discussed the modelling experiments and analyses, S.K. carried out the modelling experiments.Research.

Most head and neck cancer (HNC) patients are resistant to cetuximab, an antibody against the epidermal growth factor receptor. Such therapy resistance is known to be mediated, in part, by stromal cells surrounding the tumor cells; however, the mechanisms underlying such a resistance phenotype remain unclear. To identify the mechanisms of cetuximab resistance in an unbiased manner, RNA-sequencing (RNA-seq) of HNC patient-derived xenografts (PDXs) was performed. Comparing the gene expression of HNC-PDXs before and after treatment with cetuximab indicated that the transforming growth factor-beta (TGF-beta) signaling pathway was upregulated in the stromal cells of PDXs that progressed on cetuximab treatment (CetuximabProg-PDX). However, in PDXs that were extremely sensitive to cetuximab (CetuximabSen-PDX), the TGF-beta pathway was downregulated in the stromal compartment. Histopathological analysis of PDXs showed that TGF-beta-activation was detected in cancer-associated fibroblasts (CAFs) of CetuximabProg-PDX. These TGF-beta-activated CAFs were sufficient to limit cetuximab efficacy in vitro and in vivo. Moreover, blocking the TGF-beta pathway using the SMAD3 inhibitor, SIS3, enhanced cetuximab efficacy and prevented the progression of CetuximabProg-PDX. Altogether, our findings indicate that TGF-beta-activated CAFs play a role in limiting cetuximab efficacy in HNC.

Approximately 40% of human messenger RNAs (mRNAs) contain upstream open reading frames (uORFs) in their 5′ untranslated regions. Some of these uORF sequences, thought to attenuate scanning ribosomes or lead to mRNA degradation, were recently shown to be translated, although the function of the encoded peptides remains unknown. Here, we show a uORF-encoded peptide that exhibits kinase inhibitory functions. This uORF, upstream of the protein kinase C-eta (PKC-η) main ORF, encodes a peptide (uPEP2) containing the typical PKC pseudosubstrate motif present in all PKCs that autoinhibits their kinase activity. We show that uPEP2 directly binds to and selectively inhibits the catalytic activity of novel PKCs but not of classical or atypical PKCs. The endogenous deletion of uORF2 or its overexpression in MCF-7 cells revealed that the endogenously translated uPEP2 reduces the protein levels of PKC-η and other novel PKCs and restricts cell proliferation. Functionally, treatment of breast cancer cells with uPEP2 diminished cell survival and their migration and synergized with chemotherapy by interfering with the response to DNA damage. Furthermore, in a xenograft of MDA-MB-231 breast cancer tumor in mice models, uPEP2 suppressed tumor progression, invasion, and metastasis. Tumor histology showed reduced proliferation, enhanced cell death, and lower protein expression levels of novel PKCs along with diminished phosphorylation of PKC substrates. Hence, our study demonstrates that uORFs may encode biologically active peptides beyond their role as translation regulators of their downstream ORFs. Together, we point to a unique function of a uORF-encoded peptide as a kinase inhibitor, pertinent to cancer therapy.

Deposition of ubiquitin conjugates on inclusion bodies composed of protein aggregates is a definitive cytopathological hallmark of neurodegenerative diseases. We show that accumulation of ubiquitin on polyQ IB, associated with Huntington’s disease, is correlated with extensive depletion of nuclear ubiquitin and histone de-ubiquitination. Histone ubiquitination plays major roles in chromatin regulation and DNA repair. Accordingly, we observe that cells expressing IB fail to respond to radiomimetic DNA damage, to induce gamma-H2AX phosphorylation and to recruit 53BP1 to damaged foci. Interestingly ubiquitin depletion, histone de-ubiquitination and impaired DNA damage response are not restricted to PolyQ aggregates and are associated with artificial aggregating luciferase mutants. The longevity of brain neurons depends on their capacity to respond to and repair extensive ongoing DNA damage. Impaired DNA damage response, even modest one, could thus lead to premature neuron aging and mortality.

An understanding of the mechanisms underlying acquired resistance to cetuximab is urgently needed to improve cetuximab efficacy in patients with head and neck squamous cell carcinoma (HNSCC). Here, we present a clinical observation that MET pathway activation constitutes the mechanism of acquired resistance to cetuximab in a patient with HNSCC. Specifically, RNA sequencing and mass spectrometry analysis of cetuximab‐sensitive (CetuxSen) and cetuximab‐resistant (CetuxRes) tumors indicated MET amplification and overexpression in the CetuxRes tumor compared to the CetuxSen lesion. Stimulation of MET in HNSCC cell lines was sufficient to reactivate the MAPK pathway and to confer resistance to cetuximab in vitro and in vivo. In addition to the direct role of MET in reactivation of the MAPK pathway, MET stimulation abrogates the well‐known cetuximab‐induced compensatory feedback loop of HER2/HER3 expression. Mechanistically, we showed that the overexpression of HER2 and HER3 following cetuximab treatment is mediated by the ETS homologous transcription factor (EHF), and is suppressed by MET/MAPK pathway activation. Collectively, our findings indicate that evaluation of MET and HER2/HER3 in response to cetuximab in HNSCC patients can provide the rationale of successive line of treatment.