Endothelial cells activate the cancer stem cell‐associated NANOGP8 pathway in colorectal cancer cells in a paracrine fashion

Abstract

In colorectal cancer (CRC), cancer stem cells (CSCs) have been hypothesized to mediate cell survival and chemoresistance. Previous studies from our laboratory described a role for liver parenchymal endothelial cells (LPECs) in mediating the CSC phenotype in CRC cells in a paracrine/angiocrine fashion. The objectives of this study were to determine whether endothelial cells (ECs) from different organs can induce the CSC phenotype in CRC cells and to elucidate the signaling pathways involved. We treated a newly developed CRC cell line (HCP-1) and established CRC cell lines (HT29 and SW480) with conditioned medium (CM) from primary ECs isolated from nonmalignant liver, lung, colon mucosa, and kidney. Our results showed that CM from ECs from all organs increased the number of CSCs, as determined by sphere formation, and protein levels of NANOG and OCT4 in CRC cells. With the focus of further elucidating the role of the liver vascular network in mediating the CSC phenotype, we demonstrated that CM from LPECs increased resistance to 5-fluorouracil in CRC cells. Moreover, we showed that LPEC CM specifically induced NANOGP8 expression in CRC cells by specific enzyme digestion and a luciferase reporter assay using a vector containing the NANOGP8 promoter. Lastly, we found that LPEC CM-induced NANOGP8 expression and sphere formation were mediated by AKT activation. Our studies demonstrated a paracrine role for ECs in regulating the CSC phenotype and chemoresistance in CRC cells by AKT-mediated induction of NANOGP8. These studies suggest a more specific approach to target CSCs by blocking the expression of NANOGP8 in cancer cells.

Endothelial Cells Promote Colorectal Cancer Cell Survival by Activating the HER3-AKT Pathway in a Paracrine Fashion

“…To determine the effects of ECs on CRC cell survival (including cell viability and chemoresistance), we used primary liver EC lines (LPEC-1 and LPEC-6) that our laboratory established from non-malignant liver tissues (10). CM containing EC-secreted factors was harvested and added to CRC cells (HCP-1, HT29 and SW480), with CM from CRC cells themselves serving as control CM.…”

“…The colorectal cancer (CRC) cell lines SW480, HT29, HCT116, RKO, SW48 and Caco2 were purchased from American Type Culture Collection (ATCC, Manassas, VA). The H uman C RC P rimary cell line (HCP-1), luciferase-labeled HCP-1 cells, L iver P arenchymal E ndothelial C ell (LPEC-1 and LPEC-6) lines, ECs from lung (lung ECs), and ECs from colon mucosa (colon ECs) were established in our laboratory (10,11). CRC cells were cultured in MEM supplemented with 5% FBS (Atlanta Biologicals, Atlanta, GA), vitamins (1x), nonessential amino acids (1x), penicillin-streptomycin antibiotics (1 x), sodium pyruvate (1x), and L-glutamine (1x), all from Invitrogen (Carlsbad, CA).…”

“…When the HER3 inhibitor AZD8931 was used, cells were pretreated with AZD8931 in 1% FBS medium overnight, and then cultured with or without 5-FU and AZD8931 in CM. Cell apoptosis was determined using the FITC Annexin V Apoptosis Detection Kit I from BD Biosciences (San Jose, CA) as described before (10). In brief, single suspended cells were double-stained with FITC Annexin V and propidium iodide and analyzed by fluorescence-activated cell sorting.…”

“…In the past few years, our laboratory has isolated primary ECs from non-malignant liver and established an in vitro model using conditioned medium (CM) from these primary ECs to study their effects on CRC cells. With this model, we previously demonstrated that ECs secrete soluble factors in CM that, in turn, increase the cancer stem cell (CSC) phenotype in CRC cells in a paracrine fashion (10,11). In these prior studies, we showed that incubation of CM from liver ECs activated CSC-associated pathways (such as NOTCH and NANOG) and induced CSC-associated functions (including sphere formation, resistance to chemotherapy, and potential to metastasize) in CRC cell.…”

Differential sequences of exosomal NANOG DNA as a potential diagnostic cancer marker

“…The NANOG family is comprised of the original NANOG gene and ten associated pseudogenes (P1-P10). Recently, researchers have shown increased expression levels of NANOGP8 in glioblastoma cancer stem cells and have suggested that NANOGP8 may participate in the reprogramming of normal cells into cancerous states [ 10 , 11 ]. Furthermore, various studies have also reported increased expression levels of NANOG in cancer stem cells in other types of cancers, such as breast and lung cancers, supporting that NANOG and NANOGP8 are established cancer markers [ 12 , 13 , 14 , 15 ].…”