Wnt secretion is required to maintain high levels of Wnt activity in colon cancer cells

Abstract

Aberrant regulation of the Wnt/β-catenin pathway has an important role during the onset and progression of colorectal cancer, with over 90% of cases of sporadic colon cancer featuring mutations in APC or β-catenin. However, it has remained a point of controversy whether these mutations are sufficient to activate the pathway or require additional upstream signals. Here we show that colorectal tumours express elevated levels of Wnt3 and Evi/Wls/GPR177. We found that in colon cancer cells, even in the presence of mutations in APC or β-catenin, downstream signalling remains responsive to Wnt ligands and receptor proximal signalling. Furthermore, we demonstrate that truncated APC proteins bind β-catenin and key components of the destruction complex. These results indicate that cells with mutations in APC or β-catenin depend on Wnt ligands and their secretion for a sufficient level of β-catenin signalling, which potentially opens new avenues for therapeutic interventions by targeting Wnt secretion via Evi/Wls.

ERAD‐dependent control of the Wnt secretory factor Evi

“…Taken together, our data indicate that CGRRF1 and UBE2J2 are part of an ubiquitination complex that can regulate Evi abundance (Fig EV5F). CGRRF1 expression is reduced in colon and endometrial cancer (Fig EV5G; TCGA, 2012), which correlates with increased Evi protein levels (Voloshanenko et al , 2013; Stewart et al , 2015) and a lack of transcriptional upregulation of Evi as assessed by TCGA expression data (Fig EV5H; TCGA, 2012). …”

“…Increased Evi protein abundance coincides with elevated Wnt3 expression in colorectal cancer (Fig EV1A; TCGA, 2012; Voloshanenko et al , 2013). However, transcriptome profiles of 456 colon adenocarcinoma samples showed no increase in Evi mRNA levels (Fig EV1B; TCGA, 2012).…”

“…Since several Wnt proteins are upregulated in colorectal cancer (TCGA, 2012; Voloshanenko et al , 2013), we assumed that an increase in Wnt proteins might affect the levels of the cargo receptor Evi, allowing the cells to adjust to the molecular requirements for Wnt secretion. To test this hypothesis, we transiently increased Wnt levels in HEK293T cells and observed an elevation in Evi protein levels upon Wnt3A expression, but not upon expression of a secreted control protein, IGFBP5‐V5 (Fig 1B).…”

“…To confirm the role of Porcn function in Evi regulation without manipulating Wnt expression, we monitored Evi in the colon cancer cell line HCT116, that depends on Wnt secretion (Voloshanenko et al , 2013) and in the melanoma cell line A375, that expresses Wnt5A and Wnt10B endogenously (Yang et al , 2012). LGK974 treatment of HCT116 and A375 cells reduced Evi protein levels in a time‐dependent manner and depleted Evi below the detection limit after 24–48 h (Fig 2E) without reducing Evi mRNA levels (Fig EV2C and D).…”

“…Evi is then recycled via the retromer complex back to the Golgi and ER (Coudreuse, 2006; Belenkaya et al , 2008; Franch‐Marro et al , 2008; Pan et al , 2008; Port et al , 2008; Yang et al , 2008; Yu et al , 2014). The levels of both Wnt and Evi proteins are often aberrantly regulated in cancer (Augustin et al , 2012; Voloshanenko et al , 2013; Stewart et al , 2015), which contributes to high Wnt activity during tumorigenesis.…”