De novo and acquired resistance, largely attributed to genetic alterations, are barriers to effective anti-EGFR therapy. We generated cetuximab-resistant cells following prolonged cetuximab exposure to cetuximab-sensitive colorectal cancer cells in three-dimensional culture. Through whole exome sequencing and transcriptional profiling, we found overexpression of lncRNA MIR100HG and two embedded miRNAs, miR-100 and miR-125b, in the absence of known genetic events linked to cetuximab resistance. MIR100HG and miR-100/125b overexpression was also observed in cetuximab-resistant colorectal cancer and head and neck squamous cell cancer cell lines and in tumors from colorectal cancer patients that progressed on cetuximab. miR-100/125b coordinately represses five Wnt/β-catenin negative regulators, resulting in increased Wnt signaling, and Wnt inhibition in cetuximab-resistant cells restored cetuximab responsiveness. We describe a double-negative feedback loop between MIR100HG and GATA6, whereby GATA6 represses MIR100HG, but this repression is relieved by miR-125b targeting of GATA6. These studies identify a clinically actionable, epigenetic cause of cetuximab resistance.

Given the disposability of somatic tissue, selection can favor a higher mutation rate in the early segregating soma than in germline, as seen in some animals. Although in plants intra-organismic mutation rate heterogeneity is poorly resolved, the same selectionist logic can predict a lower rate in shoot than in root and in longer-lived terminal tissues (e.g., leaves) than in ontogenetically similar short-lived ones (e.g., petals), and that mutation rate heterogeneity should be deterministic with no significant differences between biological replicates. To address these expectations, we sequenced 754 genomes from various tissues of eight plant species. Consistent with a selectionist model, the rate of mutation accumulation per unit time in shoot apical meristem is lower than that in root apical tissues in perennials, in which a high proportion of mutations in shoots are themselves transmissible, but not in annuals, in which somatic mutations tend not to be transmissible. Similarly, the number of mutations accumulated in leaves is commonly lower than that within a petal of the same plant, and there is no more heterogeneity in accumulation rates between replicate branches than expected by chance. High mutation accumulation in runners of strawberry is, we argue, the exception that proves the rule, as mutation transmission patterns indicate that runner has a restricted germline. However, we also find that in vitro callus tissue has a higher mutation rate (per unit time) than the wild-grown comparator, suggesting nonadaptive mutational “fragility”. As mutational fragility does not obviously explain why the shoot—root difference varies with plant longevity, we conclude that some mutation rate variation between tissues is consistent with selectionist theory but that a mechanistic null of mutational fragility should be considered.

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...

Introduction: Metastasis and drug resistance contribute substantially to the poor prognosis of colorectal cancer (CRC) patients. However, the epigenetic regulatory mechanisms by which CRC develops metastatic and drug-resistant characteristics remain unclear. This study aimed to investigate the role of miR-302a in the metastasis and molecular-targeted drug resistance of CRC and elucidate the underlying molecular mechanisms.Methods: miR-302a expression in CRC cell lines and patient tissue microarrays was analyzed by qPCR and fluorescence in situ hybridization. The roles of miR-302a in metastasis and cetuximab (CTX) resistance were evaluated both in vitro and in vivo. Bioinformatic prediction algorithms and luciferase reporter assays were performed to identify the miR-302a binding regions in the NFIB and CD44 3'-UTRs. A chromatin immunoprecipitation assay was performed to examine NFIB occupancy in the ITGA6 promoter region. Immunoblotting was performed to identify the EGFR-mediated pathways altered by miR-302a.Results: miR-302a expression was frequently reduced in CRC cells and tissues, especially in CTX-resistant cells and patient-derived xenografts. The decreased miR-302a levels correlated with poor overall CRC patient survival. miR-302a overexpression inhibited metastasis and restored CTX responsiveness in CRC cells, whereas miR-302a silencing exerted the opposite effects. NFIB and CD44 were identified as novel targets of miR-302a. miR-302a inhibited the metastasis-promoting effect of NFIB that physiologically activates ITGA6 transcription. miR-302a restored CTX responsiveness by suppressing CD44-induced cancer stem cell-like properties and EGFR-mediated MAPK and AKT signaling. These results are consistent with clinical observations indicating that miR-302a expression is inversely correlated with the expression of its targets in CRC specimens.Conclusions: Our findings show that miR-302a acts as a multifaceted regulator of CRC metastasis and CTX resistance by targeting NFIB and CD44, respectively. Our study implicates miR-302a as a candidate prognostic predictor and a therapeutic agent in CRC.

Depletion of soil nutrients is a major cause of decline in productivity of forest plantations in successive rotations. Biochar amendment in agricultural systems has been shown to yield various beneficial effects, including increasing soil phosphorus (P) availability. However, the direct and indirect effects of biochar addition on forest soil P dynamics have largely been unexplored. The objective of this study was to examine how biochar produced from harvest residue (leaves and woodchips) affect the P dynamics in second rotation Cunninghamia lanceolata (Chinese fir) plantation soil. An incubation experiment which involved mixing of forest soil with 1% or 3% w/w leaf or woodchip biochar, pyrolyzed at 300 °C or 600 °C, was conducted for 80 days at 20 °C. After 7, 40 and 80 days of incubation, soil samples were analyzed for total and available P, inorganic and organic P pools, and soil phosphatase activity. At the end of the incubation period, bacterial community composition and diversity were analyzed by 16S rDNA sequencing. The leaf biochar produced at both pyrolysis temperatures was more alkaline and had significantly higher soluble P, nitrogen and calcium contents than the woodchip biochar. Soil total and available P increased significantly in all leaf biochar treatments after 80 days incubation compared to the untreated control soil, but the woodchip biochar treatments had no significant effects.