Endothelial Cells Promote the Colorectal Cancer Stem Cell Phenotype through a Soluble Form of Jagged-1

SummaryWe report a paracrine effect whereby endothelial cells (ECs) promote the cancer stem cell (CSC) phenotype of human colorectal cancer (CRC) cells. We showed that, without direct cell-cell contact, ECs secrete factors that promoted the CSC phenotype in CRC cells via Notch activation. In human CRC specimens, CD133 and Notch intracellular domain-positive CRC cells colocalized in perivascular regions. An EC-derived, soluble form of Jagged-1, via ADAM17 proteolytic activity, led to Notch activation in CRC cells in a paracrine manner; these effects were blocked by immunodepletion of Jagged-1 in EC-conditioned medium or blockade of ADAM17 activity. Collectively, ECs play an active role in promoting Notch signaling and the CSC phenotype by secreting soluble Jagged-1

GHS-R in brown fat potentiates differential thermogenic responses under metabolic and thermal stresses

In response to cold or diet, fatty acids are dissipated into heat through uncoupling protein 1 (UCP1) in brown adipose tissue (BAT). This process is termed non-shivering thermogenesis, which is important for body temperature maintenance and contributes to obesity pathogenesis. Thermogenic enhancement has been considered a promising anti-obesity strategy. Ghrelin and its receptor Growth Hormone Secretagogue Receptor (GHS-R) have critical roles in energy intake, nutrient sensing, and lipid metabolism. We previously reported that global Ghsr-knockout mice have increased energy expenditure due to enhanced thermogenesis. To determine the site of action for GHS-R mediated thermogenesis, we generated brown adipocyte-specific Ghsr knockout mice (UCP1-CreER/Ghsrf/f) and assessed thermogenic responses under regular diet (RD) fed homeostatic metabolic state or high-fat diet (HFD) fed metabolically-impaired obese state, under normal or cold housing environment. Under a RD-feeding, UCP1-CreER/Ghsrf/f mice showed increased body fat and a slightly elevated core body temperature under cold but not under normal temperature. Consistently, the expression of thermogenic genes in BAT of RD-fed UCP1-CreER/Ghsrf/f mice was increased in reposes to cold. Under HFD feeding, HFD-fed UCP1-CreER/Ghsrf/f mice showed no difference in body fat or body temperature under either normal or cold exposure. Interestingly, the expression of thermogenic genes in BAT of HFD-fed UCP1-CreER/Ghsrf/f mice was upregulated under normal temperature but downregulated under cold exposure. Overall, our data show that GHS-R has cell-autonomous effect in brown adipocytes, and GHS-R regulates BAT thermogenic activity in a temperature- and metabolic state-dependent manner. The thermogenic effect of GHS-R in BAT is more pronounced in cold environment and differentially variable based on metabolic state; under cold exposure, GHS-R inhibition in BAT activates thermogenesis under homeostatic state but suppresses thermogenesis under obese state. Our finding collectively suggests that GHS-R in BAT, acting as a “metabolic thermostat”, differentially regulates thermogenesis in response to different metabolic and thermal stimuli.ISSN:1932-620

Cytospin-A Regulates Colorectal Cancer Cell Division and Migration by Modulating Stability of Microtubules and Actin Filaments

Proteins that interact with cytoskeletal elements play important roles in cell division and are potentially important targets for therapy in cancer. Cytospin-A (CYTSA), a protein known to interact with actin and microtubules, has been previously described to be important in various developmental disorders, including oblique facial clefting. We hypothesized that CYTSA plays an important role in colorectal cancer (CRC) cell division. The effects of CYTSA depletion on CRC cell proliferation were analyzed using cell growth assays, microscopic analyses of live and fixed cells, and time-lapse imaging. CYTSA depletion led to inhibition of cell proliferation, significant increases in CRC cell death, and accumulation of doublet cells during and following cell division. Depletion of CYTSA also resulted in strong inhibition of CRC cell migration and invasion. Mechanistically, CYTSA depletion resulted in significant decreases in the stability of microtubules and altered polymerization of actin filaments in CRC cells. Finally, bioinformatic analyses were performed to determine the correlation between CYTSA expression and survival of patients with CRC. Interestingly, a strong correlation between high CYTSA expression and poor survival was observed in the TCGA adenocarcinoma data set but not in an independent data set. Since inhibiting CYTSA significantly reduces CRC cell proliferation, migration, and invasion, targeting CYTSA may be a potential novel therapeutic option for patients with metastatic CRC

Murine macrophage CM enhanced the SHH signaling pathway.

<p><b>(A)</b> CSC transcription factor expression with 48 h of CM treatment in CT26 and HCP-1 cells. <b>(B)</b> Western blot analysis of SHH secretion in CRC cells and RAW cells. <b>(C)</b> Western blot analysis shows knockdown of SHH at 48 h after the indicated siRNA transfections. <b>(D</b> and <b>E)</b> Effects of knockdown SHH-mediated sphere formation in CT26 and HCP-1 cells (*P < 0.01). <b>(F)</b> HCP-1 cells were cultured with LPS-activated RAW CM or LPS-activated primary macrophage CM with or without a SMO inhibitor and the sphere-forming assay was performed (*P < 0.01).</p

Macrophage conditioned medium promotes colorectal cancer stem cell phenotype via the hedgehog signaling pathway

<div><p>Background</p><p>There is conflicting data on the role of macrophages in colorectal cancer (CRC); some studies have shown that macrophages can exert an anti-tumor effect whereas others show that macrophages are tumor promoting. We sought to determine the role of conditioned medium (CM) from macrophages, in particular classically activated macrophages, on the development of the CSC phenotype in CRC cells, which is believed to mediate tumor growth and chemoresistance.</p><p>Methods</p><p>Murine (CT26) and human (HCP-1) CRC cell lines were treated with CM from lipopolysaccharide (LPS)-activated murine macrophages. The CSC population was assessed using the sphere-forming assay and aldehyde dehydrogenase assay. Chemoresistance studies were performed using the MTT assay. CSC transcription factors and SHH protein were analyzed by Western blotting.</p><p>Results</p><p>The results showed that LPS-activated macrophage CM induced the CSC phenotype in CRC cells. Further studies showed that the CSC phenotype was mediated by the sonic hedgehog (SHH)-Gli signaling pathway, which is known to drive self-renewal; these effects were blocked by depletion of SHH in macrophage CM. In addition, LPS-activated macrophage CM enhanced chemoresistance.</p><p>Conclusions</p><p>LPS-activated macrophages play an active role in promoting the CSC phenotype through activation of the SHH-Gli signaling pathway in CRC cells.</p></div

Murine macrophage Conditioned Media (CM) promotes the stem cell phenotype of CRC cells.

<p><b>(A</b> and <b>B)</b> CT26 and HCP-1 cells were cultured with LPS-activated macrophage CM or control CM, and a sphere-forming assay was performed (*P < 0.01). <b>(C</b> and <b>D)</b> Aldefluor-positive cell population was determined (*P < 0.001).</p

Murine macrophage CM promotes chemoresistance of CRC cells.

<p><b>(A</b> and <b>B)</b> CM pretreated CT26 or HCP-1 cells were incubated with 5-FU or SN38 in control CM or RAW+LPS CM and cell viability was determined by MTT assay (*P < 0.05; *P < 0.01 respectively).</p