p53 wild-type colorectal cancer cells that express a fetal gene signature are associated with metastasis and poor prognosis.

Current therapy against colorectal cancer (CRC) is based on DNA-damaging agents that remain ineffective in a proportion of patients. Whether and how non-curative DNA damage-based treatment affects tumor cell behavior and patient outcome is primarily unstudied. Using CRC patient-derived organoids (PDO)s, we show that sublethal doses of chemotherapy (CT) does not select previously resistant tumor populations but induces a quiescent state specifically to TP53 wildtype (WT) cancer cells, which is linked to the acquisition of a YAP1-dependent fetal phenotype. Cells displaying this phenotype exhibit high tumor-initiating and metastatic activity. Nuclear YAP1 and fetal traits are present in a proportion of tumors at diagnosis and predict poor prognosis in patients carrying TP53 WT CRC tumors. We provide data indicating the higher efficacy of CT together with YAP1 inhibitors for eradication of therapy resistant TP53 WT cancer cells. Together these results identify fetal conversion as a useful biomarker for patient prognosis and therapy prescription.

Dynamic chromatin association of IκBα is regulated by acetylation and cleavage of histone H4.

IκBs exert principal functions as cytoplasmic inhibitors of NF-kB transcription factors. Additional roles for IκB homologues have been described, including chromatin association and transcriptional regulation. Phosphorylated and SUMOylated IκBα (pS-IκBα) binds to histones H2A and H4 in the stem cell and progenitor cell compartment of skin and intestine, but the mechanisms controlling its recruitment to chromatin are largely unknown. Here, we show that serine 32-36 phosphorylation of IκBα favors its binding to nucleosomes and demonstrate that p-IκBα association with H4 depends on the acetylation of specific H4 lysine residues. The N-terminal tail of H4 is removed during intestinal cell differentiation by proteolytic cleavage by trypsin or chymotrypsin at residues 17-19, which reduces p-IκBα binding. Inhibition of trypsin and chymotrypsin activity in HT29 cells increases p-IκBα chromatin binding but, paradoxically, impaired goblet cell differentiation, comparable to IκBα deletion. Taken together, our results indicate that dynamic binding of IκBα to chromatin is a requirement for intestinal cell differentiation and provide a molecular basis for the understanding of the restricted nuclear distribution of p-IκBα in specific stem cell compartments.

IKKα Kinase Regulates the DNA Damage Response and Drives Chemo-resistance in Cancer.

Phosphorylated IKKα(p45) is a nuclear active form of the IKKα kinase that is induced by the MAP kinases BRAF and TAK1 and promotes tumor growth independent of canonical NF-κB signaling. Insights into the sources of IKKα(p45) activation and its downstream substrates in the nucleus remain to be defined. Here, we discover that IKKα(p45) is rapidly activated by DNA damage independent of ATM-ATR, but dependent on BRAF-TAK1-p38-MAPK, and is required for robust ATM activation and efficient DNA repair. Abolishing BRAF or IKKα activity attenuates ATM, Chk1, MDC1, Kap1, and 53BP1 phosphorylation, compromises 53BP1 and RIF1 co-recruitment to sites of DNA lesions, and inhibits 53BP1-dependent fusion of dysfunctional telomeres. Furthermore, IKKα or BRAF inhibition synergistically enhances the therapeutic potential of 5-FU and irinotecan to eradicate chemotherapy-resistant metastatic human tumors in vivo. Our results implicate BRAF and IKKα kinases in the DDR and reveal a combination strategy for cancer treatment.

Manic Fringe deficiency imposes Jagged1 addiction to intestinal tumor cells.

Delta ligands regulate Notch signaling in normal intestinal stem cells, while Jagged1 activates Notch in intestinal adenomas carrying active ß-catenin. We used the ApcMin/+ mouse model, tumor spheroid cultures, and patient-derived orthoxenografts to address this divergent ligand-dependent Notch function and its implication in disease. We found that intestinal-specific Jag1 deletion or antibody targeting Jag1 prevents tumor initiation in mice. Addiction to Jag1 is concomitant with the absence of Manic Fringe (MFNG) in adenoma cells, and its ectopic expression reverts Jag1 dependence. In 239 human colorectal cancer patient samples, MFNG imposes a negative correlation between Jag1 and Notch, being high Jag1 in the absence of MFNG predictive of poor prognosis. Jag1 antibody treatment reduces patient-derived tumor orthoxenograft growth without affecting normal intestinal mucosa. Our data provide an explanation to Jag1 dependence in cancer, and reveal that Jag1-Notch1 interference provides therapeutic benefit in a subset of colorectal cancer and FAP syndrome patients.

IKKα is required in the intestinal epithelial cells for tumour stemness.

BACKGROUND: Colorectal cancer is a common cause of death in developed countries. Progression from adenoma to invasive carcinoma requires accumulation of mutations starting with the Adenomatous Polyposis Coli (Apc) gene. NF-κB signalling is a key element in cancer, mainly related to the activity of IKKß. IKKα kinase also participates in this process by mechanisms that are primarily unknown. METHODS: We generated a compound mouse model with mutation in Apc and lacking intestinal epithelial IKKα, produced intestinal organoids and tumour spheroids with different genetic backgrounds, and performed immunohistochemistry and RNA-seq analysis. RESULTS: Deficiency of IKKα prevents adenoma formation, with adenomas lacking IKKα showing reduced proliferation. In contrast, IKKα status did not affect normal intestinal function. The same divergent phenotype was found in the organoid-spheroid model. We also found that epithelial IKKα controls stemness, proliferation and apoptosis-related expression. CONCLUSIONS: IKKα is a potential therapeutic target for Apc mutant colorectal cancer patients.

Transcriptional profiling of NCI/ADR-RES cells unveils a complex network of signaling pathways and molecular mechanisms of drug resistance.

BACKGROUND: Ovarian cancer has the highest mortality rate among all the gynecological cancers. This is mostly due to the resistance of ovarian cancer to current chemotherapy regimens. Therefore, it is of crucial importance to identify the molecular mechanisms associated with chemoresistance. METHODS: NCI/ADR-RES is a multidrug-resistant cell line that is a model for the study of drug resistance in ovarian cancer. We carried out a microarray-derived transcriptional profiling analysis of NCI/ADR-RES to identify differentially expressed genes relative to its parental OVCAR-8. RESULTS: Gene-expression profiling has allowed the identification of genes and pathways that may be important for the development of drug resistance in ovarian cancer. The NCI/ADR-RES cell line has differential expression of genes involved in drug extrusion, inactivation, and efficacy, as well as genes involved in the architectural and functional reorganization of the extracellular matrix. These genes are controlled through different signaling pathways, including MAPK-Akt, Wnt, and Notch. CONCLUSION: Our findings highlight the importance of using orthogonal therapies that target completely independent pathways to overcome mechanisms of resistance to both classical chemotherapeutic agents and molecularly targeted drugs.

NF-κB Members Left Home: NF-κB-Independent Roles in Cancer.

Nuclear factor-κB (NF-κB) has been long considered a master regulator of inflammation and immune responses. Additionally, aberrant NF-κB signaling has been linked with carcinogenesis in many types of cancer. In recent years, the study of NF-κB members in NF-κB unrelated pathways provided novel attractive targets for cancer therapy, specifically linked to particular pathologic responses. Here we review specific functions of IκB kinase complexes (IKKs) and IκBs, which have distinctly tumor promoting or suppressing activities in cancer. Understanding how these proteins are regulated in a tumor-related context will provide new opportunities for drug development.

Activating transcription factor 3 is crucial for antitumor activity and to strengthen the antiviral properties of Onconase.

Onconase is a ribonuclease that presents both antitumor and antiviral properties linked to its ribonucleolytic activity and represents a new class of RNA-damaging drugs. It has reached clinical trials for the treatment of several cancers and human papilloma virus warts. Onconase targets different RNAs in the cell cytosol but Onconase-treated cells present features that are different from a simple arrest of protein synthesis. We have used microarray-derived transcriptional profiling to identify Onconase-regulated genes in two ovarian cancer cell lines (NCI/ADR-RES and OVCAR-8). RT-qPCR analyses have confirmed the microarray findings. We have identified a network of up-regulated genes implicated in different signaling pathways that may explain the cytotoxic effects exerted by Onconase. Among these genes, activating transcription factor 3 (ATF3) plays a central role in the key events triggered by Onconase in treated cancer cells that finally lead to apoptosis. This mechanism, mediated by ATF3, is cell-type independent. Up-regulation of ATF3 may also explain the antiviral properties of this ribonuclease because this factor is involved in halting viral genome replication, keeping virus latency or preventing viral oncogenesis. Finally, Onconase-regulated genes are different from those affected by nuclear-directed ribonucleases.

A nuclear-directed human pancreatic ribonuclease (PE5) targets the metabolic phenotype of cancer cells.

Ribonucleases represent a new class of antitumor RNA-damaging drugs. However, many wild-type members of the vertebrate secreted ribonuclease family are not cytotoxic because they are not able to evade the cytosolic ribonuclease inhibitor. We previously engineered the human pancreatic ribonuclease to direct it to the cell nucleus where the inhibitor is not present. The best characterized variant is PE5 that kills cancer cells through apoptosis mediated by the p21(WAF1/CIP1) induction and the inactivation of JNK. Here, we have used microarray-derived transcriptional profiling to identify PE5 regulated genes on the NCI/ADR-RES ovarian cancer cell line. RT-qPCR analyses have confirmed the expression microarray findings. The results show that PE5 cause pleiotropic effects. Among them, it is remarkable the down-regulation of multiple genes that code for enzymes involved in deregulated metabolic pathways in cancer cells.

Generation of new cytotoxic human ribonuclease variants directed to the nucleus.

Ribonucleases are promising agents for use in anticancer therapy. Engineering a nuclear localization signal into the sequence of the human pancreatic ribonuclease has been revealed as a new strategy to endow this enzyme with cytotoxic activity against tumor cells. We previously described a cytotoxic human pancreatic ribonuclease variant, named PE5, which is able to cleave nuclear RNA, inducing the apoptosis of cancer cells and reducing the amount of P-glycoprotein in different multidrug-resistant cell lines. These results open the opportunity to use this ribonuclease in combination with other chemotherapeutics. In this work, we have investigated how to improve the properties of PE5 as an antitumor drug candidate. When attempting to develop a recombinant protein as a drug, two of the main desirable attributes are minimum immunogenicity and maximum potency. The improvements of PE5 have been designed in both senses. First, in order to reduce the potential immunogenicity of the protein, we have studied which residues mutated on PE5 can be reverted to those of the wild-type human pancreatic ribonuclease sequence without affecting its cytotoxicity. Second, we have investigated the effect of introducing an additional nuclear localization signal at different sites of PE5 in an effort to obtain a more cytotoxic enzyme. We show that the nuclear localization signal location is critical for the cytotoxicity. One of these variants, named NLSPE5, presents about a 10-fold increase in cytotoxicity respective to PE5. This variant induces apoptosis and kills the cells using the same mechanism as PE5.