Overcoming adaptive resistance in mucoepidermoid carcinoma through inhibition of the IKK-β/IκBα/NFκB axis.
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ABSTRACT:
SUBMITTER: Wagner VP
PROVIDER: S-EPMC5341961 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:This chapter provides a short review of various biophysical experiments that have been applied to the inhibitor of kappa B, IκBα and its binding partner, nuclear factor kappa B, or NFκB. The picture that emerges from amide hydrogen/deuterium exchange, NMR and binding kinetics experiments is one in which parts of both proteins are "fuzzy" in the free-state and some parts remain "fuzzy" in the NFκB-IκBα complex. The NFκB family of transcription factors responds to inflammatory cytokines with rapid transcriptional activation, in which NFκB enters the nucleus and binds DNA. Just as rapidly as transcription is activated, it is subsequently repressed by newly synthesized IκBα?that also enters the nucleus and removes NFκB from the DNA. Because IκBα?is an ankyrin repeat protein, it's "fuzziness" can be controlled by mutagenesis to stabilized the folded state. Experimental comparison with such stabilized mutants helps provide evidence that much of the system control depends on the "fuzziness" of IκBα.
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Project description:Upregulation of protein neddylation occurs in numerous types of human cancer, including liver cancer. MLN4924, a potent neddylation‑inhibiting pharmacological agent, demonstrates anticancer ability in numerous cancers. However, the sensitivity of MLN4924 in liver cancer remains unsatisfactory due to factors causing resistance. RT‑qPCR and western blotting were utilized to assess the mRNA and protein levels of genes, respectively. Cell Counting Kit‑8 assay and colony formation assays were employed to assess cell viability and proliferation. The pathway of protein degradation and stability were determined by western blotting after treatment with MG132 and cycloheximide. An immunoprecipitation assay was utilized to detect the ubiquitination of protein. An in vitro ubiquitination assay was used to determine the ubiquitin linkage. To the best of our knowledge, the present study was the first to demonstrate that NF‑κB inhibitor α (IκBα) downregulation and subsequent inflammation in response to MLN4924 limited the antitumor potential of MLN4924. Ectopic expression of IκBα enhanced the antitumor potential of MLN4924 in liver cancer cells. Moreover, the results of the present study demonstrated that MLN4924 decreased IκBα via promoting the K48 linkage of ubiquitin to IκBα. Mechanistic studies demonstrated that MLN4924 enhanced the protein stability of β‑transducin repeat‑containing protein (β‑TrCP), promoting the ubiquitination of IκBα, which led to the ubiquitin‑mediated degradation of IκBα. In addition, the results of the present study also demonstrated that β‑TrCP knockdown markedly inhibited MLN4924 from suppressing the growth of liver cancer cells, via attenuating MLN4924‑mediated IκBα downregulation and inflammation. Collectively, these results indicated that the β‑TrCP/IκBα/inflammation pathway may act as a novel resistance factor of MLN4924, and targeting β‑TrCP may be beneficial for the treatment of liver cancer.
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Project description:We recently discovered that IκBα enhances the rate of release of nuclear factor kappa B (NFκB) from DNA target sites in a process we have termed molecular stripping. Coarse-grained molecular dynamics simulations of the stripping pathway revealed two mechanisms for the enhanced release rate: the negatively charged PEST region of IκBα electrostatically repels the DNA, and the binding of IκBα appears to twist the NFκB heterodimer so that the DNA can no longer bind. Here, we report amide hydrogen/deuterium exchange data that reveal long-range allosteric changes in the NFκB (RelA-p50) heterodimer induced by DNA or IκBα binding. The data suggest that the two Ig-like subdomains of each Rel-homology region, which are connected by a flexible linker in the heterodimer, communicate in such a way that when DNA binds to the N-terminal DNA-binding domains, the nuclear localization signal becomes more highly exchanging. Conversely, when IκBα binds to the dimerization domains, amide exchange throughout the DNA-binding domains is decreased as if the entire domain is becoming globally stabilized. The results help understand how the subtle mechanism of molecular stripping actually occurs.
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Project description:Mucoepidermoid carcinoma (MEC) is the most frequent malignant salivary gland cancer. Response to chemoradiotherapy is modest, and therefore radical surgery remains the standard-of-care. Emerging evidence suggests that Interleukin (IL)-6 signaling correlates with the survival of cancer stem cells and resistance to therapy. Here, we investigated whether inhibition of IL-6 receptor (IL-6R) signaling with tocilizumab (humanized anti-human IL-6R antibody) sensitizes MEC to chemotherapy using human mucoepidermoid carcinoma cell lines (UM-HMC) and correspondent xenograft models. In vitro, we observed that tocilizumab inhibited STAT3 phosphorylation but had no measurable effect in MEC cell viability (UM-HMC-1,-3A,-3B). In contrast, the anti-tumor effect of single agent tocilizumab on MEC xenografts was comparable to paclitaxel or cisplatin. Combination of tocilizumab with cisplatin or paclitaxel enhanced the inhibitory effect of chemotherapy on xenograft growth (P < 0.05), time to failure (P < 0.01), decreased vascular endothelial growth factor (VEGF) expression and tumor microvessel density (P < 0.05) without added systemic toxicities. Notably, tocilizumab decreased the fraction of MEC cancer stem cells (ALDH(high)CD44(high)) in vitro, and prevented paclitaxel-induced increase in the fraction of cancer stem cells in vivo (P < 0.05). Collectively, these findings demonstrate that tocilizumab enhances the anti-tumor effect of conventional chemotherapy in preclinical models of mucoepidermoid carcinoma, and suggest that patients might benefit from combination therapy with an inhibitor of IL-6R signaling and chemotherapeutic agent such as paclitaxel.
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Project description:Stress-response transcription factors such as NFκB turn on hundreds of genes and must have a mechanism for rapid cessation of transcriptional activation. We recently showed that the inhibitor of NFκB signaling, IκBα, dramatically accelerates the dissociation of NFκB from transcription sites, a process we have called "stripping." To test the role of the IκBα C-terminal PEST (rich in proline, glutamic acid, serine, and threonine residues) sequence in NFκB stripping, a mutant IκBα was generated in which five acidic PEST residues were mutated to their neutral analogs. This IκBα(5xPEST) mutant was impaired in stripping NFκB from DNA and formed a more stable intermediate ternary complex than that formed from IκBα(WT) because DNA dissociated more slowly. NMR and amide hydrogen-deuterium exchange mass spectrometry showed that the IκBα(5xPEST) appears to be "caught in the act of stripping" because it is not yet completely in the folded and NFκB-bound state. When the mutant was introduced into cells, the rate of postinduction IκBα-mediated export of NFκB from the nucleus decreased markedly.
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Project description:The brain-derived neurotrophic factor (BDNF)/tyrosine receptor kinase B (TrkB) pathway was previously associated with key oncogenic outcomes in a number of adenocarcinomas. The aim of our study was to determine the role of this pathway in mucoepidermoid carcinoma (MEC). Three MEC cell lines (UM-HMC-2, H253 and H292) were exposed to Cisplatin, the TrkB inhibitor, ANA-12 and a combination of these drugs. Ultrastructural changes were assessed through transmission electron microscopy; scratch and Transwell assays were used to assess migration and invasion; and a clonogenic assay and spheroid-forming assay allowed assessment of survival and percentage of cancer stem cells (CSC). Changes in cell ultrastructure demonstrated Cisplatin cytotoxicity, while the effects of ANA-12 were less pronounced. Both drugs, used individually and in combination, delayed MEC cell migration, invasion and survival. ANA-12 significantly reduced the number of CSC, but the Cisplatin effect was greater, almost eliminating this cell population in all MEC cell lines. Interestingly, the spheroid forming capacity recovered, following the combination therapy, as compared to Cisplatin alone. Our studies allowed us to conclude that the TrkB inhibition, efficiently impaired MEC cell migration, invasion and survival in vitro, however, the decrease in CSC number, following the combined treatment of ANA-12 and Cisplatin, was less than that seen with Cisplatin alone; this represents a limiting factor.
| S-EPMC7759804 | biostudies-literature
Project description:PurposeUnique cells characterized by multipotency, self-renewal, and high tumorigenic potential have been recently discovered in mucoepidermoid carcinomas. These cells are defined by high aldehyde dehydrogenase activity and high CD44 expression (ALDHhighCD44high) and function as cancer stem cells (CSC). It has been recently shown that p53 regulates cell differentiation, suggesting that induction of p53 by therapeutic blockade of the MDM2-p53 interaction may constitute a novel strategy to ablate CSCs. Here, we evaluated the effect of a small-molecule inhibitor of MDM2-p53 interaction (MI-773) on the fraction of CSCs in mucoepidermoid carcinoma.Experimental designHuman mucoepidermoid carcinoma cells (UM-HMC-1,-3A,-3B) were used to assess the effect of MI-773 on cell survival, cell cycle, fraction of CSCs, and expression of p53, p21, MDM2, and Bmi-1 (key regulator of self-renewal). Mice bearing xenograft tumors generated with these mucoepidermoid carcinoma cells were treated with MI-773 to determine the effect of MDM2-p53 inhibition on CSCs in vivo.ResultsMDM2 is highly expressed in human mucoepidermoid carcinoma tissues. MI-773 induced expression of p53 and its downstream targets p21 and MDM2, caused G1 cell-cycle arrest, and induced mucoepidermoid carcinoma tumor cell apoptosis in vitro. Importantly, a marked decrease in expression of Bmi-1 and in the fraction of ALDHhighCD44high (CSCs) was caused by MI-773 in vitro and in mice harboring mucoepidermoid carcinoma xenografts.ConclusionsCollectively, these data demonstrate that MI-773 reduces the fraction of CSCs, suggesting that patients with mucoepidermoid carcinoma might benefit from therapeutic inhibition of the MDM2-p53 interaction.
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Project description:Mucoepidermoid carcinoma (MEC) represents 10-15% of salivary neoplasms. Due to their low incidence, it is challenging to conduct clinical trials and develop treatment guidelines. Although surgery is the most common approach for a resectable tumor, various treatment options such as chemotherapy, radiotherapy, and immunotherapy have been investigated. There is a need to implement a standardized treatment protocol to effectively manage MEC as it is a common histological subtype. Furthermore, it has become essential to assess chromosomal and genetic abnormalities recently identified with MEC, including alterations of CDKN2A, TP53, CDKN2B, BAP1, etc. These mutations are involved in the transformation of low-grade tumors to high-grade tumors, presenting a vital tool for evaluating the aggressive behavior of this carcinoma. Detailed immunohistochemical and translocation studies can help develop targeted therapies and monitor treatment response. Therefore, biomarker-driven research will immensely improve the outcome, especially in advanced cases. Based on thorough histology and chromosomal translocations, a more personalized treatment plan can improve the overall disease outcome. The purpose of this article is to elaborate on the current treatment advancements, particularly chemotherapy and targeted therapy, as an effective treatment modality for the management of MEC and highlight the comparison with traditional treatment approaches.
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Project description:Mucoepidermoid carcinoma (MEC) of the breast is an extremely rare salivary gland-type tumor characterized by epidermoid, basaloid, intermediate, and/or mucinous cells arranged in solid and cystic patterns. Despite their triple-negative phenotype, breast MECs are generally considered low-risk malignancies but their biology is largely unexplored; therefore, guidelines for clinical management are lacking. Here, we sought to characterize the molecular landscape of breast MECs. Thirteen cases were histologically reviewed, characterized for tumor-infiltrating lymphocytes (TILs), and were subjected to immunohistochemistry for programmed death-ligand 1 (PD-L1, clone 22C3), EGFR, and amphiregulin (AREG). Rearrangements in MAML2 and EWSR1 were investigated by fluorescent in situ hybridization. Targeted next-generation sequencing of 161 genes was performed on eight cases. Most MECs had low histological grade (n = 10, 77%), with the presence of TILs (n = 9/12; 75%) and PD-L1 combined positive score ranging from 10 to 20 (n = 4/6; 67%). All cases showed EGFR and AREG overexpression and were fusion negative. Enrichment of genetic alterations was observed in PI3K/AKT/mTOR and cell cycle regulation pathways, while only one case harbored TP53 mutations. This is the first study providing extensive molecular data on breast MECs and the largest collection of cases available to date in the literature. Breast MECs lack TP53 mutations found in high-grade forms of triple-negative breast cancers and MAML2 or EWSR1 rearrangements pathognomonic of salivary MECs. Triple-negativity and PD-L1 positivity suggest a window of opportunity for immunotherapy in these patients. The EGFR/AREG axis activation, coupled with the mutational patterns in PI3K/AKT/mTOR and cell cycle pathways warrants caution in considering MECs as low-risk neoplasms.
| S-EPMC10225218 | biostudies-literature
Project description:BackgroundMucoepidermoid carcinoma (MEC) can be classified into low-, intermediate-, and high-grade tumors based on its histological features. MEC is mainly composed of three cell types (squamous or epidermoid, mucous and intermediate cells), which correlates with the histological grade and reflects its clinical behavior. Most cancers exhibit reduced methylation of repetitive sequences such as Long INterspersed Element-1 (LINE-1) and Alu elements. However, to date very little information is available on the LINE-1 and Alu methylation status in MEC. The aim of this study was to investigate LINE-1 and Alu element methylation in MEC and compare if key differences in the methylation status exist between the three different cell types, and adjacent normal salivary gland cells, to see if this may reflect the histological grade.MethodsLINE-1 and Alu element methylation of 24 MEC, and 14 normal salivary gland tissues were compared using Combine Bisulfite Restriction Analysis (COBRA). Furthermore, the three different cell types from MEC samples were isolated for enrichment by laser capture microdissection (LCM), essentially to see if COBRA was likely to increase the predictive value of LINE-1 and Alu element methylation.ResultsLINE-1 and Alu element methylation levels were significantly different (p<0.001) between the cell types, and showed a stepwise decrease from the adjacent normal salivary gland to the intermediate, mucous and squamous cells. The reduced methylation levels of LINE-1 were correlated with a poorer histological grade. In addition, MEC tissue showed a significantly lower level of LINE-1 and Alu element methylation overall compared to normal salivary gland tissue (p<0.001).ConclusionsOur findings suggest that LINE-1 methylation differed among histological grade mucoepidermoid carcinoma. Hence, this epigenetic event may hold value for MEC diagnosis and prognostic prediction.
| S-EPMC3610265 | biostudies-literature