PXR Modulates the Prostate Cancer Cell Response to Afatinib by Regulating the Expression of the Monocarboxylate Transporter SLC16A1.

Resistance to castration is a crucial issue in the treatment of metastatic prostate cancer. Kinase inhibitors (KIs) have been tested as potential alternatives, but none of them are approved yet. KIs are subject of extensive metabolism at both the hepatic and the tumor level. Here, we studied the role of PXR (Pregnane X Receptor), a master regulator of metabolism, in the resistance to KIs in a prostate cancer setting. We confirmed that PXR is expressed in prostate tumors and is more frequently detected in advanced forms of the disease. We showed that stable expression of PXR in 22Rv1 prostate cancer cells conferred a resistance to dasatinib and a higher sensitivity to erlotinib, dabrafenib, and afatinib. Higher sensitivity to afatinib was due to a ~ 2-fold increase in its intracellular accumulation and involved the SLC16A1 transporter as its pharmacological inhibition by BAY-8002 suppressed sensitization of 22Rv1 cells to afatinib and was accompanied with reduced intracellular concentration of the drug. We found that PXR could bind to the SLC16A1 promoter and induced its transcription in the presence of PXR agonists. Together, our results suggest that PXR could be a biomarker of response to kinase inhibitors in castration-resistant prostate cancers.

Metastatic colorectal cancer cells maintain the TGFß program and use TGFBI to fuel angiogenesis.

Colorectal cancer (CRC) cells are traditionally considered unresponsive to TGFß due to mutations in the receptors and/or downstream signaling molecules. TGFß influences CRC cells only indirectly via stromal cells, such as cancer-associated fibroblasts. However, CRC cell ability to directly respond to TGFß currently remains unexplored. This represents a missed opportunity for diagnostic and therapeutic interventions. Methods: We examined whether cancer cells from primary CRC and liver metastases respond to TGFß by inducing TGFß-induced protein ig-h3 (TGFBI) expression, and the contribution of canonical and non-canonical TGFß signaling pathways to this effect. We then investigated in vitro and in vivo TGFBI impact on metastasis formation and angiogenesis. Using patient serum samples and an orthotopic mouse model of CRC liver metastases we assessed the diagnostic/tumor targeting value of novel antibodies against TGFBI. Results: Metastatic CRC cells, such as circulating tumor cells, directly respond to TGFß. These cells were characterized by the absence of TGFß receptor mutations and the frequent presence of p53 mutations. The pro-tumorigenic program orchestrated by TGFß in CRC cells was mediated through TGFBI, the expression of which was positively regulated by non-canonical TGFß signaling cascades. TGFBI inhibition was sufficient to significantly reduce liver metastasis formation in vivo. Moreover, TGFBI pro-tumorigenic function was linked to its ability to stimulate angiogenesis. TGFBI levels were higher in serum samples from untreated patients with CRC than in patients who were receiving chemotherapy. A radiolabeled anti-TGFBI antibody selectively targeted metastatic lesions in vivo, underscoring its diagnostic and therapeutic potential. Conclusions: TGFß signaling in CRC cells directly contributes to their metastatic potential and stromal cell-independence. Proteins downstream of activated TGFß, such as TGFBI, represent novel diagnostic and therapeutic targets for more specific anti-metastatic therapies.

HP1s modulate the S-Adenosyl Methionine synthesis pathway in liver cancer cells.

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer in adults. Among the altered pathways leading to HCC, an increasing role is attributed to abnormal epigenetic regulation. Members of the Heterochromatin Protein (HP1) 1 family are key players in chromatin organisation, acting as docking sites for chromatin modifiers. Here, we inactivated HP1α in HepG2 human liver carcinoma cells and showed that HP1α participated in cell proliferation. HP1α-depleted cells have a global decrease in DNA methylation and consequently a perturbed chromatin organisation, as exemplified by the reactivation of transcription at centromeric and pericentromeric regions, eventhough the protein levels of chromatin writers depositing methylation marks, such as EZH2, SETDB1, SUV39H1, G9A and DNMT3A remained unaltered. This decrease was attributed mainly to a low S-Adenosyl Methionine (SAM) level, a cofactor involved in methylation processes. Furthermore, we showed that this decrease was due to a modification in the Methionine adenosyl transferase 2A RNA (MAT2A) level, which modifies the ratio of MAT1A/MAT2A, two enzymes that generate SAM. Importantly, HP1α reintroduction into HP1α-depleted cells restored the MAT2A protein to its initial level. Finally, we demonstrated that this transcriptional deregulation of MAT2A in HP1α-depleted cells relied on a lack of recruitment of HP1ß and HP1γ to MAT2A promoter where an improper non-CpG methylation site was promoted in the vicinity of the transcription start site where HP1ß and HP1γ bound. Altogether, these results highlight an unanticipated link between HP1 and the SAM synthesis pathway, and emphasise emerging functions of HP1s as sensors of some aspects of liver cell metabolism.

The mouse HP1 proteins are essential for preventing liver tumorigenesis.

Chromatin organization is essential for appropriate interpretation of the genetic information. Here, we demonstrated that the chromatin-associated proteins HP1 are dispensable for hepatocytes survival but are essential within hepatocytes to prevent liver tumor development in mice with HP1ß being pivotal in these functions. Yet, we found that the loss of HP1 per se is not sufficient to induce cell transformation but renders cells more resistant to specific stress such as the expression of oncogenes and thus in fine, more prone to cell transformation. Molecular characterization of HP1-Triple KO premalignant livers and BMEL cells revealed that HP1 are essential for the maintenance of heterochromatin organization and for the regulation of specific genes with most of them having well characterized functions in liver functions and homeostasis. We further showed that some specific retrotransposons get reactivated upon loss of HP1, correlating with overexpression of genes in their neighborhood. Interestingly, we found that, although HP1-dependent genes are characterized by enrichment H3K9me3, this mark does not require HP1 for its maintenance and is not sufficient to maintain gene repression in absence of HP1. Finally, we demonstrated that the loss of TRIM28 association with HP1 recapitulated several phenotypes induced by the loss of HP1 including the reactivation of some retrotransposons and the increased incidence of liver cancer development. Altogether, our findings indicate that HP1 proteins act as guardians of liver homeostasis to prevent tumor development by modulating multiple chromatin-associated events within both the heterochromatic and euchromatic compartments, partly through regulation of the corepressor TRIM28 activity.

Notch inhibition overcomes resistance to tyrosine kinase inhibitors in EGFR-driven lung adenocarcinoma.

EGFR-mutated lung adenocarcinoma patients treated with gefitinib and osimertinib show a therapeutic benefit limited by the appearance of secondary mutations, such as EGFRT790M and EGFRC797S. It is generally assumed that these secondary mutations render EGFR completely unresponsive to the inhibitors, but contrary to this, we uncovered here that gefitinib and osimertinib increased STAT3 phosphorylation (p-STAT3) in EGFRT790M and EGFRC797S tumoral cells. Interestingly, we also found that concomitant Notch inhibition with gefitinib or osimertinib treatment induced a p-STAT3-dependent strong reduction in the levels of the transcriptional repressor HES1. Importantly, we showed that tyrosine kinase inhibitor-resistant tumors, with EGFRT790M and EGFRC797S mutations, were highly responsive to the combined treatment of Notch inhibitors with gefitinib or osimertinib, respectively. Finally, in patients with EGFR mutations treated with tyrosine kinase inhibitors, HES1 protein levels increased during relapse and correlated with shorter progression-free survival. Therefore, our results offer a proof of concept for an alternative treatment to chemotherapy in lung adenocarcinoma osimertinib-treated patients after disease progression.

Coprs inactivation leads to a derepression of LINE1 transposons in spermatocytes.

Repression of retrotransposons is essential for genome integrity during germ cell development and is tightly controlled through epigenetic mechanisms. In primordial germ cells, protein arginine N-methyltransferase (Prmt5) is involved in retrotransposon repression by methylating Piwi proteins, which is part of the piRNA pathway. Here, we show that in mice, genetic inactivation of coprs (which is highly expressed in testis and encodes a histone-binding protein required for the targeting of Prmt5 activity) affects the maturation of spermatogonia to spermatids. Mass spectrometry analysis revealed the presence of Miwi in testis protein lysates immunoprecipitated with an anti-Coprs antibody. The observed deregulation of Miwi and pachytene pre-piRNAs levels and the derepression of LINE1 repetitive sequences observed in coprs-/- mice suggest that Coprs is implicated in genome surveillance mechanisms.

The Wnt-target gene Dlk-1 is regulated by the Prmt5-associated factor Copr5 during adipogenic conversion.

Protein arginine methyl transferase 5 (Prmt5) regulates various differentiation processes, including adipogenesis. Here, we investigated adipogenic conversion in cells and mice in which Copr5, a Prmt5- and histone-binding protein, was genetically invalidated. Compared to control littermates, the retroperitoneal white adipose tissue (WAT) of Copr5 KO mice was slightly but significantly reduced between 8 and 16 week/old and contained fewer and larger adipocytes. Moreover, the adipogenic conversion of Copr5 KO embryoid bodies (EB) and of primary embryo fibroblasts (Mefs) was markedly delayed. Differential transcriptomic analysis identified Copr5 as a negative regulator of the Dlk-1 gene, a Wnt target gene involved in the control of adipocyte progenitors cell fate. Dlk-1 expression was upregulated in Copr5 KO Mefs and the Vascular Stromal Fraction (VSF) of Copr5 KO WAT. Chromatin immunoprecipitation (ChIP) show that the ablation of Copr5 has impaired both the recruitment of Prmt5 and ß-catenin at the Dlk-1 promoter. Overall, our data suggest that Copr5 is involved in the transcriptional control exerted by the Wnt pathway on early steps of adipogenesis.

PRMT5-mediated histone H4 arginine-3 symmetrical dimethylation marks chromatin at G + C-rich regions of the mouse genome.

Symmetrical dimethylation on arginine-3 of histone H4 (H4R3me2s) has been reported to occur at several repressed genes, but its specific regulation and genomic distribution remained unclear. Here, we show that the type-II protein arginine methyltransferase PRMT5 controls H4R3me2s in mouse embryonic fibroblasts (MEFs). In these differentiated cells, we find that the genome-wide pattern of H4R3me2s is highly similar to that in embryonic stem cells. In both the cell types, H4R3me2s peaks are detected predominantly at G + C-rich regions. Promoters are consistently marked by H4R3me2s, independently of transcriptional activity. Remarkably, H4R3me2s is mono-allelic at imprinting control regions (ICRs), at which it marks the same parental allele as H3K9me3, H4K20me3 and DNA methylation. These repressive chromatin modifications are regulated independently, however, since PRMT5-depletion in MEFs resulted in loss of H4R3me2s, without affecting H3K9me3, H4K20me3 or DNA methylation. Conversely, depletion of ESET (KMT1E) or SUV420H1/H2 (KMT5B/C) affected H3K9me3 and H4K20me3, respectively, without altering H4R3me2s at ICRs. Combined, our data indicate that PRMT5-mediated H4R3me2s uniquely marks the mammalian genome, mostly at G + C-rich regions, and independently from transcriptional activity or chromatin repression. Furthermore, comparative bioinformatics analyses suggest a putative role of PRMT5-mediated H4R3me2s in chromatin configuration in the nucleus.

The histone-binding protein COPR5 is required for nuclear functions of the protein arginine methyltransferase PRMT5.

Protein arginine methyltransferase 5 (PRMT5) targets nuclear and cytoplasmic proteins. Here, we identified a nuclear protein, called cooperator of PRMT5 (COPR5), involved in the nuclear functions of PRMT5. COPR5 tightly binds to PRMT5, both in vitro and in living cells, but not to other members of the PRMT family. PRMT5 bound to COPR5 methylates histone H4 (R3) preferentially when compared with histone H3 (R8), suggesting that COPR5 modulates the substrate specificity of nuclear PRMT5-containing complexes, at least towards histones. Markedly, recombinant COPR5 binds to the amino terminus of histone H4 and is required to recruit PRMT5 to reconstituted nucleosomes in vitro. Consistently, COPR5 depletion in cells strongly reduces PRMT5 recruitment on chromatin at the PRMT5 target gene cyclin E1 (CCNE1) in vivo. Moreover, both COPR5 depletion and overexpression affect CCNE1 promoter expression. We propose that COPR5 is an important chromatin adaptor for PRMT5 to function on a subset of its target genes.

Coactivator-associated arginine methyltransferase 1 (CARM1) is a positive regulator of the Cyclin E1 gene.

The Cyclin E1 gene (CCNE1) is an ideal model to explore the mechanisms that control the transcription of cell cycle-regulated genes whose expression rises transiently before entry into S phase. E2F-dependent regulation of the CCNE1 promoter was shown to correlate with changes in the level of H3-K9 acetylation/methylation of nucleosomal histones positioned at the transcriptional start site region. Here we show that, upon growth stimulation, the same region is subject to variations of H3-R17 and H3-R26 methylation that correlate with the recruitment of coactivator-associated arginine methyltransferase 1 (CARM1) onto the CCNE1 and DHFR promoters. Accordingly, CARM1-deficient cells lack these modifications and present lowered levels and altered kinetics of CCNE1 and DHFR mRNA expression. Consistently, reporter gene assays demonstrate that CARM1 functions as a transcriptional coactivator for their E2F1/DP1-stimulated expression. CARM1 recruitment at the CCNE1 gene requires activator E2Fs and ACTR, a member of the p160 coactivator family that is frequently overexpressed in human breast cancer. Finally, we show that grade-3 breast tumors present coelevated mRNA levels of ACTR and CARM1, along with their transcriptional target CCNE1. All together, our results indicate that CARM1 is an important regulator of the CCNE1 gene.

Negative regulation of transcription by the type II arginine methyltransferase PRMT5.

We have identified previously a repressor element in the transcription start site region of the cyclin E1 promoter that periodically associates with an atypical, high molecular weight E2F complex, termed CERC. Purification of native CERC reveals the presence of the type II arginine methyltransferase PRMT5, which can mono- or symetrically dimethylate arginine residues in proteins. Chromatin immunoprecipitations (ChIPs) show that PRMT5 is associated specifically with the transcription start site region of the cyclin E1 promoter. ChIP analyses also show that this correlates with the presence on the same promoter region of arginine-methylated proteins including histone H4, an in vitro substrate of PRMT5. Consistent with its presence within the repressor complex, forced expression of PRMT5 negatively affects cyclin E1 promoter activity and cellular proliferation, effects that require its methyltransferase activity. These data provide the first direct experimental evidence that a type II arginine methylase is involved in the control of transcription and proliferation.

Identification of the first Rho-GEF inhibitor, TRIPalpha, which targets the RhoA-specific GEF domain of Trio.

The Rho-guanine nucleotide exchange factors (Rho-GEFs) remodel the actin cytoskeleton via their Rho-GTPase targets and affect numerous physiological processes such as transformation and cell motility. They are therefore attractive targets to design specific inhibitors that may have therapeutic applications. Trio contains two Rho-GEF domains, GEFD1 and GEFD2, which activate the Rac and RhoA pathways, respectively. Here we have used a genetic screen in yeast to select in vivo peptides coupled to thioredoxin, called aptamers, that could inhibit GEFD2 activity. One aptamer, TRIAPalpha (TRio Inhibitory APtamer), specifically blocks GEFD2-exchange activity on RhoA in vitro. The corresponding peptide sequence, TRIPalpha, inhibits TrioGEFD2-mediated activation of RhoA in intact cells and specifically reverts the neurite retraction phenotype induced by TrioGEFD2 in PC12 cells. Thus TRIPalpha is the first Rho-GEF inhibitor isolated so far, and represents an important step in the design of inhibitors for the expanding family of Rho-GEFs.