Three-dimensional culture system identifies a new mode of cetuximab resistance and disease-relevant genes in colorectal cancer

Abstract

We previously reported that single cells from a human colorectal cancer (CRC) cell line (HCA-7) formed either hollow single-layered polarized cysts or solid spiky masses when plated in 3D in type-I collagen. To begin in-depth analyses into whether clonal cysts and spiky masses possessed divergent properties, individual colonies of each morphology were isolated and expanded. The lines thus derived faithfully retained their parental cystic and spiky morphologies and were termed CC (cystic) and SC (spiky), respectively. Although both CC and SC expressed EGF receptor (EGFR), the EGFRneutralizing monoclonal antibody, cetuximab, strongly inhibited growth of CC, whereas SC was resistant to growth inhibition, and this was coupled to increased tyrosine phosphorylation of MET and RON. Addition of the dual MET/RON tyrosine kinase inhibitor, crizotinib, restored cetuximab sensitivity in SC. To further characterize these two lines, we performed comprehensive genomic and transcriptomic analysis of CC and SC in 3D. One of the most upregulated genes in CC was the tumor suppressor 15-PGDH/HPGD, and the most up-regulated gene in SC was versican (VCAN) in 3D and xenografts. Analysis of a CRC tissue microarray showed that epithelial, but not stromal, VCAN staining strongly correlated with reduced survival, and combined epithelial VCAN and absent HPGD staining portended a poorer prognosis. Thus, with this 3D system, we have identified a mode of cetuximab resistance and a potential prognostic marker in CRC. As such, this represents a potentially powerful system to identify additional therapeutic strategies and disease-relevant genes in CRC and possibly other solid tumors.colorectal cancer | versican | HPGD | 3D culture | cetuximab resistance T raditionally, epithelial cells have been cultured on plastic as a flat monolayer, precluding formation of their characteristic apico-basolateral structural organization. Pioneering work by Mina Bissell and Joan Brugge has shown that select breast epithelial cell lines can be grown in 3D in Matrigel as polarizing cysts with intact apico-basolateral polarity (1-3). These 3D cultures have been used to study oncogene-induced transformation and are shown to better predict in vivo behavior than 2D cultures (4, 5). Similar work in colonic epithelial cells has lagged behind; a notable exception is the work of Alan Hall and colleagues with Caco-2 cells that form uniform polarizing cysts in 3D Matrigel (6).We sought to identify human colorectal cancer (CRC) lines that exhibit apico-basolateral polarity in a better-defined 3D environment than Matrigel, which is a complex, incompletely defined extracellular matrix secreted by Engelbreth-Holm-Swarm mouse sarcoma cells (7). We previously observed that a human CRC line, HCA-7, cultured in type-1 collagen, gave rise to colonies consisting of unilamellar cysts with intact apico-basolateral polarity or less frequent colonies composed of irregular solid masses of cells (8). We derived CC and SC lines from cystic and spiky colonies, respectively. When injected sub...

Hotspots of aberrant enhancer activity in fibrolamellar carcinoma reveal molecular mechanisms of oncogenesis and intrinsic drug resistance

“…Our analyses show that a large proportion of the genes that are associated with FLC-specific enhancers function in drug resistance, including LINC00473 (Han et al, 2018;Wang et al, 2018), VCAN (Li et al, 2017), and CA12 (Boyd et al, 2017;Doyen et al, 2013;Kopecka et al, 2016;Yoo et al, 2010). This suggests that high expression of these genes may be responsible for the strong drug resistance phenotype observed in FLC.…”

“…3E), respectively; FAM19A5 because it has the greatest number of significantly correlated enhancers (Fig. 4G); TESC and VCAN because they have previously been strongly implicated in drug resistance Li et al, 2017;Man et al, 2014), a salient feature of FLC (Maniaci et al, 2009;Torbenson, 2012); CA12 and OAT Stacked bargraph examining the 10 FLC-specific enhancer hotspots that did not overlap with super enhancers in SEdb. Individual enhancers from each enhancer hotspot were examined for overlap with any enhancer from ENCODE, FANTOM5, and NIH Roadmap Epigenomics databases.…”

Proceedings of the 16th Annual UT-KBRIN Bioinformatics Summit 2016: bioinformatics

“…In this presentation, Dr. Liu focused on the two aims of integration of multi-dimensional data, including understanding the relationships between the different types of data and understanding both the latent and observable phenotypes. She discussed integrative techniques they have used for transcriptomics, proteomics, and miRNA [8] as well as other methods for integrating multiomics data with clinical applications, such as the determination of the cause of resistance to chemotherapeutics in colon cancer [9,10]. Bruce Ramshaw (University of Tennessee, Knoxville) followed with the presentation "Complex Systems Science Applied to Health Care."…”

Evidence for GALNT12 as a moderate penetrance gene for colorectal cancer

“…Moreover, GALNT12 is selectively expressed in intestinal epithelia and frequently downregulated in CRCs (Guo et al., , Guo et al., ). Furthermore, GALNT12 has been identified as one of the key genes facilitating normal morphology and polarization in colon epithelial cells (Li et al., ). GALNT12 is a key enzyme catalyzing the initiation of mucin‐type O ‐glycosylation (Bennett et al., ); a pathway shown to play a critical role in CRC progression (Pinho & Reis, ; Stowell et al., ).…”