Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival

International audienceBACKGROUND: Neurotrophin receptors were initially identified in neural cells. They were recently detected in some cancers in association with invasiveness, but the function of these tyrosine kinase receptors was not previously investigated in colorectal cancer (CRC) cells. METHODS AND FINDINGS: We report herein that human CRC cell lines synthesize the neural growth factor Brain-derived neurotrophic factor (BDNF) under stress conditions (serum starvation). In parallel, CRC cells expressed high- (TrkB) and low-affinity (p75(NTR)) receptors at the plasma membrane, whereas TrkA and TrkC, two other high affinity receptors for NGF and NT-3, respectively, were undetectable. We demonstrate that BDNF induced cell proliferation and had an anti-apoptotic effect mediated through TrkB, as assessed by K252a, a Trk pharmacologic inhibitor. It suppressed both cell proliferation and survival of CRC cells that do not express TrkA nor TrkC. In parallel to the increase of BDNF secretion, sortilin, a protein acting as a neurotrophin transporter as well as a co-receptor for p75(NTR), was increased in the cytoplasm of primary and metastatic CRC cells, which suggests that sortilin could regulate neurotrophin transport in these cells. However, pro-BDNF, also detected in CRC cells, was co-expressed with p75(NTR) at the cell membrane and co-localized with sortilin. In contrast to BDNF, exogenous pro-BDNF induced CRC apoptosis, which suggests that a counterbalance mechanism is involved in the control of CRC cell survival, through sortilin as the co-receptor for p75(NTR), the high affinity receptor for pro-neurotrophins. Likewise, we show that BDNF and TrkB transcripts (and not p75(NTR)) are overexpressed in the patients' tumors by comparison with their adjacent normal tissues, notably in advanced stages of CRC. CONCLUSION: Taken together, these results highlight that BDNF and TrkB are essential for CRC cell growth and survival in vitro and in tumors. This autocrine loop could be of major importance to define new targeted therapies

Cancer stem cell sorting from colorectal cancer cell lines by sedimentation field flow fractionation.

International audienceRecently, cancer stem cells (CSCs) have been identified in many types of cancers, such as colorectal cancer (CRC). CSCs seem to be involved in initiation, growth, and tumor metastasis, as well as in radio- and chemotherapy failures. CSCs appears as new biological targets for cancer therapy, requiring the development of noninvasive cell sorting methods. In this study, we used sedimentation field flow fractionation (SdFFF) to prepare enriched populations of CSCs from eight cell lines corresponding to different CRC grades. On the basis of phenotypic and functional characterizations, "hyperlayer" elution resulted in a fraction overexpressing CSC markers (CD44, CD166, EpCAM) for all cell lines. CSCs were eluted in the last fraction for seven out of eight cell lines, but in the first for HCT116. These results suggest, according to the literature, that two different pools of CSCs exist, quiescent and activated, which can both be sorted by SdFFF. Moreover, according to CSC properties, enriched fractions are able to form colonies

New ex-ovo colorectal-cancer models from different SdFFF-sorted tumor-initiating cells

International audienceDespite effective treatments, relapse of colorectal cancer (CRC) is frequent, in part caused by the existence of tumor-initiating cells (TICs). Different subtypes of TICs, quiescent and activated, coexist in tumors, defining the tumor aggressiveness and therapeutic response. These subtypes have been sorted by hyperlayer sedimentation field-flow fractionation (SdFFF) from WiDr and HCT116 cell lines. On the basis of a new strategy, including TIC SdFFF sorting, 3D Matrigel amplification, and grafting of corresponding TIC colonies on the chick chorioallantoic membrane (CAM), specific tumor matrices could be obtained. If tumors had similar architectural structure with vascularization by the host system, they had different proliferative indices in agreement with their initial quiescent or activated state. Protein analysis also revealed that tumors obtained from a population enriched for “activated” TICs lost “stemness” properties and became invasive. In contrast, tumors obtained from a population enriched for “quiescent” TICs kept their stemness properties and seemed to be less proliferative and invasive. Then, it was possible to produce different kinds of tumor which could be used as selective supports to study carcinogenesis and therapy sensitivity

Relationship between pro-BDNF, sortilin, p75^NTR and apoptosis.

<p>(A, B) Sortilin as a coreceptor of p75^NTR. Double staining (yellow) of sortilin (red) and p75^NTR (green) in SW480 cells (A) and SW620 (B) after 24 h of serum deprivation. (C) Colocalization of pro-BDNF and sortilin. Confocal microscopy study of a WiDr cells stained with an anti-pro-BDNF Ab (green) and an anti-sortilin Ab (red), and double staining (yellow) after 24 h of serum deprivation. (D, E) apoptotic ratios after 24 h serum deprivation alone (0% FCS) or combined with recombinant Pro-BDNF (0% Pro-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001, when compared with serum-free condition alone (0% FCS).</p

BDNF release and effect of exogenous BDNF, pro-BDNF, TrkB inhibitor (K252a) and neutralizing anti-BDNF on apoptosis and proliferation in metastatic CRC cell lines.

<p>Results are expressed as relative units of proliferating cells (EDU) or apoptotic ratios of soluble nucleosomes detected by ELISA Cell Death (ECD) after 24 to 72 hours (H) of serum deprivation (0% FCS). Mean ± SEM of at least three independent experiments.</p><p>*, <i>p</i><0.05;</p><p>**, <i>p</i><0.01;</p><p>***, <i>p</i><0.001, compared with serum-free condition alone (0% FCS).</p

Sortilin expression by CRC cell lines.

<p>(A) Sortilin detection by RT-PCR of total RNA extracted from cells cultured in 10% FCS and after 24–72 h serum starvation. Expression was controlled with specific primers for its extracellular (Sortilin EC) and intracellular (Sortilin IC) parts. A Representative result from at least three independent experiments. (B) Assessment by western blotting of sortilin expression (in reference to actin) in total cellular protein extracted from studied cell lines cultured under basal condition and after 24–72 h of serum deprivation. According to densitometric analyses, quantification showed a significant increased expression of sortilin in cultured cells. Histograms are means ± SEM of at least three independent experiments. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001, when compared with basal culture condition (10% FCS). (C) Confocal microscopy of SW620 cells stained with an anti-BDNF Ab (green) and an anti-Sortilin Ab (red) and double staining (merged) in basal culture condition and after 24 h serum starvation. Relative quantification was assessed by green and red fluorescence surface plot. Images were representative for at least three independent experiments. Scale bars, 10 µm. Similar results were observed with the three other lines (data not shown).</p

Hallmarks in colorectal cancer: Angiogenesis and cancer stem-like cells

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Membranous and cytoplasmic expression of BDNF and TrkB depending on culture conditions.

<p>Confocal microscopy of WiDr (A, B) and COLO 205 (C, D) cells, stained with an anti-BDNF Ab (red), anti-TrkB mAb (green) or both (overlay) cultured with 10% FCS (A, C) or after 24-h serum deprivation (B, D). Under basal culture conditions (10% FCS), TrkB and BDNF were sequestered in the cytoplasm (arrows) in WiDr (A) and COLO 205 (C) cells. The same staining patterns were obtained with the two other cell lines (data not shown). After serum starvation relocation to the cell membrane and colocalization of TrkB and BDNF (yellow in merged, arrows) were detected in WiDr (B) and COLO 205 (D). Images were representative for at least three to five independent experiments.</p

BDNF and its receptors are expressed in colorectal cancer tissues.

<p>RT-PCR analysis of BDNF (A), its two high TrkB145 and TrkB95 (B, C, respectively), and low p75^NTR.(D) affinity receptors in total mRNA extracted from 16 surgically resected primary and metastatic colon adenocarcinoma specimens. <i>N</i>, non-tumor tissue (n = 16); <i>T</i>, tumor tissue (n = 16); <i>NCT</i>, noncancerous tissue (n = 4). Histograms, mean percentage of each amplified mRNA/GAPDH expression of band intensities evaluated by densitometry. Statistical significance:* <i>p</i><0.05; ** <i>p</i><0.01.</p