HIF-1 alpha-independent hypoxia-induced rapid PTK6 stabilization is associated with increased motility and invasion

© 2014 Landes Bioscience. PTK6/Brk is a non-receptor tyrosine kinase overexpressed in cancer. Here we demonstrate that cytosolic PTK6 is rapidly and robustly induced in response to hypoxic conditions in a HIF-1-independent manner. Furthermore, a proportion of hypoxic PTK6 subsequently re-localized to the cell membrane. We observed that the rapid stabilization of PTK6 is associated with a decrease in PTK6 ubiquitylation and we have identified c-Cbl as a putative PTK6 E3 ligase in normoxia. The consequences of hypoxia-induced PTK6 stabilization and subcellular re-localization to the plasma membrane include increased cell motility and invasion, suggesting PTK6 targeting as a therapeutic approach to reduce hypoxia-regulated metastatic potential. This could have particular significance for breast cancer patients with triple negative disease

Improving Breast Cancer Treatment Specificity Using Aptamers Obtained by 3D Cell-SELEX

Three-dimensional spheroids of non-malignant MCF10A and malignant SKBR3 breast cells were used for subsequent 3D Cell-SELEX to generate aptamers for specific binding and treatment of breast cancer cells. Using 3D Cell-SELEX combined with Next-Generation Sequencing and bioinformatics, ten abundant aptamer families with specific structures were identified that selectively bind to SKBR3, and not to MCF10A cells. Multivalent aptamer polymers were synthesized by co-polymerization and analyzed for binding performance as well as therapeutic efficacy. Binding performance was determined by confocal fluorescence imaging and revealed specific binding and efficient internalization of aptamer polymers into SKBR3 spheroids. For therapeutic purposes, DNA sequences that intercalate the cytotoxic drug doxorubicin were co-polymerized into the aptamer polymers. Viability tests show that the drug-loaded polymers are specific and effective in killing SKBR3 breast cancer cells. Thus, the 3D-selected aptamers enhanced the specificity of doxorubicin against malignant over non-malignant breast cells. The innovative modular DNA aptamer platform based on 3D Cell SELEX and polymer multivalency holds great promise for diagnostics and treatment of breast cancer

Tumor microenvironmental changes induced by the sulfamate carbonic anhydrase IX inhibitor S4 in a laryngeal tumor model

BACKGROUND AND PURPOSE: Carbonic anhydrase IX (CAIX) plays a pivotal role in pH homeostasis, which is essential for tumor cell survival. We examined the effect of the CAIX inhibitor 4-(3'(3",5"-dimethylphenyl)-ureido)phenyl sulfamate (S4) on the tumor microenvironment in a laryngeal tumor model by analyzing proliferation, apoptosis, necrosis, hypoxia, metabolism and CAIX ectodomain shedding. METHODS: SCCNij202 tumor bearing-mice were treated with S4 for 1, 3 or 5 days. CAIX ectodomain shedding was measured in the serum after therapy. Effects on tumor cell proliferation, apoptosis, necrosis, hypoxia (pimonidazole) and CAIX were investigated with quantitative immunohistochemistry. Metabolic transporters and enzymes were quantified with qPCR. RESULTS: CAIX ectodomain shedding decreased after treatment with S4 (p<0.01). S4 therapy did neither influence tumor cell proliferation nor the amount of apoptosis and necrosis. Hypoxia (pimonidazole) and CAIX expression were also not affected by S4. CHOP and MMP9 mRNA as a reference of intracellular pH did not change upon treatment with S4. Compensatory mechanisms of pH homeostasis at the mRNA level were not observed. CONCLUSION: As the clinical and biological meaning of the decrease in CAIX ectodomain shedding after S4 therapy is not clear, studies are required to elucidate whether the CAIX ectodomain has a paracrine or autocrine signaling function in cancer biology. S4 did not influence the amount of proliferation, apoptosis, necrosis and hypoxia. Therefore, it is unlikely that S4 can be used as single agent to influence tumor cell kill and proliferation, and to target primary tumor growth

Aberrant APOBEC3B Expression in Breast Cancer Is Linked to Proliferation and Cell Cycle Phase

APOBEC3B (A3B) is aberrantly overexpressed in a subset of breast cancers, where it associates with advanced disease, poor prognosis, and treatment resistance, yet the causes of A3B dysregulation in breast cancer remain unclear. Here, A3B mRNA and protein expression levels were quantified in different cell lines and breast tumors and related to cell cycle markers using RT-qPCR and multiplex immunofluorescence imaging. The inducibility of A3B expression during the cell cycle was additionally addressed after cell cycle synchronization with multiple methods. First, we found that A3B protein levels within cell lines and tumors are heterogeneous and associate strongly with the proliferation marker Cyclin B1 characteristic of the G2/M phase of the cell cycle. Second, in multiple breast cancer cell lines with high A3B, expression levels were observed to oscillate throughout the cell cycle and again associate with Cyclin B1. Third, induction of A3B expression is potently repressed throughout G0/early G1, likely by RB/E2F pathway effector proteins. Fourth, in cells with low A3B, induction of A3B through the PKC/ncNF-κB pathway occurs predominantly in actively proliferating cells and is largely absent in cells arrested in G0. Altogether, these results support a model in which dysregulated A3B overexpression in breast cancer is the cumulative result of proliferation-associated relief from repression with concomitant pathway activation during the G2/M phase of the cell cycle.</p

GATA transcription factors in testicular adrenal rest tumours

Testicular adrenal rest tumours (TARTs) are benign adrenal-like testicular tumours that frequently occur in male patients with congenital adrenal hyperplasia. Recently, GATA transcription factors have been linked to the development of TARTs in mice. The aim of our study was to determine GATA expression in human TARTs and other steroidogenic tissues. We determined GATA expression in TARTs (n = 16), Leydig cell tumours (LCTs; n = 7), adrenal (foetal (n = 6) + adult (n = 10)) and testis (foetal (n = 13) + adult (n = 8)). We found testis-like GATA4, and adrenal-like GATA3 and GATA6 gene expressions by qPCR in human TARTs, indicating mixed testicular and adrenal characteristics of TARTs. Currently, no marker is available to discriminate TARTs from LCTs, leading to misdiagnosis and incorrect treatment. GATA3 and GATA6 mRNAs exhibited excellent discriminative power (area under the curve of 0.908 and 0.816, respectively), while immunohistochemistry did not. GATA genes contain several CREB-binding sites and incubation with 0.1 mM dibutyryl cAMP for 4 h stimulated GATA3, GATA4 and GATA6 expressions in a human foetal testis cell line (hs181.tes). Incubation of adrenocortical cells (H295RA) with ACTH, however, did not induce GATA expression in vitro Although ACTH did not dysregulate GATA expression in the only human ACTH-sensitive in vitro model available, our results do suggest that aberrant expression of GATA transcription factors in human TARTs might be involved in TART formation

Tribbles homolog 3 denotes a poor prognosis in breast cancer and is involved in hypoxia response

Hypoxia in solid tumors is associated with treatment resistance, resulting in poor prognosis. Tribbles homolog 3 (TRIB3) is induced during hypoxia and is involved in multiple cellular pathways involved in cell survival. Here, we investigated the role of TRIB3 in breast cancer. TRIB3 mRNA expression was measured in breast tumor tissue from 247 patients and correlated with clinicopathological parameters and clinical outcome. Furthermore, we studied TRIB3 expression regulation in cell lines, xenografts tissues and human breast cancer material using Reverse transcriptase, quantitative polymerase chain reaction (RT-qPCR) and immunohistochemical staining. Finally, the effect of small interfering RNA (siRNA) mediated TRIB3 knockdown on hypoxia tolerance was assessed. Breast cancer patients with low, intermediate or high TRIB3 expression exhibited a mean disease free survival (DFS) of 80 (95% confidence interval [CI] = 74 to 86), 74 (CI = 67 to 81), and 63 (CI = 55 to 71) months respectively (P = .002, Mantel-Cox log-rank). The prognostic value of TRIB3 was limited to those patients that had received radiotherapy as part of their primary treatment (n = 179, P = .005) and remained statistically significant after correction for other clinicopathological parameters (DFS, Hazard Ratio = 1.90, CI = 1.17 to 3.08, P = .009). In breast cell lines TRIB3 expression was induced by hypoxia, nutrient starvation, and endoplasmic reticulum stress in an hypoxia inducible factor 1 (HIF-1) independent manner. TRIB3 induction after hypoxia did not increase with decreasing oxygen levels. In breast tumor xenografts and human breast cancer tissues TRIB3 co-localized with the hypoxic cell marker pimonidazole. The induction of TRIB3 by hypoxia was shown to be regulated via the PERK/ATF4/CHOP pathway of the unfolded protein response and knockdown of TRIB3 resulted in a dose-dependent increase in hypoxia sensitivity. TRIB3 is independently associated with poor prognosis of breast cancer patients, possibly through its association with tumor cell hypoxi

Optimizing the Timing of Highest Hydrocortisone Dose in Children and Adolescents With 21-Hydroxylase Deficiency

CONTEXT: Hydrocortisone treatment of young patients with 21-hydroxylase deficiency (21OHD) is given thrice daily, but there is debate about the optimal timing of the highest hydrocortisone dose, either mimicking the physiological diurnal rhythm (morning), or optimally suppressing androgen activity (evening). OBJECTIVE: We aimed to compare 2 standard hydrocortisone timing strategies, either highest dosage in the morning or evening, with respect to hormonal status throughout the day, nocturnal blood pressure (BP), and sleep and activity scores. METHODS: This 6-week crossover study included 39 patients (aged 4-19 years) with 21OHD. Patients were treated for 3 weeks with the highest hydrocortisone dose in the morning, followed by 3 weeks with the highest dose in the evening (n = 21), or vice versa (n = 18). Androstenedione (A4) and 17-hydroxyprogesterone (17OHP) levels were quantified in saliva collected at 5 am; 7 am; 3 pm; and 11 pm during the last 2 days of each treatment period. The main outcome measure was comparison of saliva 17OHP and A4 levels between the 2 treatment strategies. RESULTS: Administration of the highest dose in the evening resulted in significantly lower 17OHP levels at 5 am, whereas the highest dose in the morning resulted in significantly lower 17OHP and A4 levels in the afternoon. The 2 treatment dose regimens were comparable with respect to averaged daily hormone levels, nocturnal BP, and activity and sleep scores. CONCLUSION: No clear benefit for either treatment schedule was established. Given the variation in individual responses, we recommend individually optimizing dose distribution and monitoring disease control at multiple time points

Cathepsins B, L and cystatin C in cyst fluid of ovarian tumors

Contains fulltext : 88032.pdf (publisher's version ) (Closed access)INTRODUCTION: In cancer, an extracellular and membrane bound localization of cathepsins contribute to the invasion of tumor cells at the basement membrane. METHODS: This is the first study that explored levels of cathepsins B (CatB), L (CatL) and their inhibitor cystatin C (CysC) in the cystic fluid (CF) of ovarian tumors (n = 110). RESULTS: CF contained considerable amounts of CatB, CatL and CysC. Remarkable differences in CatB and CatL and CysC CF levels were found between different histopathological tumor subtypes. Levels of CatB and CysC were significantly higher in CF of malignant serous tumors compared to those found in benign serous tumors (p = 0.010 and p = 0.001 respectively), whereas levels of CatL were significantly higher in CF of malignant mucinous tumors compared to those found in benign mucinous tumors (p = 0.035). CatB and CysC showed a strong correlation in the group of patients with malignant serous tumors (p < 0.001; R = 0.921) suggesting that the increase in CatB might be balanced by a corresponding increase in CysC. CONCLUSION: Further studies are warranted to investigate cathepsins as possible prognostic biomarkers for the aggressiveness of ovarian cancer.1 mei 201