefore, three melanoma cell lines responded to vemurafenib under hypoxia in a cell type­specific manner, suggesting that a subset of cells provides a treatment-resistant pool, from which disease relapse may originate. These data confirmed that vemurafenib may be successful in treating the proliferating cells, whereas the non­proliferating subpopulation must be addressed by a combination of vemurafenib with other treatment strategies.

Anti-chondroitin sulfate proteoglycan 4-specific antibodies modify the effects of vemurafenib on melanoma cells differentially in normoxia and hypoxia.

Chondroitin sulfate proteoglycan 4 (CSPG4), a highly immunogenic melanoma tumor antigen, is a potential target for antibody-based immunotherapy. The mechanism by which CSPG4 affects melanoma progression is only partly understood, in particular the involvement of other receptor tyrosine kinases and the tumor microenvironment. We have previously reported on a mimotope-based vaccine against CSPG4 in a human melanoma xenograft model that resulted in reduction of tumor growth. Herein we describe the influence of hypoxia on the response to polyclonal anti-CSPG4-antibodies induced by this vaccine in combination with the BRAF inhibitor vemurafenib to enhance therapeutic efficacy by simultaneously targeting multiple signaling pathways. Melanoma cells were treated with polyclonal anti-CSPG4-antibodies and vemurafenib. Proliferation, migration and invasion were evaluated in a real-time setting in the impedance-based x-CELLigence® system. Western blotting and quantitative PCR arrays were used to determine protein and mRNA expression of hypoxia inducible factor 1α (HIF1α), carbonic anhydrase IX (CAIX) and signaling pathway proteins. A melanoma xenograft model was used to detect HIF1α and CAIX expression in vivo. Hypoxia enhanced the antiproliferative response to vemurafenib. The migration and invasion capacities of vemurafenib-treated melanoma cells were increased, in spite of vemurafenib-decreased expression of HIF1α and CAIX. Polyclonal anti-CSPG4-antibodies reduced the Transwell migration of vemurafenib-treated, BRAF V600E-mutant and CSPG4-expressing melanoma cells in hypoxia. This was associated with the downregulation of phosphorylated AKT, a kinase contributing to tumor cell migration. Our results highlight CSPG4 as a potential target for modulating treatment resistance to vemurafenib induced by the hypoxic microenvironment.

Deregulation of energetic metabolism in the clear cell renal cell carcinoma: A multiple pathway analysis based on microarray profiling.

Clear cell renal cell carcinoma (ccRCC) is the most frequent type of kidney cancer. In order to better understand the biology of ccRCC, we accomplished the gene profiling of fresh tissue specimens from 11 patients with the renal tumors (9 ccRCCs, 1 oncocytoma and 1 renal B-lymphoma), in which the tumor-related data were compared to the paired healthy kidney tissues from the same patients. All ccRCCs exhibited a considerably elevated transcription of the gene coding for carbonic anhydrase IX (CAIX). Moreover, the ccRCC tumors consistently displayed increased expression of genes encoding the glycolytic pathway enzymes, e.g. hexokinase II (HK2) and lactate dehydrogenase A (LDHA) and a decreased expression of genes for the mitochondrial electron transport chain components, indicating an overall reprogramming of the energetic metabolism in this tumor type. This appears to be accompanied by altered expression of the genes of the pH regulating machinery, including ion and lactate transporters. Immunohistochemical staining of tumor tissue sections confirmed the increased expression of CAIX, HK2 and LDHA in ccRCC, validating the microarray data and supporting their potential as the energetic metabolism-related biomarkers of the ccRCC.

Sulforaphane reduces molecular response to hypoxia in ovarian tumor cells independently of their resistance to chemotherapy.

One of the recently emerging anticancer strategies is the use of natural dietary compounds, such as sulforaphane, a cancer-chemopreventive isothiocyanate found in broccoli. Based on the growing evidence, sulforaphane acts through molecular mechanisms that interfere with multiple oncogenic pathways in diverse tumor cell types. Herein, we investigated the anticancer effects of bioavailable concentrations of sulforaphane in ovarian carcinoma cell line A2780 and its two derivatives, adriamycin-resistant A2780/ADR and cisplatin-resistant A2780/CP cell lines. Since tumor microenvironment is characterized by reduced oxygenation that induces aggressive tumor phenotype (such as increased invasiveness and resistance to chemotherapy), we evaluated the effects of sulforaphane in ovarian cancer cells exposed to hypoxia (2% O2). Using the cell-based reporter assay, we identified several oncogenic pathways modulated by sulforaphane in hypoxia by activating anticancer responses (p53, ARE, IRF-1, Pax-6 and XRE) and suppressing responses supporting tumor progression (AP-1 and HIF-1). We further showed that sulforaphane decreases the level of HIF-1α protein without affecting its transcription and stability. It can also diminish transcription and protein level of the HIF-1 target, CA IX, which protects tumor cells from hypoxia-induced pH imbalance and facilitates their migration/invasion. Accordingly, sulforaphane treatment leads to diminished pH regulation and reduced migration of ovarian carcinoma cells. These effects occur in all three ovarian cell lines suggesting that sulforaphane can overcome the chemoresistance of cancer cells. This offers a path potentially exploitable in sensitizing resistant cancer cells to therapy, and opens a window for the combined treatments of sulforaphane either with conventional chemotherapy, natural compounds, or with other small molecules.