PDF file - 55K, Table S1. Expression of cell surface stem cell markers in parental HT29 cells, its 5FUresistant 5F7 and 5F31 clonal derivatives, 5FU- and oxaliplatin-resistant HT29FU and HT29OXA subpopulations.

Supplementary Figure S1 identifies YAP/RXRalpha and YAP/RARgamma molecular complexes in HT29-YAPdc cells expressing activated nuclear YAP

Supplementary Table 2 from Autocrine Induction of Invasive and Metastatic Phenotypes by the MIF-CXCR4 Axis in Drug-Resistant Human Colon Cancer Cells

Supplementary Figure 2 from Netrin-1 Induces Apoptosis in Human Cervical Tumor Cells via the TAp73α Tumor Suppressor

Supplementary Methods, Figure Legends 1 from Autocrine Induction of Invasive and Metastatic Phenotypes by the MIF-CXCR4 Axis in Drug-Resistant Human Colon Cancer Cells

Supplementary Table S1 displays the list of antibodies, siRNAs and primers used in the study

Supplementary Figure Legends 1-11, Methods from Implication of Metastasis Suppressor NM23-H1 in Maintaining Adherens Junctions and Limiting the Invasive Potential of Human Cancer Cells

Supplementary Table S2 presents the detailed proteomics of nuclear YAP-interactome identified in HT29-YAPdc cells

Supplementary Figure S2 shows the co-occupancy by RXRalpha, RARalpha/gamma and YAP of ALDH1A3 and LGR5 proximal RAREs in control HT29 cells

Supplementary Figure S3 shows the validation of the YAP/RAR-RXR crosstalk in control and YAPdc-RKO colorectal cancer cells.

Supplementary Movie 2 from Implication of Metastasis Suppressor NM23-H1 in Maintaining Adherens Junctions and Limiting the Invasive Potential of Human Cancer Cells

PDF file - 309K, Figure S1. Expression of cancer stem cell phenotypes in 5FU-resistant 5F7 and 5F31 clones.

Supplementary Table S3 (Parts A and B) provides the classification of 115 YAP-binding partners according to several criteria related to the study

PDF file - 202K, Figure S2. Western blot analysis of c-Yes and YAP in human colon cancer cells HT29 and RKO cells treated by oxaliplatin and 5FU.

PDF file - 54K, Table S2. Effect of 5FU on the cell cycle progression in parental HT29 cells and 5FUresistant HT29 clonal derivatives 5F7 and 5F31.

Abstract The mechanisms whereby the Hippo pathway effector YAP regulates cancer cell stemness, plasticity, and chemoresistance are not fully understood. We previously showed that in 5-fluorouracil (5-FU)–resistant colorectal cancer cells, the transcriptional coactivator YAP is differentially regulated at critical transitions connected with reversible quiescence/dormancy to promote metastasis. Here, we found that experimental YAP activation in 5-FU–sensitive and 5-FU–resistant HT29 colorectal cancer cells enhanced nuclear YAP localization and the transcript levels of the retinoic acid (RA) receptors RARα/γ and RAR target genes CYP26A1, ALDH1A3, and LGR5 through RA Response Elements (RARE). In these two cell models, constitutive YAP activation reinforced the expression of the stemness biomarkers and regulators ALDH1A3, LGR5, and OCT4. Conversely, YAP silencing, RAR/RXR inhibition by the pan-RAR antagonist BMS493, and vitamin A depletion downregulated stemness traits and self-renewal. Regarding the mechanisms engaged, proximity-dependent labeling, nuclear YAP pulldown coupled with mass spectrometry, and chromatin immunoprecipitation (ChIP)/re-ChIP experiments revealed: (i) the nuclear colocalization/interaction of YAP with RARγ and RXRs; and (ii) combined genomic co-occupancy of YAP, RARα/γ, and RXRα interactomes at proximal RAREs of LGR5 and ALDH1A3 promoters. Moreover, activation of the YAP/RAR-RXR cross-talk in colorectal cancer cells promoted RAR self-activation loops via vitamin A metabolism, RA, and active RAR ligands generated by ALDH1A3. Together, our data identify YAP as a bona fide RAR-RXR transcriptional coactivator that acts through RARE-activated stemness genes. Implications: Targeting the newly identified YAP/RAR-RXR cross-talk implicated in cancer cell stemness maintenance may lead to multitarget combination therapies for patients with colorectal cancer.

Spheroids are three-dimensional cellular models with widespread basic and translational application across academia and industry. However, methodological transparency and guidelines for spheroid research have not yet been established. The MISpheroID Consortium developed a crowdsourcing knowledgebase that assembles the experimental parameters of 3,058 published spheroid-related experiments. Interrogation of this knowledgebase identified heterogeneity in the methodological setup of spheroids. Empirical evaluation and interlaboratory validation of selected variations in spheroid methodology revealed diverse impacts on spheroid metrics. To facilitate interpretation, stimulate transparency and increase awareness, the Consortium defines the MISpheroID string, a minimum set of experimental parameters required to report spheroid research. Thus, MISpheroID combines a valuable resource and a tool for three-dimensional cellular models to mine experimental parameters and to improve reproducibility.

TWIST1 is a basic helix-loop-helix transcription factor, and one of the master Epithelial-to-Mesenchymal Transition (EMT) regulators. We show that tumor suppressor miR-145-5p controls TWIST1 expression in an immortalized prostate epithelial cell line and in a tumorigenic prostate cancer-derived cell line. Indeed, shRNA-mediated miR-145-5p silencing enhanced TWIST1 expression and induced EMT-associated malignant properties in these cells. However, we discovered that the translational inhibitory effect of miR-145-5p on TWIST1 is lost in 22Rv1, another prostate cancer cell line that intrinsically expresses high levels of the CPEB1 cytoplasmic polyadenylation element binding protein. This translational regulator typically reduces TWIST1 translation efficiency by shortening the TWIST1 mRNA polyA tail. However, our results indicate that the presence of CPEB1 also interferes with the binding of miR-145-5p to the TWIST1 mRNA 3'UTR. Mechanistically, CPEB1 binding to its first cognate site either directly hampers the access to the miR-145-5p response element or redirects the cleavage/polyadenylation machinery to an intermediate polyadenylation site, resulting in the elimination of the miR-145-5p binding site. Taken together, our data support the notion that the tumor suppressive activity of miR-145-5p on TWIST1 translation, consequently on EMT, self-renewal, and migration, depends on the CPEB1 expression status of the cancer cell. A preliminary prospective study using clinical samples suggests that reconsidering the relative status of miR-145-5p/TWIST1 and CPEB1 in the tumors of prostate cancer patients may bear prognostic value.

In mammals, insulin regulates blood glucose levels and plays a key regulatory role in appetite via the hypothalamus. In contrast, chickens are characterized by atypical glucose homeostasis, with relatively high blood glucose levels, reduced glucose sensitivity of pancreatic beta cells, and large resistance to exogenous insulin. The aim of the present study was to investigate in chickens the effects of 5 h fasting and 5 h insulin immuno-neutralization on hypothalamic mRNA levels of 23 genes associated with food intake, energy balance, and glucose metabolism. We observed that insulin immune-neutralization by administration of anti-porcine insulin guinea pig serum (AI) significantly decreased food intake and increased plasma glucose levels in chickens, while 5 h fasting produced a limited and non-significant reduction in plasma glucose. In addition, 5 h fasting increased levels of NPY, TAS1R1, DIO2, LEPR, GLUT1, GLUT3, GLUT8, and GCK mRNA. In contrast, AI had no impact on the levels of any selected mRNA. Therefore, our results demonstrate that in chickens, food intake inhibition or satiety mechanisms induced by insulin immuno-neutralization do not rely on hypothalamic abundance of the 23 transcripts analyzed. The hypothalamic transcripts that were increased in the fasted group are likely components of a mechanism of adaptation to fasting in chickens.