An ER-Associated Pathway Defines Endosomal Architecture for Controlled Cargo Transport.

Through a network of progressively maturing vesicles, the endosomal system connects the cell's interior with extracellular space. Intriguingly, this network exhibits a bilateral architecture, comprised of a relatively immobile perinuclear vesicle "cloud" and a highly dynamic peripheral contingent. How this spatiotemporal organization is achieved and what function(s) it curates is unclear. Here, we reveal the endoplasmic reticulum (ER)-located ubiquitin ligase Ring finger protein 26 (RNF26) as the global architect of the entire endosomal system, including the trans-Golgi network (TGN). To specify perinuclear vesicle coordinates, catalytically competent RNF26 recruits and ubiquitinates the scaffold p62/sequestosome 1 (p62/SQSTM1), in turn attracting ubiquitin-binding domains (UBDs) of various vesicle adaptors. Consequently, RNF26 restrains fast transport of diverse vesicles through a common molecular mechanism operating at the ER membrane, until the deubiquitinating enzyme USP15 opposes RNF26 activity to allow vesicle release into the cell's periphery. By drawing the endosomal system's architecture, RNF26 orchestrates endosomal maturation and trafficking of cargoes, including signaling receptors, in space and time.

EGFR signaling controls directionality of epithelial multilayer formation upon loss of cell polarity.

Apical-basal polarity is maintained by distinct protein complexes that reside in membrane junctions, and polarity loss in monolayered epithelial cells can lead to formation of multilayers, cell extrusion, and/or malignant overgrowth. Yet, how polarity loss cooperates with intrinsic signals to control directional invasion toward neighboring epithelial cells remains elusive. Using the Drosophila ovarian follicular epithelium as a model, we found that posterior follicle cells with loss of lethal giant larvae (lgl) or Discs large (Dlg) accumulate apically toward germline cells, whereas cells with loss of Bazooka (Baz) or atypical protein kinase C (aPKC) expand toward the basal side of wildtype neighbors. Further studies revealed that these distinct multilayering patterns in the follicular epithelium were determined by epidermal growth factor receptor (EGFR) signaling and its downstream target Pointed, a zinc-finger transcription factor. Additionally, we identified Rho kinase as a Pointed target that regulates formation of distinct multilayering patterns. These findings provide insight into how cell polarity genes and receptor tyrosine kinase signaling interact to govern epithelial cell organization and directional growth that contribute to epithelial tumor formation.

miR-107 reverses the multidrug resistance of gastric cancer by targeting the CGA/EGFR/GATA2 positive feedback circuit.

Chemotherapy is still the main therapeutic strategy for gastric cancer (GC). However, most patients eventually acquire multidrug resistance (MDR). Hyperactivation of the EGFR signaling pathway contributes to MDR by promoting cancer cell proliferation and inhibiting apoptosis. We previously identified the secreted protein CGA as a novel ligand of EGFR and revealed a CGA/EGFR/GATA2 positive feedback circuit that confers MDR in GC. Herein, we outline a microRNA-based treatment approach for MDR reversal that targets both CGA and GATA2. We observed increased expression of CGA and GATA2 and increased activation of EGFR in GC samples. Bioinformatic analysis revealed that miR-107 could simultaneously target CGA and GATA2, and the low expression of miR-107 was correlated with poor prognosis in GC patients. The direct interactions between miR-107 and CGA or GATA2 were validated by luciferase reporter assays and Western blot analysis. Overexpression of miR-107 in MDR GC cells increased their susceptibility to chemotherapeutic agents, including fluorouracil, adriamycin, and vincristine, in vitro. Notably, intratumor injection of the miR-107 prodrug enhanced MDR xenograft sensitivity to chemotherapies in vivo. Molecularly, targeting CGA and GATA2 with miR-107 inhibited EGFR downstream signaling, as evidenced by the reduced phosphorylation of ERK and AKT. These results suggest that miR-107 may contribute to the development of a promising therapeutic approach for the treatment of MDR in GC.

Diarylheptanoid 35d overcomes EGFR TKI resistance by inducing hsp70-mediated lysosomal degradation of EGFR in EGFR-mutant lung adenocarcinoma.

Epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma (LUAD) patients often respond to EGFR tyrosine kinase inhibitors (TKIs) initially but eventually develop resistance to TKIs. The switch of EGFR downstream signaling from TKI-sensitive to TKI-insensitive is a critical mechanism-driving resistance to TKIs. Identification of potential therapies to target EGFR effectively is a potential strategy to treat TKI-resistant LUADs. In this study, we developed a small molecule diarylheptanoid 35d, a curcumin derivative, that effectively suppressed EGFR protein expression, killed multiple TKI-resistant LUAD cells in vitro, and suppressed tumor growth of EGFR-mutant LUAD xenografts with variant TKI-resistant mechanisms including EGFR C797S mutations in vivo. Mechanically, 35d triggers heat shock protein 70-mediated lysosomal pathway through transcriptional activation of several components in the pathway, such as HSPA1B, to induce EGFR protein degradation. Interestingly, higher HSPA1B expression in LUAD tumors associated with longer survival of EGFR-mutant, TKI-treated patients, suggesting the role of HSPA1B on retarding TKI resistance and providing a rationale for combining 35d with EGFR TKIs. Our data showed that combination of 35d significantly inhibits tumor reprogression on osimertinib and prolongs mice survival. Overall, our results suggest 35d as a promising lead compound to suppress EGFR expression and provide important insights into the development of combination therapies for TKI-resistant LUADs, which could have translational potential for the treatment of this deadly disease.

Impaired cholesterol efflux in retinal pigment epithelium of individuals with juvenile macular degeneration.

Macular degeneration (MD) is characterized by the progressive deterioration of the macula and represents one of the most prevalent causes of blindness worldwide. Abnormal intracellular accumulation of lipid droplets and pericellular deposits of lipid-rich material in the retinal pigment epithelium (RPE) called drusen are clinical hallmarks of different forms of MD including Doyne honeycomb retinal dystrophy (DHRD) and age-related MD (AMD). However, the appropriate molecular therapeutic target underlying these disorder phenotypes remains elusive. Here, we address this knowledge gap by comparing the proteomic profiles of induced pluripotent stem cell (iPSC)-derived RPEs (iRPE) from individuals with DHRD and their isogenic controls. Our analysis and follow-up studies elucidated the mechanism of lipid accumulation in DHRD iRPE cells. Specifically, we detected significant downregulation of carboxylesterase 1 (CES1), an enzyme that converts cholesteryl ester to free cholesterol, an indispensable process in cholesterol export. CES1 knockdown or overexpression of EFEMP1R345W, a variant of EGF-containing fibulin extracellular matrix protein 1 that is associated with DHRD and attenuated cholesterol efflux and led to lipid droplet accumulation. In iRPE cells, we also found that EFEMP1R345W has a hyper-inhibitory effect on epidermal growth factor receptor (EGFR) signaling when compared to EFEMP1WT and may suppress CES1 expression via the downregulation of transcription factor SP1. Taken together, these results highlight the homeostatic role of cholesterol efflux in iRPE cells and identify CES1 as a mediator of cholesterol efflux in MD.

Cooperative regulation of adherens junction expansion through epidermal growth factor receptor activation.

The mechanisms controlling the dynamics of expansion of adherens junctions are significantly less understood than those controlling their static properties. Here, we report that for suspended cell aggregates, the time to form a new junction between two cells speeds up with the number of junctions that the cells are already engaged in. Upon junction formation, the activation of epidermal growth factor receptor (EGFR) distally affects the actin turnover dynamics of the free cortex of the cells. The 'primed' actin cortex results in a faster expansion of the subsequent new junctions. In such aggregates, we show that this mechanism results in a cooperative acceleration of the junction expansion dynamics (kinetype) but does not alter the cell contractility, and hence the final junction size (phenotype). This article has an associated First Person interview with the first author of the paper.

Nanopore-mediated protein delivery enabling three-color single-molecule tracking in living cells.

Multicolor single-molecule tracking (SMT) provides a powerful tool to mechanistically probe molecular interactions in living cells. However, because of the limitations in the optical and chemical properties of currently available fluorophores and the multiprotein labeling strategies, intracellular multicolor SMT remains challenging for general research studies. Here, we introduce a practical method employing a nanopore-electroporation (NanoEP) technique to deliver multiple organic dye-labeled proteins into living cells for imaging. It can be easily expanded to three channels in commercial microscopes or be combined with other in situ labeling methods. Utilizing NanoEP, we demonstrate three-color SMT for both cytosolic and membrane proteins. Specifically, we simultaneously monitored single-molecule events downstream of EGFR signaling pathways in living cells. The results provide detailed resolution of the spatial localization and dynamics of Grb2 and SOS recruitment to activated EGFR along with the resultant Ras activation.

Genetic ablation of Smoothened in pancreatic fibroblasts increases acinar-ductal metaplasia.

The contribution of the microenvironment to pancreatic acinar-to-ductal metaplasia (ADM), a preneoplastic transition in oncogenic Kras-driven pancreatic cancer progression, is currently unclear. Here we show that disruption of paracrine Hedgehog signaling via genetic ablation of Smoothened (Smo) in stromal fibroblasts in a Kras(G12D) mouse model increased ADM. Smo-deleted fibroblasts had higher expression of transforming growth factor-α (Tgfa) mRNA and secreted higher levels of TGFα, leading to activation of EGFR signaling in acinar cells and increased ADM. The mechanism involved activation of AKT and noncanonical activation of the GLI family transcription factor GLI2. GLI2 was phosphorylated at Ser230 in an AKT-dependent fashion and directly regulated Tgfa expression in fibroblasts lacking Smo Additionally, Smo-deleted fibroblasts stimulated the growth of Kras(G12D)/Tp53(R172H) pancreatic tumor cells in vivo and in vitro. These results define a non-cell-autonomous mechanism modulating Kras(G12D)-driven ADM that is balanced by cross-talk between Hedgehog/SMO and AKT/GLI2 pathways in stromal fibroblasts.

Pancreatic fibroblasts smoothen their activities via AKT-GLI2-TGFα.

Pancreatic stromal fibroblasts provide structural support. Activated fibroblasts are critical in the tumor microenvironment. In this issue of Genes & Development, Liu and colleagues (pp. 1943-1955) unravel the finding that depletion of Smoothened (Smo) in pancreatic stromal fibroblasts results in AKT activation and noncanonical GLI2 activation with subsequent TGFα secretion, activation of EGFR in pancreatic epithelial cells, and augmentation of acinar-ductal metaplasia. Additionally, Smo-mediated signaling has proproliferative effects on pancreatic tumor cells.

Resistance to hormone therapy in breast cancer cells promotes autophagy and EGFR signaling pathway.

Breast cancer is the leading cause of cancer deaths for women worldwide. Endocrine therapies represent the cornerstone for hormone-dependent breast cancer treatment. However, in many cases, endocrine resistance is induced with poor prognosis for patients. In the current study, we have developed MCF-7 cell lines resistant to fulvestrant (MCF-7Fulv) and tamoxifen (MCF-7Tam) aiming at investigating mechanisms underlying resistance. Both resistant cell lines exerted lower proliferation capacity in two-dimensional (2-D) cultures but retain estrogen receptor α (ERα) expression and proliferate independent of the presence of estrogens. The established cell lines tend to be more aggressive exhibiting advanced capacity to form colonies, increased expression of epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and heterodimerization of ERBB family receptors and activation of EGFR downstream pathways like MEK/ERK1/2 and PI3K/AKT. Tyrosine kinase inhibitors tested against resistant MCF-7Fulv and MCF-7Tam cells showed moderate efficacy to inhibit cell proliferation, except for lapatinib, which concomitantly inhibits both EGFR and HER2 receptors and strongly reduced cell proliferation. Furthermore, increased autophagy was observed in resistant MCF-7Fulv and MCF-7Tam cells as shown by the presence of autophagosomes and increased Beclin-1 levels. The increased autophagy in resistant cells is not associated with increased apoptosis, suggesting a cytoprotective role for autophagy that may favor cells' survival and aggressiveness. Thus, by exploiting those underlying mechanisms, new targets could be established to overcome endocrine resistance.NEW & NOTEWORTHY The development of resistance to hormone therapy caused by both fulvestrant and tamoxifen promotes autophagy with concomitant apoptosis evasion, rendering cells capable of surviving and growing. The fact that resistance also triggers ERBB family signaling pathways, which are poorly inhibited by tyrosine kinase inhibitors might attribute to cells' aggressiveness. It is obvious that the development of endocrine therapy resistance involves a complex interplay between deregulated ERBB signaling and autophagy that may be considered in clinical practice.

ST6Gal-I-mediated sialylation of the epidermal growth factor receptor modulates cell mechanics and enhances invasion.

Heterogeneity within the glycocalyx influences cell adhesion mechanics and signaling. However, the role of specific glycosylation subtypes in influencing cell mechanics via alterations of receptor function remains unexplored. It has been shown that the addition of sialic acid to terminal glycans impacts growth, development, and cancer progression. In addition, the sialyltransferase ST6Gal-I promotes epidermal growth factor receptor (EGFR) activity, and we have shown EGFR is an 'allosteric mechano-organizer' of integrin tension. Here, we investigated the impact of ST6Gal-I on cell mechanics. Using DNA-based tension gauge tether probes of variable thresholds, we found that high ST6Gal-I activity promotes increased integrin forces and spreading in Cos-7 and OVCAR3, OVCAR5, and OV4 cancer cells. Further, employing inhibitors and function-blocking antibodies against ß1, ß3, and ß5 integrins and ST6Gal-I targets EGFR, tumor necrosis factor receptor, and Fas cell surface death receptor, we validated that the observed phenotypes are EGFR-specific. We found that while tension, contractility, and adhesion are extracellular-signal-regulated kinase pathway-dependent, spreading, proliferation, and invasion are phosphoinositide 3-kinase-Akt serine/threonine kinase dependent. Using total internal reflection fluorescence microscopy and flow cytometry, we also show that high ST6Gal-I activity leads to sustained EGFR membrane retention, making it a key regulator of cell mechanics. Our findings suggest a novel sialylation-dependent mechanism orchestrating cellular mechanics and enhancing cell motility via EGFR signaling.

Tubular cell transcriptional intermediary factor 1γ deficiency exacerbates kidney injury-induced tubular cell polyploidy and fibrosis.

Tubulointerstitial fibrosis is considered the final convergent pathway of progressive chronic kidney diseases (CKD) regardless of etiology. However, mechanisms underlying kidney injury-induced fibrosis largely remain unknown. Recent studies have indicated that transcriptional intermediary factor 1γ (TIF1γ) inhibits the progression of fibrosis in other organs. Here, we found that TIF1γ was highly expressed in the cytoplasm and nucleus of the kidney proximal tubule. Interestingly, we found tubular TIF1γ expression was decreased in patients with CKD, including those with diabetes, hypertension, and IgA nephropathy, and in mouse models with experimental kidney fibrosis (unilateral ureteral obstruction [UUO], folic acid nephropathy [FAN], and aristolochic acid-induced nephrotoxicity). Tubule-specific knock out of TIF1γ in mice exacerbated UUO- and FAN-induced tubular cell polyploidy and subsequent fibrosis, whereas overexpression of kidney TIF1γ protected mice against kidney fibrosis. Mechanistically, in tubular epithelial cells, TIF1γ exerted an antifibrotic role via transforming growth factor-ß (TGF-ß)-dependent and -independent signaling. TIF1γ hindered TGF-ß signaling directly by inhibiting the formation and activity of the transcription factor Smad complex in tubular cells, and we discovered that TIF1γ suppressed epidermal growth factor receptor (EGFR) signaling upstream of TGF-ß signaling in tubular cells by ubiquitylating EGFR at its lysine 851/905 sites thereby promoting EGFR internalization and lysosomal degradation. Pharmacological inhibition of EGFR signaling attenuated exacerbated polyploidization and the fibrotic phenotype in mice with tubule deletion of TIF1γ. Thus, tubular TIF1γ plays an important role in kidney fibrosis by suppressing profibrotic EGFR and TGF-ß signaling. Hence, our findings suggest that maintaining homeostasis of tubular TIF1γ may be a new therapeutic option for treating tubulointerstitial fibrosis and subsequent CKD.

Secretory phospholipase (sPLA2-IIA) regulates breast cancer stem cells differentiation and metastatic potential.

Breast cancer is the second most cancer worldwide in females. The primary factor responsible for tumor recurrence is the presence of breast cancer stem cells (BCSCs), which escape the chemo-radiotherapy. In this study, we have investigated the role of Secretory phospholipase-A2 Group 2A (sPLA2-IIA) that is overexpressed in BCSCs of MCF7 and MDA-MB-231 breast cancer cell lines. Further, overexpression of sPLA2-IIA revealed an increased EGFR/JNK/c-JUN/c-FOS signaling in BCSCs, while sPLA2-IIA knockdown significantly reduced the percentage of BCSCs and decreased signaling in both the cell lines. Importantly, sPLA2-IIA knockdown showed differentiation of BCSCs. Strikingly, PET imaging showed a decreased metastatic potential of BCSCs. Our study revealed a novel role of sPLA2-IIA in regulating BCSCs, which play a crucial role in regulating the differentiation and metastatic potential of BCSCs.

The ETS-transcription factor Pointed is sufficient to regulate the posterior fate of the follicular epithelium.

The Janus-kinase/signal transducer and activator of transcription (JAK/STAT) pathway regulates the anterior posterior axis of the Drosophila follicle cells. In the anterior, it activates the bone morphogenetic protein (BMP) signaling pathway through expression of the BMP ligand decapentaplegic (dpp). In the posterior, JAK/STAT works with the epidermal growth factor receptor (EGFR) pathway to express the T-box transcription factor midline (mid). Although MID is necessary for establishing the posterior fate of the egg chamber, we show that it is not sufficient to determine a posterior fate. The ETS-transcription factor pointed (pnt) is expressed in an overlapping domain to mid in the follicle cells. This study shows that pnt is upstream of mid and that it is sufficient to induce a posterior fate in the anterior end, which is characterized by the induction of mid, the prevention of the stretched cells formation and the abrogation of border cell migration. We demonstrate that the anterior BMP signaling is abolished by PNT through dpp repression. However, ectopic DPP cannot rescue the anterior fate formation, suggesting additional targets of PNT participate in the posterior fate determination.

Quantitative analyses of EGFR localization and trafficking dynamics in the follicular epithelium.

To bridge the gap between qualitative and quantitative analyses of the epidermal growth factor receptor (EGFR) in tissues, we generated an sfGFP-tagged EGF receptor (EGFR-sfGFP) in Drosophila The homozygous fly appears similar to wild type with EGFR expression and activation patterns that are consistent with previous reports in the ovary, early embryo, and imaginal discs. Using ELISA, we quantified an average of 1100, 6200 and 2500 receptors per follicle cell (FC) at stages 8/9, 10 and ≥11 of oogenesis, respectively. Interestingly, the spatial localization of the EGFR to the apical side of the FCs at early stages depended on the TGFα-like ligand Gurken. At later stages, EGFR localized to basolateral positions of the FCs. Finally, we followed the endosomal localization of EGFR in the FCs. The EGFR colocalized with the late endosome, but no significant colocalization of the receptor was found with the early endosome. The EGFR-sfGFP fly is an exciting new resource for studying cellular localization and regulation of EGFR in tissues.

Egfr signaling promotes juvenile hormone biosynthesis in the German cockroach.

BACKGROUND: In insects, an interplay between the activities of distinct hormones, such as juvenile hormone (JH) and 20-hydroxyecdysone (20E), regulates the progression through numerous life history hallmarks. As a crucial endocrine factor, JH is mainly synthesized in the corpora allata (CA) to regulate multiple physiological and developmental processes, including molting, metamorphosis, and reproduction. During the last century, significant progress has been achieved in elucidating the JH signal transduction pathway, while less progress has been made in dissecting the regulatory mechanism of JH biosynthesis. Previous work has shown that receptor tyrosine kinase (RTK) signaling regulates hormone biosynthesis in both insects and mammals. Here, we performed a systematic RNA interference (RNAi) screening to identify RTKs involved in regulating JH biosynthesis in the CA of adult Blattella germanica females. RESULTS: We found that the epidermal growth factor receptor (Egfr) is required for promoting JH biosynthesis in the CA of adult females. The Egf ligands Vein and Spitz activate Egfr, followed by Ras/Raf/ERK signaling, and finally activation of the downstream transcription factor Pointed (Pnt). Importantly, Pnt induces the transcriptional expression of two key enzyme-encoding genes in the JH biosynthesis pathway: juvenile hormone acid methyltransferase (JHAMT) and methyl farnesoate epoxidase (CYP15A1). Dual-luciferase reporter assay shows that Pnt is able to activate a promoter region of Jhamt. In addition, electrophoretic mobility shift assay confirms that Pnt directly binds to the - 941~ - 886 nt region of the Jhamt promoter. CONCLUSIONS: This study reveals the detailed molecular mechanism of Egfr signaling in promoting JH biosynthesis in the German cockroach, shedding light on the intricate regulation of JH biosynthesis during insect development.

Absence of Scaffold Protein Tks4 Disrupts Several Signaling Pathways in Colon Cancer Cells.

Tks4 is a large scaffold protein in the EGFR signal transduction pathway that is involved in several cellular processes, such as cellular motility, reactive oxygen species-dependent processes, and embryonic development. It is also implicated in a rare developmental disorder, Frank-ter Haar syndrome. Loss of Tks4 resulted in the induction of an EMT-like process, with increased motility and overexpression of EMT markers in colorectal carcinoma cells. In this work, we explored the broader effects of deletion of Tks4 on the gene expression pattern of HCT116 colorectal carcinoma cells by transcriptome sequencing of wild-type and Tks4 knockout (KO) cells. We identified several protein coding genes with altered mRNA levels in the Tks4 KO cell line, as well as a set of long non-coding RNAs, and confirmed these changes with quantitative PCR on a selected set of genes. Our results show a significant perturbation of gene expression upon the deletion of Tks4, suggesting the involvement of different signal transduction pathways over the well-known EGFR signaling.

Annexin A1 promotes the progression of bladder cancer via regulating EGFR signaling pathway.

BACKGROUND: Bladder cancer (BLCA) is one of the most common malignancies worldwide. One of the main reasons for the unsatisfactory management of BLCA is the complex molecular biological mechanism. Annexin A1 (ANXA1), a Ca2+-regulated phospholipid-binding protein, has been demonstrated to be implicated in the progression and prognosis of many cancers. However, the expression pattern, biological function and mechanism of ANXA1 in BLCA remain unclear. METHODS: The clinical relevance of ANXA1 in BLCA was investigated by bioinformatics analysis based on TCGA and GEO datasets. Immunohistochemical (IHC) analysis was performed to detect the expression of ANXA1 in BLCA tissues, and the relationships between ANXA1 and clinical parameters were analyzed. In vitro and in vivo experiments were conducted to study the biological functions of ANXA1 in BLCA. Finally, the potential mechanism of ANXA1 in BLCA was explored by bioinformatics analysis and verified by in vitro and in vivo experiments. RESULTS: Bioinformatics and IHC analyses indicated that a high expression level of ANXA1 was strongly associated with the progression and poor prognosis of patients with BLCA. Functional studies demonstrated that ANXA1 silencing inhibited the proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of BLCA cells in vitro, and suppressed the growth of xenografted bladder tumors in vivo. Mechanistically, loss of ANXA1 decreased the expression and phosphorylation level of EGFR and the activation of downstream signaling pathways. In addition, knockdown of ANXA1 accelerated ubiquitination and degradation of P-EGFR to downregulate the activation of EGFR signaling. CONCLUSIONS: These findings indicate that ANXA1 is a reliable clinical predictor for the prognosis of BLCA and promotes proliferation and migration by activating EGFR signaling in BLCA. Therefore, ANXA1 may be a promising biomarker for the prognosis of patients with BLCA, thus shedding light on precise and personalized therapy for BLCA in the future.

An oleanolic acid derivative, K73-03, inhibits pancreatic cancer cells proliferation in vitro and in vivo via blocking EGFR/Akt pathway.

Oleanolic acid (OA) and its derivatives show potent anticancer function. Pancreatic cancer (PC) is the fourth core motive of cancer-related deaths worldwide. Epidermal growth factor receptor (EGFR) has been implicated in PC and has been validated as a therapeutic target. Our study demonstrated that K73-03, an OA derivative, was identified as a potent inhibitor of EGFR by using reverse pharmacophore screening and molecular dynamics simulation assays. Moreover, Western blot analysis showed that K73-03 markedly suppressed the levels of phosphorylated-EGFR (p-EGFR) and phosphorylated-Akt (p-Akt). The inhibitory effect of K73-03 on PC cells was assessed in vitro and in vivo. Mechanistically, K73-03 effectively inhibited the cell proliferation of PC cells, and induced apoptosis and autophagy of ASPC-1 cells in a dose-dependent manner. Additionally, pretreatment with chloroquine, an autophagy inhibitor, significantly inhibited K73-03-induced autophagy and enhanced K73-03-induced apoptotic cell death. K73-03 also strongly repressed ASPC-1 cells xenograft growth in vivo. Thus, all these findings provided new clues about OA analog K73-03 as an effective anticancer agent targeted EGFR against ASPC-1 cells, it is worth further evaluation in the future.

Heteronemin and Tetrac Induce Anti-Proliferation by Blocking EGFR-Mediated Signaling in Colorectal Cancer Cells.

Overexpressed EGFR and mutant K-Ras play vital roles in therapeutic resistance in colorectal cancer patients. To search for an effective therapeutic protocol is an urgent task. A secondary metabolite in the sponge Hippospongia sp., Heteronemin, has been shown to induce anti-proliferation in several types of cancers. A thyroxine-deaminated analogue, tetrac, binds to integrin αvß3 to induce anti-proliferation in different cancers. Heteronemin- and in combination with tetrac-induced antiproliferative effects were evaluated. Tetrac enhanced heteronemin-induced anti-proliferation in HT-29 cells (KRAS WT CRC) and HCT-116 cells (KRAS MT CRC). Heteronemin and tetrac arrested cell cycle in different phases. Combined treatment increased the cell accumulation in sub-G1 and S phases. The combined treatment also induced the inactivation of EGFR signaling and downregulated the phosphorylated ERK1/2 protein in both cell lines. Heteronemin and the combination showed the downregulation of the phosphorylated and total PI3K protein in HT-29 cells (KRAS WT CRC). Results by NanoString technology and RT-qPCR revealed that heteronemin and combined treatment suppressed the expression of EGFR and downstream genes in HCT-116 cells (KRAS MT CRC). Heteronemin or combined treatment downregulated genes associated with cancer progression and decreased cell motility. Heteronemin or the combined treatment suppressed PD-L1 expression in both cancer cell lines. However, only tetrac and the combined treatment inhibited PD-L1 protein accumulation in HT-29 cells (KRAS WT CRC) and HCT-116 cells (KRAS MT CRC), respectively. In summary, heteronemin induced anti-proliferation in colorectal cancer cells by blocking the EGFR-dependent signal transduction pathway. The combined treatment further enhanced the anti-proliferative effect via PD-L1 suppression. It can be an alternative strategy to suppress mutant KRAS resistance for anti-EGFR therapy.