Summary Esophageal adenocarcinoma (EAC) is the most prevalent esophageal cancer type in the United States. TNFα/mTOR pathway is known to mediate the development of EAC. Additionally, aberrant activation of Gli1, downstream effector of hedgehog pathway, has been observed in EAC. In this study, we found that activated mTOR/S6K1 pathway promotes Gli1 transcriptional activity and oncogenic function through S6K1-mediated Gli1 phosphorylation at Ser84, which releases Gli1 from its endogenous inhibitor, SuFu. Moreover, elimination of S6K1 activation by mTOR pathway inhibitor enhances the killing effects of the hedgehog pathway inhibitor. Together, our results established a crosstalk between mTOR/S6K1 and the hedgehog pathways, which provides not only a mechanism for SMO-independent Gli1 activation but also a rationale for combination therapy for EAC.

Pro-inflammatory cytokines produced in the tumor microenvironment facilitate tumor development and metastatic progression. In particular, TNF-α promotes cancer invasion and angiogenesis associated with epithelial-mesenchyme transition (EMT), however, the mechanisms underlying its induction of EMT in cancer cells remain unclear. Here we show that EMT and cancer stemness properties induced by chronic treatment with TNFα̣ are mediated by the upregulation of the transcriptional repressor Twist1. Exposure to TNF-α rapidly induced Twist1 mRNA and protein expression in normal breast epithelial and breast cancer cells. Both IKK-β and NF-κB p65 were required for TNF-α-induced expression of Twist1, suggesting the involvement of canonical NF-κB signaling. In support of this likelihood, we defined a functional NF-κB binding site in the Twist1 promoter and overexpression of p65 was sufficient to induce transcriptional upregulation of Twist1 along with EMT in mammary epithelial cells. Conversely, suppressing Twist1 expression abrogated p65-induced cell migration, invasion, EMT and stemness properties, establishing that Twist1 is required for NF-κB to induce these aggressive phenotypes in breast cancer cells. Taken together, our results establish a signaling axis through which the tumor microenvironment elicits Twist1 expression to promote cancer metastasis. We suggest that targeting NF-κB-mediated Twist1 upregulation may offer an effective a therapeutic strategy for breast cancer treatment.

MicroRNAs (miRNAs) are generated by two-step processing to yield small RNAs that negatively regulate target gene expression at the post-transcriptional level1. Deregulation of miRNAs has been linked to diverse pathological processes, including cancer2,3. Recent studies have also implicated miRNAs in the regulation of cellular response to a spectrum of stresses4, such as hypoxia, which is frequently encountered in the poorly angiogenic core of a solid tumour5. However, the upstream regulators of miRNA biogenesis machineries remain obscure, raising the question of how tumour cells efficiently coordinate and impose specificity on miRNA expression and function in response to stresses. Here we show that epidermal growth factor receptor (EGFR), which is the product of a well-characterized oncogene in human cancers, suppresses the maturation of specific tumour-suppressor-like miRNAs in response to hypoxic stress through phosphorylation of argonaute 2 (AGO2) at Tyr 393. The association between EGFR and AGO2 is enhanced by hypoxia, leading to elevated AGO2-Y393 phosphorylation, which in turn reduces the binding of Dicer to AGO2 and inhibits miRNA processing from precursor miRNAs to mature miRNAs. We also identify a long-loop structure in precursor miRNAs as a critical regulatory element in phospho-Y393-AGO2-mediated miRNA maturation. Furthermore, AGO2-Y393 phosphorylation mediates EGFR-enhanced cell survival and invasiveness under hypoxia, and correlates with poorer overall survival in breast cancer patients. Our study reveals a previously unrecognized function of EGFR in miRNA maturation and demonstrates how EGFR is likely to function as a regulator of AGO2 through novel post-translational modification. These findings suggest that modulation of miRNA biogenesis is important for stress response in tumour cells and has potential clinical implications.

SUMMARY IκB kinase β (IKKβ) is involved in tumor development and progression through activation of the nuclear factor (NF)–κB pathway. However, the molecular mechanism that regulates IKKβ degradation remains largely unknown. Here, we show that a Cullin 3 (CUL3)–based ubiquitin ligase, Kelch-like ECH-associated protein 1 (KEAP1), is responsible for IKKβ ubiquitination. Depletion of KEAP1 led to the accumulation and stabilization of IKKβ and to up-regulation of NF-κB–derived tumor angiogenic factors. A systematic analysis of the CUL3, KEAP1, and RBX1 genomic loci revealed a high percentage of genome loss and missense mutations in human cancers that failed to facilitate IKKβ degradation. Our results suggest that the dysregulation of KEAP1-mediated IKKβ ubiquitination may contribute to tumorigenesis.

Myeloid cell leukemia-1 (Mcl-1), a Bcl-2-like antiapoptotic protein, plays a role in cell immortalization and chemoresistance in a number of human malignancies. A peptidylprolyl cis/trans isomerase, Pin1 is involved in many cellular events, such as cell cycle progression, cell proliferation, and differentiation through isomerizing prophosphorylated substrates. It has been reported that down-regulation of Pin1 induces apoptosis, and that Erk phosphorylates and upregulates Mcl-1; however, the underlying mechanisms for the two phenomena are not clear yet. Here, we showed that Pin 1 stabilizes Mcl-1, which is required for Mcl-1 posphorylation by Erk. First, we found expression of Mcl-1 and Pin1 were positively correlated and associated with poor survival in human breast cancer. We then showed that Erk could phosphorylate Mcl-1 at two consensus residues, Thr 92 and 163, which is required for the association of Mcl-1 and Pin1, resulting in stabilization of Mcl-1. Moreover, Pin1 is also required for the up-regulation of Mcl-1 by Erk activation. Based on this newly identified mechanism of Mcl-1 stabilization, two strategies were used to overcome Mcl-1-mediated chemoresistance: inhibiting Erk by Sorafenib, an approved clinical anticancer drug, or knocking down Pin1 by using a SiRNA technique. In conclusion, the current report not only unravels a novel mechanism to link Erk/Pin1 pathway and Mcl-1-mediated chemoresistance but also provides a plausible combination therapy, Taxol (Paclitaxel) plus Sorafenib, which was shown to be effective in killing breast cancer cells. [Cancer Res 2008;68(15):6109-17]

Oncogenic signaling reprograms cancer cell metabolism to augment the production of glycolytic metabolites in favor of tumor growth. The ability of cancer cells to evade immunosurveillance and the role of metabolic regulators in T cell functions suggest that oncogene-induced metabolic reprogramming may be linked to immune escape. Epidermal growth factor (EGF) signaling, frequently dysregulated in triple-negative breast cancer (TNBC), is also associated with increased glycolysis. Here, we demonstrated in TNBC cells that EGF signaling activates the first step in glycolysis, but impedes the last step, leading to an accumulation of metabolic intermediates in this pathway. Furthermore, we showed that one of these intermediates, fructose 1,6 bisphosphate (F1,6BP), directly binds to and enhances the activity of the EGF receptor (EGFR), thereby increasing lactate excretion which leads to inhibition of local cytotoxic T cell activity. Notably, combining the glycolysis inhibitor 2-deoxy-D-glucose (2-DG) with the EGFR inhibitor gefitinib effectively suppressed TNBC cell proliferation and tumor growth. Our results illustrate how jointly targeting the EGFR/F1,6BP signaling axis may offer an immediately applicable therapeutic strategy to treat TNBC.

Accumulating evidence indicates that endocytosis plays an essential role in the nuclear transport of the ErbB family members, such as epidermal growth factor receptor (EGFR) and ErbB-2. Nevertheless, how full-length receptors embedded in the endosomal membrane pass through the nuclear pore complexes and function as non-membrane-bound receptors in the nucleus remains unclear. Here we show that upon EGF treatment, the biotinylated cell surface EGFR is trafficked to the inner nuclear membrane (INM) through the nuclear pore complexes, remaining in a membrane-bound environment. We further find that importin ␤ regulates EGFR nuclear transport to the INM in addition to the nucleus/nucleoplasm. Unexpectedly, the well known endoplasmic reticulum associated translocon Sec61␤ is found to reside in the INM and associate with EGFR. Knocking down Sec61␤ expression reduces EGFR level in the nucleoplasm portion and accumulates it in the INM portion. Thus, the Sec61␤ translocon plays an unrecognized role in the release of the membrane-anchored EGFR from the lipid bilayer of the INM to the nucleus. The newly identified Sec61␤ function provides an alternative pathway for nuclear transport that can be utilized by membrane-embedded proteins such as full-length EGFR.Receptor tyrosine kinases (RTKs), 3 including insulin-like growth factor 1 receptor, c-Met, fibroblast growth factor receptor, vascular endothelial growth factor receptor, and the entire epidermal growth factor receptor (EGFR) family, have been shown to localize in the nucleus (1-6). Among these, both EGFR and ErbB-2 are suggested to be involved in transcriptional regulation, cell proliferation, DNA repair, DNA replication, and chemo and radio resistance (7-14). Nuclear EGFR is associated with poor clinical prognosis for breast cancer, ovarian cancer, and in oropharyngeal and esophageal squamous cell carcinomas (15-19). In addition, nuclear EGFRvIII, a constitutively activated EGFR variant, is also correlated with poor patient outcome in prostate cancer (20). In the canonical model of nuclear import, nuclear localization signal (NLS)-bearing molecules form a complex with importin ␣/␤ or importin ␤ alone. Importin ␤ is responsible for nuclear translocation through nuclear pore complexes (NPCs) by directly associating with the nucleoporins (21). Several studies have shown that importin ␤ and NLS are involved in the nuclear transport of many cell surface RTKs, including EGFR (14, 22-26), ErbB-2 (27, 28), and fibroblast growth factor receptor (29). In addition, nuclear transport of RTKs is mediated by the mechanisms involving endocytosis and endosomal sorting by associating with early endosomal proteins in the nucleus (24, 27). However, the exact mechanisms by which RTKs embedded in the endosomal membrane translocate into the nucleus through NPCs and exist as nonmembrane-bound receptors in the nucleus are still largely unknown.In eukaryotes, the membrane system of the endoplasmic reticulum (ER) is contiguous with the nuclear envelope (NE), a lipid bilayer that forms the bound...

ObjectivePeritoneal carcinomatosis (PC) occurs frequently in patients with gastric adenocarcinoma (GAC) and confers a poor prognosis. Multiplex profiling of primary GACs has been insightful but the underpinnings of PC’s development/progression remain largely unknown. We characterised exome/transcriptome/immune landscapes of PC cells from patients with GAC aiming to identify novel therapeutic targets.DesignWe performed whole-exome sequencing (WES) and whole transcriptome sequencing (RNA-seq) on 44 PC specimens (43 patients with PC) including an integrative analysis of WES, RNA-seq, immune profile, clinical and pathological phenotypes to dissect the molecular pathogenesis, identifying actionable targets and/or biomarkers and comparison with TCGA primary GACs.ResultsWe identified distinct alterations in PC versus primary GACs, such as more frequent CDH1 and TAF1 mutations, 6q loss and chr19 gain. Alterations associated with aggressive PC phenotypes emerged with increased mutations in TP53, CDH1, TAF1 and KMT2C, higher level of ‘clock-like’ mutational signature, increase in whole-genome doublings, chromosomal instability (particularly, copy number losses), reprogrammed microenvironment, enriched cell cycle pathways, MYC activation and impaired immune response. Integrated analysis identified two main molecular subtypes: ‘mesenchymal-like’ and ‘epithelial-like’ with discriminating response to chemotherapy (31% vs 71%). Patients with the less responsive ‘mesenchymal-like’ subtype had high expression of immune checkpoint T-Cell Immunoglobulin And Mucin Domain-Containing Protein 3 (TIM-3), its ligand galectin-9, V-domain Ig suppressor of T cell activation (VISTA) and transforming growth factor-β as potential therapeutic immune targets.ConclusionsWe have uncovered the unique mutational landscape, copy number alteration and gene expression profile of PC cells and defined PC molecular subtypes, which correlated with PC therapy resistance/response. Novel targets and immune checkpoint proteins have been identified with a potential to be translated into clinics.