Core-Clock Genes Regulate Proliferation and Invasion via a Reciprocal Interplay with MACC1 in Colorectal Cancer Cells

Abstract

The circadian clock coordinates the timing of several cellular processes including transcription, the cell cycle, and metabolism. Disruptions in the clock machinery trigger the abnormal regulation of cancer hallmarks, impair cellular homeostasis, and stimulate tumourigenesis. Here we investigated the role of a disrupted clock by knocking out or knocking down the core-clock (CC) genes ARNTL, PER2 or NR1D1 in cancer progression (e.g., cell proliferation and invasion) using colorectal cancer (CRC) cell lines HCT116, SW480 and SW620, from different progression stages with distinct clock phenotypes, and identified mechanistic links from the clock to altered cancer-promoting cellular properties. We identified MACC1 (metastasis-associated in colon cancer 1), a known driver for metastasis and an EMT (epithelial-to-mesenchymal transition)-related gene, to be significantly differentially expressed in CC manipulated cells and analysed the effect of MACC1 manipulation (knockout or overexpression) in terms of circadian clock phenotype as well as cancer progression. Our data points to a bi-directional MACC1-circadian clock interplay in CRC, via CC genes. In particular, knocking out MACC1 reduced the period of oscillations, while its overexpression increased it. Interestingly, we found the MACC1 protein to be circadian expressed in HCT116 WT cells, which was disrupted after the knockout of CC genes, and identified a MACC1-NR1D1 protein–protein interaction. In addition, MACC1 manipulation and CC knockout altered cell invasion properties of HCT116 cells, pointing to a regulation of clock and cancer progression in CRC, possibly via the interaction of MACC1 with core-clock genes.

MACC1-induced migration in tumors: Current state and perspective

“…Increased PTEN expression was found after MACC1 silencing in esophageal carcinoma ( 178 ), and in hepatocellular carcinoma, MACC1 induced PI3K activation ( 179 ). In line with this, several studies reported associations of MACC1 with AKT activation ( 33 , 143 , 166 , 178 – 183 ), suggesting the following MACC1 signaling: MACC1/IGFBP2/PTEN/PI3K/AKT ( 175 , 178 , 179 , 184 ). Yet, MACC1 may affect PI3K/AKT signaling also via direct interactions with YWHAE (14-3-3 epsilon), as confirmed by affinity capture-MS ( 185 ).…”

“…Therefore, the setup and data analysis can be considered rather simple and quick. Of note, there are modified versions of the Boyden chamber assay that do not need manual counting and allow for a continuous data assessment, e.g., the IncuCyte or xCELLigence systems ( 12 , 143 , 144 ). Yet, for MACC1, the standard Boyden chamber assay was the most used one (see Table 2 ).…”

Identification and characterization of the CDK1-BMAL1-UHRF1 pathway driving tumor progression

“…Consistent with BMAL1, knockdown or knockout of core clock genes (PER2 or NR1D1) results in differential expression of several key EMT (epithelial-to-mesenchymal transition) genes, including MACC, and influences cancer proliferation and migration. 43 NR1D1 downregulation resulted in impaired viability and reduced micrometastasis formation in colon cancer cells, with altered expression levels of SNAI1. 44 Overall, circadian clock network disruption contributes to tumor growth and metastasis.…”

Antiproliferative Effects of Cynara Cardunculus in Colorectal Cancer Cells Are Modulated by the Circadian Clock

“…CRC cell lines display a variety of circadian phenotypes which range from a completely disrupted clock to less dramatic alteration in circadian rhythms, which have been reported in published work [ 8 , 9 , 23 , 24 , 25 ]. This variability can also be observed when evaluating the connection between the circadian clock and cancer-related pathways, and the effects are often specific for certain cell lines, as well as specific core-clock gene disruptions [ 8 , 38 ]. Although we focus on the HCT116 cell line and its derived core-clock KO cell lines in this study, future studies should include other CRC cell lines for possible generalizations of the treatment effect of C. cardunculus in CRC.…”