Heng Huan Lee

p53 regulates epithelial–mesenchymal transition and stem cell properties through modulating miRNAs

The epithelial–mesenchymal transition (EMT) has recently been linked to stem cell phenotype. However, the molecular mechanism underlying EMT and regulation of stemness remains elusive. Here, using genomic approaches, we show that tumour suppressor p53 has a role in regulating both EMT and EMT-associated stem cell properties through transcriptional activation of the microRNA miR-200c. p53 transactivates miR-200c through direct binding to the miR-200c promoter. Loss of p53 in mammary epithelial cells leads to decreased expression of miR-200c and activates the EMT programme, accompanied by an increased mammary stem cell population. Re-expressing miR-200c suppresses genes that mediate EMT and stemness properties and thereby reverts the mesenchymal and stem-cell-like phenotype caused by loss of p53 to a differentiated epithelial cell phenotype. Furthermore, loss of p53 correlates with a decrease in the level of miR-200c, but an increase in the expression of EMT and stemness markers, and development of a high tumour grade in a cohort of breast tumours. This study elucidates a role for p53 in regulating EMT–MET (mesenchymal–epithelial transition) and stemness or differentiation plasticity, and reveals a potential therapeutic implication to suppress EMT-associated cancer stem cells through activation of the p53–miR-200c pathway.

The translocon Sec61β localized in the inner nuclear membrane transports membrane-embedded EGF receptor to the nucleus

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 β regulates EGFR nuclear transport to the INM in addition to the nucleus/nucleoplasm. Unexpectedly, the well known endoplasmic reticulum associated translocon Sec61β is found to reside in the INM and associate with EGFR. Knocking down Sec61β expression reduces EGFR level in the nucleoplasm portion and accumulates it in the INM portion. Thus, the Sec61β 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 Sec61β function provides an alternative pathway for nuclear transport that can be utilized by membrane-embedded proteins such as full-length EGFR.

p53 Acetylation Is Crucial for Its Transcription-independent Proapoptotic Functions

Acetylation of p53 at carboxyl-terminal lysine residues enhances its transcriptional activity associated with cell cycle arrest and apoptosis. Here we demonstrate that p53 acetylation at Lys-320/Lys-373/Lys-382 is also required for its transcription-independent functions in BAX activation, reactive oxygen species production, and apoptosis in response to the histone deacetylase inhibitors (HDACi) suberoylanilide hydroxamic acid and LAQ824. Knock-out of p53 markedly reduced HDACi-induced apoptosis. Unexpectedly, expression of transactivation-deficient p53 variants sensitized p53-null cells to HDACi-mediated BAX-dependent apoptosis, whereas knockdown of endogenous mutant p53 in cancer cells reduced HDACi-mediated cytotoxicity. Evaluation of the mechanisms controlling this response led to the discovery of a novel interaction between p53 and Ku70. The association between these two proteins was acetylation-independent, but acetylation of p53 could prevent and disrupt the Ku70-BAX complex and enhance apoptosis. These results suggest a new mechanism of acetylated p53 transcription-independent regulation of apoptosis.

COPI-mediated retrograde trafficking from the Golgi to the ER regulates EGFR nuclear transport.

Emerging evidence indicates that cell surface receptors, such as the entire epidermal growth factor receptor (EGFR) family, have been shown to localize in the nucleus. A retrograde route from the Golgi to the endoplasmic reticulum (ER) is postulated to be involved in the EGFR trafficking to the nucleus; however, the molecular mechanism in this proposed model remains unexplored. Here, we demonstrate that membrane-embedded vesicular trafficking is involved in the nuclear transport of EGFR. Confocal immunofluorescence reveals that in response to EGF, a portion of EGFR redistributes to the Golgi and the ER, where its NH(2)-terminus resides within the lumen of Golgi/ER and COOH-terminus is exposed to the cytoplasm. Blockage of the Golgi-to-ER retrograde trafficking by brefeldin A or dominant mutants of the small GTPase ADP-ribosylation factor, which both resulted in the disassembly of the coat protein complex I (COPI) coat to the Golgi, inhibit EGFR transport to the ER and the nucleus. We further find that EGF-dependent nuclear transport of EGFR is regulated by retrograde trafficking from the Golgi to the ER involving an association of EGFR with gamma-COP, one of the subunits of the COPI coatomer. Our findings experimentally provide a comprehensive pathway that nuclear transport of EGFR is regulated by COPI-mediated vesicular trafficking from the Golgi to the ER, and may serve as a general mechanism in regulating the nuclear transport of other cell surface receptors.

Glycosylation and stabilization of programmed death ligand-1 suppresses T-cell activity

Extracellular interaction between programmed death ligand-1 (PD-L1) and programmed cell death protein-1 (PD-1) leads to tumour-associated immune escape. Here we show that the immunosuppression activity of PD-L1 is stringently modulated by ubiquitination and N-glycosylation. We show that glycogen synthase kinase 3β (GSK3β) interacts with PD-L1 and induces phosphorylation-dependent proteasome degradation of PD-L1 by β-TrCP. In-depth analysis of PD-L1 N192, N200 and N219 glycosylation suggests that glycosylation antagonizes GSK3β binding. In this regard, only non-glycosylated PD-L1 forms a complex with GSK3β and β-TrCP. We also demonstrate that epidermal growth factor (EGF) stabilizes PD-L1 via GSK3β inactivation in basal-like breast cancer. Inhibition of EGF signalling by gefitinib destabilizes PD-L1, enhances antitumour T-cell immunity and therapeutic efficacy of PD-1 blockade in syngeneic mouse models. Together, our results link ubiquitination and glycosylation pathways to the stringent regulation of PD-L1, which could lead to potential therapeutic strategies to enhance cancer immune therapy efficacy.

Membrane-bound trafficking regulates nuclear transport of integral epidermal growth factor receptor (EGFR) and ErbB-2

Background: EGFR is translocated to the inner nuclear membrane through the INTERNET (integral trafficking from the ER to the nuclear envelope transport) pathway. Results: INTERNET regulates EGFR and ErbB-2 but not FGFR-1. Conclusion: At least two different pathways of nuclear transport exist for cell surface receptors. Significance: This provides a new direction for investigating the trafficking mechanisms of various nuclear RTKs. Nuclear localization of multiple receptor-tyrosine kinases (RTKs), such as EGF receptor (EGFR), ErbB-2, FGF receptor (FGFR), and many others, has been reported by several groups. We previously showed that cell surface EGFR is trafficked to the nucleus through a retrograde pathway from the Golgi to the endoplasmic reticulum (ER) and that EGFR is then translocated to the inner nuclear membrane (INM) through the INTERNET (integral trafficking from the ER to the nuclear envelope transport) pathway. However, the nuclear trafficking mechanisms of other membrane RTKs, apart from EGFR, remain unclear. The purpose of this study was to compare the nuclear transport of EGFR family proteins with that of FGFR-1. Interestingly, we found that digitonin permeabilization, which selectively releases soluble nuclear transporters from the cytoplasm and has been shown to inhibit nuclear transport of FGFR-1, had no effects on EGFR nuclear transport, raising the possibility that EGFR and FGFR-1 use different pathways to be translocated into the nucleus. Using the subnuclear fractionation assay, we further demonstrated that biotinylated cell surface ErbB-2, but not FGFR-1, is targeted to the INM, associating with Sec61β in the INM, similar to the nuclear trafficking of EGFR. Thus, ErbB-2, but not FGFR-1, shows a similar trafficking pathway to EGFR for translocation to the nucleus, indicating that at least two different pathways of nuclear transport exist for cell surface receptors. This finding provides a new direction for investigating the trafficking mechanisms of various nuclear RTKs.

PARP inhibitor upregulates PD-L1 expression and enhances cancer-associated immunosuppression

Purpose: To explore whether a cross-talk exists between PARP inhibition and PD-L1/PD-1 immune checkpoint axis, and determine whether blockade of PD-L1/PD-1 potentiates PARP inhibitor (PARPi) in tumor suppression. Experimental Design: Breast cancer cell lines, xenograft tumors, and syngeneic tumors treated with PARPi were assessed for PD-L1 expression by immunoblotting, IHC, and FACS analyses. The phospho-kinase antibody array screen was used to explore the underlying mechanism of PARPi-induced PD-L1 upregulation. The therapeutic efficacy of PARPi alone, PD-L1 blockade alone, or their combination was tested in a syngeneic tumor model. The tumor-infiltrating lymphocytes and tumor cells isolated from syngeneic tumors were analyzed by CyTOF and FACS to evaluate the activity of antitumor immunity in the tumor microenvironment. Results: PARPi upregulated PD-L1 expression in breast cancer cell lines and animal models. Mechanistically, PARPi inactivated GSK3β, which in turn enhanced PARPi-mediated PD-L1 upregulation. PARPi attenuated anticancer immunity via upregulation of PD-L1, and blockade of PD-L1 resensitized PARPi-treated cancer cells to T-cell killing. The combination of PARPi and anti-PD-L1 therapy compared with each agent alone significantly increased the therapeutic efficacy in vivo. Conclusions: Our study demonstrates a cross-talk between PARPi and tumor-associated immunosuppression and provides evidence to support the combination of PARPi and PD-L1 or PD-1 immune checkpoint blockade as a potential therapeutic approach to treat breast cancer. Clin Cancer Res; 23(14); 3711–20. ©2017 AACR.

PRMT1-mediated methylation of the EGF receptor regulates signaling and cetuximab response

Posttranslational modifications to the intracellular domain of the EGFR are known to regulate EGFR functions; however, modifications to the extracellular domain and their effects remain relatively unexplored. Here, we determined that methylation at R198 and R200 of the EGFR extracellular domain by protein arginine methyltransferase 1 (PRMT1) enhances binding to EGF and subsequent receptor dimerization and signaling activation. In a mouse orthotopic colorectal cancer xenograft model, expression of a methylation-defective EGFR reduced tumor growth. Moreover, increased EGFR methylation sustained signaling activation and cell proliferation in the presence of the therapeutic EGFR monoclonal antibody cetuximab. In colorectal cancer patients, EGFR methylation level also correlated with a higher recurrence rate after cetuximab treatment and reduced overall survival. Together, these data indicate that R198/R200 methylation of the EGFR plays an important role in regulating EGFR functionality and resistance to cetuximab treatment.

Dual targeting of tumor angiogenesis and chemotherapy by endostatin-cytosine deaminase-uracil phosphoribosyltransferase

Several antiangiogenic drugs targeting VEGF/VEGF receptor (VEGFR) that were approved by the Food and Drug Administration for many cancer types, including colorectal and lung cancer, can effectively reduce tumor growth. However, targeting the VEGF signaling pathway will probably influence the normal function of endothelial cells in maintaining homeostasis and can cause unwanted adverse effects. Indeed, emerging experimental evidence suggests that VEGF-targeting therapy induced less tumor cell–specific cytotoxicity, allowing residual cells to become more resistant and eventually develop a more malignant phenotype. We report an antitumor therapeutic EndoCD fusion protein developed by linking endostatin (Endo) to cytosine deaminase and uracil phosphoribosyltransferase (CD). Specifically, Endo possesses tumor antiangiogenesis activity that targets tumor endothelial cells, followed by CD, which converts the nontoxic prodrug 5-fluorocytosine (5-FC) to the cytotoxic antitumor drug 5-fluorouracil (5-FU) in the local tumor area. Moreover, selective targeting of tumor sites allows an increasing local intratumoral concentration of 5-FU, thus providing high levels of cytotoxic activity. We showed that treatment with EndoCD plus 5-FC, compared with bevacizumab plus 5-FU treatment, significantly increased the 5-FU concentration around tumor sites and suppressed tumor growth and metastasis in human breast and colorectal orthotropic animal models. In addition, in contrast to treatment with bevacizumab/5-FU, EndoCD/5-FC did not induce cardiotoxicity leading to heart failure in mice after long-term treatment. Our results showed that, compared with currently used antiangiogenic drugs, EndoCD possesses potent anticancer activity with virtually no toxic effects and does not increase tumor invasion or metastasis. Together, these findings suggest that EndoCD/5-FC could become an alternative option for future antiangiogenesis therapy. Mol Cancer Ther; 10(8); 1327–36. ©2011 AACR.

High speed digital protein interaction analysis using microfluidic single molecule detection system

The understanding of protein interaction dynamics is important for signal transduction research but current available techniques prove difficult in addressing this issue. Thus, using the microfluidic approach, we developed a digital protein analytical platform and methodology named MAPS (Microfluidic system Analyzing Protein in Single complex) that can measure the amount of target proteins and protein complexes at the digitally single molecule resolution. By counting protein events individually, this system can provide rough protein interaction ratios which will be critical for understanding signal transduction dynamics. In addition, this system only requires less than an hour to characterize the target protein sample, which is much quicker than conventional approaches. As a proof of concept, we have determined the interaction ratios of oncogenic signaling protein complexes EGFR/Src and EGFR/STAT3 before and after EGF ligand stimulation. To the best of our knowledge, this is the first time that the interaction ratio between EGFR and its downstream proteins has been characterized. The information from MAPS will be critical for the study of protein signal transduction quantitation and dynamics.