The tyrosine kinase CSK associates with FLT3 and c-Kit receptors and regulates downstream signaling.

Type III receptor tyrosine kinases (RTKs), FLT3 and c-Kit play important roles in a variety of cellular processes. A number of SH2-domain containing proteins interact with FLT3 and c-Kit and regulate downstream signaling. The SH2-domain containing non-receptor protein tyrosine kinase CSK is mainly studied in context of regulating Src family kinases. Here we present an addition role of this kinase in RTK signaling. We show that CSK interacts with FLT3 and c-Kit in a phosphorylation dependent manner. This interaction is facilitated through the SH2-domain of CSK. Under basal conditions CSK is mainly localized throughout the cytosolic compartment but upon ligand stimulation it is recruited to the inner side of cell membrane. CSK association did not alter receptor ubiquitination or phosphorylation but disrupted downstream signaling. Selective depletion of CSK using siRNA, or inhibition with CSK inhibitor, led to increased phosphorylation of Akt and Erk, but not p38, upon FLT3 ligand (FL) stimulation. Stem cell factor (SCF)-mediated Akt and Erk activation was also elevated by CSK inhibition. However, siRNA mediated CSK knockdown increased SCF stimulated Akt phosphorylation but decreased Erk phosphorylation. CSK depletion also significantly increased both FL- and SCF-induced SHC, Gab2 and SHP2 phosphorylation. Furthermore, CSK depletion contributed to oncogenic FLT3- and c-Kit-mediated cell proliferation, but not to cell survival. Thus, the results indicate that CSK association with type III RTKs, FLT3 and c-Kit can have differential impact on receptor downstream signaling

Cancer cell differentiation heterogeneity and aggressive behavior in solid tumors

The differentiation stage of tumors is a central aspect in the histopathological classification of solid malignancies. The differentiation stage is strongly associated with tumor behavior, and generally an immature tumor is more aggressive than the more differentiated counterpart. While this is common knowledge in surgical pathology, the contribution of differentiation-related gene expression and functions to tumor behavior is often overlooked in the experimental, tumor biological setting. The mechanisms by which tumor cell differentiation stages are perturbed or affected are poorly explored but have recently come into focus with the introduction.of the tumor stem cell concept. While developmental biologists view the differentiation as a unidirectional event, pathologists and tumor biologists have introduced the concept of dedifferentiation to explain phenotypic changes occurring in solid tumors. In this review we discuss the impact of the tumor cell differentiation stage as used in surgical pathology. We further discuss knowledge gained from exploring the molecular basis of the differentiation and dedifferentiation processes in neuroblastoma and breast cancer, two tumor forms where the tumor cell differentiation concept is used in the clinical diagnostic work and where the tumor stem cell theory has been applied

Influence of hypoxia on differentiation and proliferation of non-malignant and tumor cells

Solid tumors are less oxygenated than their tissue of origin. Low intra-tumor oxygen levels are associated with worse outcome, increased metastatic potential and immature phenotype in breast cancer. We have reported that tumor hypoxia correlates to low differentiation status in breast cancer. Less is known about effects of hypoxia on non-malignant cells. Here we address whether hypoxia influences the differentiation stage of non-malignant breast epithelial cells and potentially have bearing on early stages of tumorigenesis. We show that hypoxic conditions impaired acinar morphogenesis of primary and immortalized breast epithelial cells grown ex vivo on laminin-rich matrix. The hypoxic cells remained in a mitotic state, whereas proliferation ceased with acinar morphogenesis at normoxia. We found induced expression of the differentiation repressor ID1 in the undifferentiated hypoxic MCF-10A cell structures. Acinar morphogenesis was associated with global histone deacetylation whereas the hypoxic breast epithelial cells showed sustained global histone acetylation, which is generally associated with active transcription and an undifferentiated proliferative state. Despite expression of EPOR we show here that rhEPO stimulation of breast cancer cells has no apparent effect on cell growth in vitro. When knocking down EPOR we found a decreased proliferation in ERα+ breast cancer cells only. This inhibitory effect on proliferation appeared to be mediated via a decrease in ERα-activity. EPOR knockdown further improved Tamoxifen effects on ERα-activity in one of the cell lines tested. These results are in accordance with our previous observation that high EPOR expression in tumors correlates to impaired Tamoxifen response in breast cancer patients. We previously demonstrated that small cell lung carcinoma (SCLC) cells lack expression of HIF-2α protein, whereas HIF-1α is expressed at both acute and prolonged hypoxia. Searching for a role of HIF-1 in hypoxic SCLC cells, we investigated metabolic pathways and HIF-1 dependence. When repressing HIF1A expression, the ATP levels were not affected and SCLC cell viability did not decrease by glucose deprivation. In contrast, glutamine withdrawal drastically decreased the number of cells and increased cell death, still in a HIF-independent fashion. The tested SCLC cells are MYC or MYCL amplified and MYC overexpression is known to stimulate glutamine metabolism. Our results might further suggest that Myc overexpression overrides the need of HIF-1α expression and activity in the response of SCLC cells to hypoxia by inducing glutaminolysis and lipogenesis

Propagation of functional estrogen receptor positive normal human breast cells in 3D cultures.

PurposeUnderstanding how differentiation, microenvironment, and hormonal milieu influence human breast cell susceptibility to malignant transformation will require the use of physiologically relevant in vitro systems. We sought to develop a 3D culture model that enables the propagation of normal estrogen receptor alpha (ER) + cells.MethodsWe tested soluble factors and protocols for the ability to maintain progenitor and ER + cells in cultures established from primary cells. Optimized conditions were then used to profile estrogen-induced gene expression changes in cultures from three pathology-free individuals.ResultsLong-term representation of ER + cells was optimal in medium that included three different TGFβ/activin receptor-like kinase inhibitors. We found that omitting the BMP signaling antagonist, Noggin, enhanced the responsiveness of the PGR gene to estradiol exposure without altering the proportions of ER + cells in the cultures. Profiling of estradiol-exposed cultures showed that while all the cultures showed immediate and robust induction of PGR, LRP2, and IGFB4, other responses varied qualitatively and quantitatively across specimens.ConclusionsWe successfully identified conditions for the maintenance and propagation of functional ER + cells from normal human breast tissues. We propose that these 3D cultures will overcome limitations of conventional 2D cultures of partially or fully transformed cell lines by sustaining normal endocrine function and growth regulation of the cell populations that comprise intact breasts

Myc-induced glutaminolysis bypasses HIF-driven glycolysis in hypoxic small cell lung carcinoma cells

We previously demonstrated that small cell lung carcinoma (SCLC) cells lack HIF-2α protein expression, whereas HIF-1α in these cells is expressed at both acute and prolonged hypoxia. Here we show that low HIF2A expression correlates with high expression of MYC genes. Knockdown of HIF1A expression had no or limited effect on cell survival and growth in vitro. Unexpectedly, hypoxic ATP levels were not affected by HIF-1α knockdown and SCLC cell viability did not decrease upon glucose deprivation. In line with these in vitro data, xenograft tumor-take and growth were not significantly affected by repressed HIF1A expression. Glutamine withdrawal drastically decreased SCLC cell proliferation and increased cell death at normoxia and hypoxia in a HIF-independent fashion and the dependence on glutaminolysis was linked to amplification of either MYC or MYCL. Downregulation of GLS expression, regulating the first step of the glutaminolysis pathway, in MYC/MYCL overexpressing SCLC cells resulted in both impaired growth and increased cell death. Our results suggest that MYC/MYCL overexpression in SCLC cells overrides the need of HIF-1 activity in response to hypoxia by inducing glutaminolysis and lipogenesis. Targeting the glutaminolysis pathway might hence be a novel approach to selectively kill MYC amplified SCLC cells in vivo

EPO-independent functional EPO receptor in breast cancer enhances estrogen receptor activity and promotes cell proliferation.

The main function of Erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis. Recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. However, clinical trials have indicated that rhEPO treatment might promote tumor progression and has a negative effect on patient survival. In addition, EPOR expression has been detected in several cancer forms. Using a newly produced anti-EPOR antibody that reliably detects the full-length isoform of the EPOR we show that breast cancer tissue and cells express the EPOR protein. rhEPO stimulation of cultured EPOR expressing breast cancer cells did not result in increased proliferation, overt activation of EPOR (receptor phosphorylation) or a consistent activation of canonical EPOR signaling pathway mediators such as JAK2, STAT3, STAT5, or AKT. However, EPOR knockdown experiments suggested functional EPO receptors in estrogen receptor positive (ERα(+)) breast cancer cells, as reduced EPOR expression resulted in decreased proliferation. This effect on proliferation was not seen in ERα negative cells. EPOR knockdown decreased ERα activity further supports a mechanism by which EPOR affects proliferation via ERα-mediated mechanisms. We show that EPOR protein is expressed in breast cancer cells, where it appears to promote proliferation by an EPO-independent mechanism in ERα expressing breast cancer cells

Hypoxic conditions induce a cancer-like phenotype in human breast epithelial cells.

INTRODUCTION: Solid tumors are less oxygenated than their tissue of origin. Low intra-tumor oxygen levels are associated with worse outcome, increased metastatic potential and immature phenotype in breast cancer. We have reported that tumor hypoxia correlates to low differentiation status in breast cancer. Less is known about effects of hypoxia on non-malignant cells. Here we address whether hypoxia influences the differentiation stage of non-malignant breast epithelial cells and potentially have bearing on early stages of tumorigenesis. METHODS: Normal human primary breast epithelial cells and immortalized non-malignant mammary epithelial MCF-10A cells were grown in a three-dimensional overlay culture on laminin-rich extracellular matrix for up to 21 days at normoxic or hypoxic conditions. Acinar morphogenesis and expression of markers of epithelial differentiation and cell polarization were analyzed by immunofluorescence, immunohistochemistry, qPCR and immunoblot. RESULTS: In large ductal carcinoma in situ patient-specimens, we find that epithelial cells with high HIF-1α levels and multiple cell layers away from the vasculature are immature compared to well-oxygenated cells. We show that hypoxic conditions impaired acinar morphogenesis of primary and immortalized breast epithelial cells grown ex vivo on laminin-rich matrix. Normoxic cultures formed polarized acini-like spheres with the anticipated distribution of marker proteins associated with mammary epithelial polarization e.g. α6-integrin, laminin 5 and Human Milk Fat Globule/MUC1. At hypoxia, cells were not polarized and the sub-cellular distribution pattern of the marker proteins rather resembled that reported in vivo in breast cancer. The hypoxic cells remained in a mitotic state, whereas proliferation ceased with acinar morphogenesis at normoxia. We found induced expression of the differentiation repressor ID1 in the undifferentiated hypoxic MCF-10A cell structures. Acinar morphogenesis was associated with global histone deacetylation whereas the hypoxic breast epithelial cells showed sustained global histone acetylation, which is generally associated with active transcription and an undifferentiated proliferative state

Promoter-associated proteins of EPAS1 identified by enChIP-MS – A putative role of HDX as a negative regulator

Presence of perivascular neuroblastoma cells with high expression of hypoxia inducible factor (HIF)-2α correlates with distant metastasis and aggressive disease. Regulation of HIFs are traditionally considered to occur post-translationally, but we have recently shown that HIF-2α is unconventionally regulated also at the transcriptional level in neuroblastoma cells. Regulatory factors binding directly to EPAS1 (encoding HIF-2α) to promote transcription are yet to be defined. Here, we employ the novel CRISPR/Cas9-based engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) – mass spectrometry (MS) methodology to, in an unbiased fashion, identify proteins that associate with the EPAS1 promoter under normoxic and hypoxic conditions. Our enChIP analysis resulted in 27 proteins binding to the EPAS1 promoter in neuroblastoma cells. In agreement with a general hypoxia-driven downregulation of gene transcription, the majority (24 out of 27) of proteins dissociate from the promoter at hypoxia. Among them were several nucleosome-associated proteins suggesting a general opening of chromatin as one explanation to induced EPAS1 transcription at hypoxia. Of particular interest from the list of released factors at hypoxia was the highly divergent homeobox (HDX) transcription factor, that we show inversely correlates with HIF-2α in neuroblastoma cells. We propose a putative model where HDX negatively regulates EPAS1 expression through a release-of-inhibition mechanism

Hunting for Protein Markers of Hypoxia by Combining Plasma Membrane Enrichment with a New Approach to Membrane Protein Analysis

Nontransient hypoxia is strongly associated with malignant lesions, resulting in aggressive behavior and resistance to treatment. We present an analysis of mRNA and protein expression changes in neuroblastoma cell lines occurring upon the transition from normoxia to hypoxia. The correlation between mRNA and protein level changes was poor, although some known hypoxia-driven genes and proteins correlated well. We present previously undescribed membrane proteins expressed under hypoxic conditions that are candidates for evaluation as biomarkers