Nuclear export signal mutation of epidermal growth factor receptor enhances malignant phenotypes of cancer cells.

Nuclear epidermal growth factor receptor (EGFR) has been shown to be correlated with drug resistance and a poor prognosis in patients with cancer. Previously, we have identified a tripartite nuclear localization signal (NLS) within EGFR. To comprehensively determine the functions and underlying mechanism of nuclear EGFR and its clinical implications, we aimed to explore the nuclear export signal (NES) sequence of EGFR that is responsible for interacting with the exportins. We combined in silico prediction with site-directed mutagenesis approaches and identified a putative NES motif of EGFR, which is located in amino acid residues 736-749. Mutation at leucine 747 (L747) in the EGFR NES led to increased nuclear accumulation of the protein via a less efficient release of the exportin CRM1. Interestingly, L747 with serine (L747S) and with proline (L747P) mutations were found in both tyrosine kinase inhibitor (TKI)-treated and -naïve patients with lung cancer who had acquired or de novo TKI resistance and a poor outcome. Reconstituted expression of the single NES mutant EGFRL747P or EGFRL747S, but not the dual mutant along with the internalization-defective or NLS mutation, in lung cancer cells promoted malignant phenotypes, including cell migration, invasiveness, TKI resistance, and tumor initiation, supporting an oncogenic role of nuclear EGFR. Intriguingly, cells with germline expression of the NES L747 mutant developed into B cell lymphoma. Mechanistically, nuclear EGFR signaling is required for sustaining nuclear activated STAT3, but not for Erk. These findings suggest that EGFR functions are compartmentalized and that nuclear EGFR signaling plays a crucial role in tumor malignant phenotypes, leading to tumorigenesis in human cancer.

DDR1-induced neutrophil extracellular traps drive pancreatic cancer metastasis.

Pancreatic ductal adenocarcinoma (PDAC) tumors are characterized by a desmoplastic reaction resulting in dense deposition of collagen that is known to promote cancer progression. A central mediator of protumorigenic collagen signaling is the receptor tyrosine kinase discoid domain receptor 1 (DDR1). DDR1 is a critical driver of a mesenchymal and invasive cancer cell PDAC phenotype. Previous studies have demonstrated that genetic or pharmacologic inhibition of DDR1 reduces PDAC tumorigenesis and metastasis. Here, we investigated whether DDR1 signaling has cancer cell nonautonomous effects that promote PDAC progression and metastasis. We demonstrate that collagen-induced DDR1 activation in cancer cells is a major stimulus for CXCL5 production, resulting in the recruitment of tumor-associated neutrophils (TANs), the formation of neutrophil extracellular traps (NETs), and subsequent cancer cell invasion and metastasis. Moreover, we have identified that collagen-induced CXCL5 production was mediated by a DDR1/PKCθ/SYK/NF-κB signaling cascade. Together, these results highlight the critical contribution of the collagen I-DDR1 interaction in the formation of an immune microenvironment that promotes PDAC metastasis.

GSK3ß inactivation promotes the oncogenic functions of EZH2 and enhances methylation of H3K27 in human breast cancers.

During the process of tumorigenesis, inactivation of tumor suppressors is a critical step. EZH2, a histone methyltransferase, promotes cell growth and migration through catalyzing trimethylation of histone H3 at Lys 27 (H3K27me3) and plays an important role in tumorigenesis. Its expression can be controlled by phosphorylation. However, the regulation of EZH2 activity by tumor suppressor kinase is not well understood. In this study, we show that glycogen synthase kinase 3 beta (GSK3ß) negatively regulates H3K27 trimethylation. We also validate that GSKß physically interacts with EZH2, and their interaction occurs in the cytosol. GSK3ß phosphorylates EZH2 at Ser363 and Thr367 in vitro, and activating GSK3ß upregulates Thr367 phosphorylationin vivo. Cells expressing GSK3ß-non-phosphorylatable mutant EZH2 have higher H3K27 trimethylation and enhanced ability of cell migration and anchorage-independent growth. Inactivation of GSK3ß as measured by its phosphorylation at Ser9 is positively correlated with higher level of H3K27 trimethylation in tumor tissues from breast cancer patients. Our study indicated that GSK3ß phosphorylates EZH2 at Ser363 and Thr367, resulting in reduced H3K27 trimethylation and biological activity of EZH2 in breast cancer.

Tyrosine 370 phosphorylation of ATM positively regulates DNA damage response.

Ataxia telangiectasia mutated (ATM) mediates DNA damage response by controling irradiation-induced foci formation, cell cycle checkpoint, and apoptosis. However, how upstream signaling regulates ATM is not completely understood. Here, we show that upon irradiation stimulation, ATM associates with and is phosphorylated by epidermal growth factor receptor (EGFR) at Tyr370 (Y370) at the site of DNA double-strand breaks. Depletion of endogenous EGFR impairs ATM-mediated foci formation, homologous recombination, and DNA repair. Moreover, pretreatment with an EGFR kinase inhibitor, gefitinib, blocks EGFR and ATM association, hinders CHK2 activation and subsequent foci formation, and increases radiosensitivity. Thus, we reveal a critical mechanism by which EGFR directly regulates ATM activation in DNA damage response, and our results suggest that the status of ATM Y370 phosphorylation has the potential to serve as a biomarker to stratify patients for either radiotherapy alone or in combination with EGFR inhibition.

EGFR modulates DNA synthesis and repair through Tyr phosphorylation of histone H4.

Posttranslational modifications of histones play fundamental roles in many biological functions. Specifically, histone H4-K20 methylation is critical for DNA synthesis and repair. However, little is known about how these functions are regulated by the upstream stimuli. Here, we identify a tyrosine phosphorylation site at Y72 of histone H4, which facilitates recruitment of histone methyltransferases (HMTases), SET8 and SUV4-20H, to enhance its K20 methylation, thereby promoting DNA synthesis and repair. Phosphorylation-defective histone H4 mutant is deficient in K20 methylation, leading to reduced DNA synthesis, delayed cell cycle progression, and decreased DNA repair ability. Disrupting the interaction between epidermal growth factor receptor (EGFR) and histone H4 by Y72 peptide significantly reduced tumor growth. Furthermore, EGFR expression clinically correlates with histone H4-Y72 phosphorylation, H4-K20 monomethylation, and the Ki-67 proliferation marker. These findings uncover a mechanism by which EGFR transduces signal to chromatin to regulate DNA synthesis and repair.

STAT3 regulates the proliferation and differentiation of rabbit limbal epithelial cells via a ΔNp63-dependent mechanism.

PURPOSE: To explore the roles of STAT3 in the regulation of ΔNp63-dependent proliferation and differentiation of rabbit limbal keratinocytes. METHODS: siRNAs were designed to specifically suppress the expression of STAT3 and ΔNp63, and their effects on limbal epithelial cell proliferation and differentiation were examined. Ectopically expressed ΔNp63 was used to compensate for the decreased endogenous ΔNp63. Immunoblot was used to examine the expressions of STAT3, ΔNp63, K3, integrin ß1, and involucrin. RESULTS: Limbal tissue expresses higher level of phosphorylated and nuclear translocated STAT3 compared with that of the cornea. Knockdown of STAT3 expression reduces the expression of ΔNp63, inhibits the expansion of limbal epithelial outgrowth, suppresses the expression of integrin ß1, and promotes the expression of involucrin. CONCLUSIONS: STAT3 enhances the proliferation of limbal keratinocytes through a ΔNp63-dependent mechanism. Suppression of this pathway inhibits cell proliferation with a concomitant increase of cell differentiation.

The growth-promoting effect of KGF on limbal epithelial cells is mediated by upregulation of DeltaNp63alpha through the p38 pathway.

Corneal epithelial stem cells are thought to reside in the limbus, the transition zoon between cornea and conjunctiva. Keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) are two paracrine factors that regulate the proliferation, migration and differentiation of the limbal epithelial cells; however, the underlying mechanisms are still poorly understood. In an ex vivo limbal explant culture, we found that KGF is a more potent growth stimulator for the epithelial outgrowth than HGF. Immunofluorescence studies of the epithelial outgrowth from cells treated with HGF or KGF showed similar expression patterns of keratin-3 and keratin-14. Interestingly, p63 was highly expressed in KGF-treated limbal epithelial sheets but not in those treated with HGF. Kinase inhibitor studies showed that induction of DeltaNp63alpha expression by KGF is mediated via the p38 pathway. The effect of KGF on limbal epithelial outgrowth was significantly reduced when endogenous DeltaNp63alpha was suppressed, suggesting that KGF-induced limbal epithelial outgrowth is dependent on the expression of DeltaNp63alpha. Our findings strongly suggest that limbal keratocytes regulate limbal epithelial cell growth and differentiation through a KGF paracrine loop, with DeltaNp63alpha expression as one of the downstream targets.

Regulation of limbal keratinocyte proliferation and differentiation by TAp63 and DeltaNp63 transcription factors.

PURPOSE: To examine the effects of TAp63 and DeltaNp63 on the proliferation and differentiation of rabbit limbal keratinocytes cultured on human amniotic membrane. METHOD: Real-time Q-RT-PCR was used to quantify the relative abundance of TAp63 and DeltaNp63 transcripts in limbal, peripheral corneal, and central corneal epithelia. Antisense oligonucleotides were designed specifically to suppress the expression of TAp63 or DeltaNp63 in limbal keratinocytes, and their effects on cell proliferation and differentiation were examined. Immunofluorescence was used to examine the expressions of p63 and keratin-3 and -14. RESULTS: The expressions of TAp63 and DeltaNp63 transcripts appeared to be site specific. TAp63 was expressed at the highest level in limbus, decreased by approximately 10-fold in peripheral cornea and was undetectable in the central cornea. DeltaNp63 was also expressed at the highest level in limbus, decreased by approximately 35% in peripheral cornea, and was undetectable in the central cornea. Suppression of TAp63 expression inhibited limbal keratinocyte proliferation but promoted differentiation. Suppression of DeltaNp63 expression also inhibited cell proliferation but had no obvious effect on cell differentiation. CONCLUSIONS: TAp63 and DeltaNp63 affect the proliferation of limbal keratinocytes by a different mechanism. The inhibition by TAp63 antisense oligos appeared to be secondary to the promotion of cell differentiation. In contrast, the inhibition by DeltaNp63 antisense oligos appeared to be independent of cell differentiation.

Age-related expressions of p63 and other keratinocyte stem cell markers in rat cornea.

In this study, we examined the postnatal expression patterns of p63 and other keratinocyte stem cell markers in the rat cornea in an attempt to determine the markers that best represent characteristics of corneal keratinocyte stem cells. We show that the expression of p63 in the rat cornea is unique and differs from that observed in humans. It changes with age, from central cornea-positive, peripheral cornea-positive, and limbus-positive, to central cornea-positive, peripheral cornea-positive, and limbus-negative, and finally to central cornea-negative, peripheral cornea-positive, and limbus-negative, as examined by immunohistochemical staining. However, when a more sensitive staining method was used, the limbus was also shown to be positive for p63, indicating a lower level of expression than that of the peripheral cornea. The basal layer of the rat limbal epithelium is the site where beta-catenin+, K14+, PCNA-, and K3- cells reside. This cell layer is also the site where slow-cycling cells are located. In contrast with observations made in humans, our results clearly indicate that p63 is expressed in stem cells and young transient amplifying cells of the rat cornea, with higher levels of expression in the latter.