Advanced biosensors for nanomaterial-based detection of transforming growth factor alpha and beta, a class of major polypeptide regulators.

Transforming growth factors (TGFs) regulate several cellular processes including, differentiation, growth, migration, extracellular matrix production, and apoptosis. TGF alpha (TGF-α) is a heterogeneous molecule containing 160 amino acid residues. It is a potent angiogenesis promoter that is activated by JAK-STAT signaling. Whereas TGF beta (TGF-ß) consists of 390-412 amino acids. Smad and non-Smad signaling both occur in TGF beta. It is linked to immune cell activation, differentiation, and proliferation. It also triggers pre-apoptotic responses and inhibits cell proliferation. Both growth factors have a promising role in the development and homeostasis of tissues. Defects such as autoimmune diseases and cancer develop mechanisms to modulate checkpoints of the immune system resulting in altered growth factors profile. An accurate amount of these growth factors is essential for normal functioning, but an exceed or fall behind the normal level is alarming as it is linked to several disorders. This demands techniques for TGF-α and TGF-ß profiling to effectively diagnose diseases, monitor their progression, and assess the efficacy of immunotherapeutic drugs. Quantitative detection techniques including the emergence of biosensing technology seem to accomplish the purpose. Until the present time, few biosensors have been designed in the context of TGF-α and TGF-ß for disease detection, analyzing receptor binding, and interaction with carriers. In this paper, we have reviewed the physiology of transforming growth factor alpha and beta, including the types, structure, function, latent/active forms, signaling, and defects caused. It involves the description of biosensors on TGF-α and TGF-ß, advances in technology, and future perspectives.

Upregulation by duloxetine of the transforming growth factor-α-induced migration of hepatocellular carcinoma cells via enhancement of the c-Jun N-terminal kinase activity.

Duloxetine, a selective reuptake inhibitor for serotonin and norepinephrine, is a medication widely used for major depression. Currently, duloxetine is also recommended for pain related to chemotherapy-induced peripheral neuropathy or cancer. Previously, we showed that transforming growth factor-α (TGF-α) induces the migration of human hepatocellular carcinoma (HCC)-derived HuH7 cells through the activation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK) and AKT. In the present study, we investigate whether duloxetine affects cell migration and its mechanism. Duloxetine significantly enhanced the TGF-α-induced migration of HuH7 cells. Fluvoxamine and sertraline, specific inhibitors of serotonin reuptake, also upregulated the TGF-α-induced cell migration. On the contrary, reboxetine, a specific norepinephrine reuptake inhibitor, failed to affect cell migration. Duloxetine significantly amplified the TGF-α-stimulated phosphorylation of JNK, but not p38 MAPK and AKT. In addition, fluvoxamine and sertraline, but not reboxetine, enhanced the phosphorylation of JNK. SP600125, a JNK inhibitor, suppressed the enhancement by duloxetine, fluvoxamine, or sertraline of TGF-α-induced migration of HuH7 cells. Taken together, our results strongly suggest that duloxetine strengthens the TGF-α-induced activation of JNK via inhibition of serotonin reuptake in HCC cells, leading to the enhancement of cell migration.

EGF, TGF-α and Amphiregulin Differently Regulate Endometrium-Derived Mesenchymal Stromal/Stem Cells.

The prototypical receptor tyrosine kinase epidermal growth factor receptor (EGFR) is regulated by a set of its ligands, which determines the specificity of signaling and intracellular fate of the receptor. The EGFR signaling system is well characterized in immortalized cell lines such as HeLa derived from tumor tissues, but much less is known about EGFR function in untransformed multipotent stromal/stem cells (MSCs). We compared the effect of epidermal growth factor (EGF), transforming growth factor-α (TGF-α) and amphiregulin (AREG) on physiological responses in endometrial MSCs (enMSC) and HeLa cells. In addition, using Western blotting and confocal microscopy, we studied the internalization and degradation of EGFR stimulated by the three ligands in these cell lines. We demonstrated that unlike HeLa, EGF and TGF-α, but not AREG, stimulated enMSC proliferation and prevented decidual differentiation in an EGFR-dependent manner. In HeLa cells, EGF targeted EGFR for degradation, while TGF-α stimulated its recycling. Surprisingly, in enMSC, both ligands caused EGFR degradation. In both cell lines, AREG-EGFR internalization was not registered. In HeLa cells, EGFR was degraded within 2 h, restoring its level in 24 h, while in enMSC, degradation took more than 4-8 h, and the low EGFR level persisted for several days. This indicates that EGFR homeostasis in MSCs may differ significantly from that in immortalized cell lines.

The Induction of Parathyroid Cell Differentiation from Human Induced Pluripotent Stem Cells Promoted Via TGF-α/EGFR Signaling.

The parathyroid gland plays an essential role in mineral and bone metabolism. Cultivation of physiological human parathyroid cells has yet to be established and the method by which parathyroid cells differentiate from pluripotent stem cells remains uncertain. Therefore, it has been hard to clarify the mechanisms underlying the onset of parathyroid disorders, such as hyperparathyroidism. In this study, we developed a new method of parathyroid cell differentiation from human induced pluripotent stem (iPS) cells. Parathyroid cell differentiation occurred in accordance with embryologic development. Differentiated cells, which expressed the parathyroid hormone, adopted unique cell aggregation similar to the parathyroid gland. In addition, these differentiated cells were identified as calcium-sensing receptor (CaSR)/epithelial cell adhesion molecule (EpCAM) double-positive cells. Interestingly, stimulation with transforming growth factor-α (TGF-α), which is considered a causative molecule of parathyroid hyperplasia, increased the CaSR/EpCAM double-positive cells, but this effect was suppressed by erlotinib, which is an epidermal growth factor receptor (EGFR) inhibitor. These results suggest that TGF-α/EGFR signaling promotes parathyroid cell differentiation from iPS cells in a similar manner to parathyroid hyperplasia.

Rescue of neurogenesis and age-associated cognitive decline in SAMP8 mouse: Role of transforming growth factor-alpha.

Neuropathological aging is associated with memory impairment and cognitive decline, affecting several brain areas including the neurogenic niche of the dentate gyrus of the hippocampus (DG). In the healthy brain, homeostatic mechanisms regulate neurogenesis within the DG to facilitate the continuous generation of neurons from neural stem cells (NSC). Nevertheless, aging reduces the number of activated neural stem cells and diminishes the number of newly generated neurons. Strategies that promote neurogenesis in the DG may improve cognitive performance in the elderly resulting in the development of treatments to prevent the progression of neurological disorders in the aged population. Our work is aimed at discovering targeting molecules to be used in the design of pharmacological agents that prevent the neurological effects of brain aging. We study the effect of age on hippocampal neurogenesis using the SAMP8 mouse as a model of neuropathological aging. We show that in 6-month-old SAMP8 mice, episodic and spatial memory are impaired; concomitantly, the generation of neuroblasts and neurons is reduced and the generation of astrocytes is increased in this model. The novelty of our work resides in the fact that treatment of SAMP8 mice with a transforming growth factor-alpha (TGFα) targeting molecule prevents the observed defects, positively regulating neurogenesis and improving cognitive performance. This compound facilitates the release of TGFα in vitro and in vivo and activates signaling pathways initiated by this growth factor. We conclude that compounds of this kind that stimulate neurogenesis may be useful to counteract the neurological effects of pathological aging.

Role of Hepatocyte Growth Regulators in Liver Regeneration.

We have studied whether growth factors, cytokines, hormones, neurotransmitters, and local hormones (autacoids) promote the proliferation of hepatic parenchymal cells (i.e., hepatocytes) using in vitro primary cultured hepatocytes. The indicators used for this purpose include changes in DNA synthesis activity, nuclear number, cell number, cell cycle, and gene expression. In addition, the intracellular signaling pathways from the plasma membrane receptors to the nucleus have been examined in detail for representative growth-promoting factors that have been found to promote DNA synthesis and cell proliferation of hepatocytes. In examining intracellular signaling pathways, the effects of specific inhibitors of presumed signaling factors involved have been pharmacologically confirmed, and the phosphorylation activities of the signaling factors (e.g., RTK, ERK, mTOR, and p70 S6K) have been evaluated. As a result, it has been found that there are many factors that promote the proliferation of hepatocytes (e.g., HGF, EGF, TGF-α, IL-1ß, TNF-α, insulin, growth hormone (GH), prostaglandin (PG)), and serotonin (5-HT)), while there are very few factors (e.g., TGF-ß1 and glucocorticoids) that inhibit the effects of growth-promoting factors. We have also found that 5-HT and GH promote the proliferation of hepatocytes via different autocrine factors (e.g., TGF-α and IGF-I, respectively). Using primary cultured hepatocytes, it will be possible to further study the molecular and cellular aspects of liver regeneration.

A TGF-α and TGF-ß1 Poloxamer-based micelle/hydrogel composite: A promising novel candidate for the treatment of anosmia.

Anosmia is the inability to smell or loss of the sense of smell. It can reduce your ability to detect the smell of smoke, gas leaks, or spoiled food, as well as hinder the quality of life related to social interactions and feelings of well-being. In the current study, a drug delivery composite was designed to cure anosmia and its efficiency in delivering transforming growth factor alpha (TGF-α) and transforming growth factor beta 1 (TGF-ß1) to the nasal cavity was evaluated. Bovine serum albumin (BSA) was used as a model protein for encapsulation into Poloxamers 407 micelles. For the optimization of the BSA-micelle formulation, a two-parameter five-level central composite design (CCD) was applied. The BSA-micelle was optimized with a particle size of 41 nm, drug loading of 8%, and encapsulation efficiency of 74%. Further, the BSA-micelle was characterized by FESEM, TEM, and FTIR. The analysis of release profile suggested high-paced free BSA release compared to the gradual and prolonged release of BSA-micelle/hydrogel and BSA-micelles. The cytotoxicity assay demonstrated the safety of TGF-α and TGF-ß1-micelles/hydrogel. Moreover, it was observed that TGF-α and TGF-ß1 within the hydrogels promote cellular viability and human olfactory ectomesenchymal stem cell OE-MSCs proliferation. In conclusion, According to the results of our study, the TGF-α and TGF-ß1-micelle/hydrogel-based delivery system provides a suitable alternative for anosmia treatment.

Stimulatory effects of TGFα in granulosa cells of bovine small antral follicles.

Intraovarian growth factors play a vital role in influencing the fate of ovarian follicles. They affect proliferation and apoptosis of granulosa cells (GC) and can influence whether small antral follicles continue their growth or undergo atresia. Transforming growth factor-alpha (TGFα), an oocyte-derived growth factor, is thought to regulate granulosa cell function; yet its investigation has been largely overshadowed by emerging interest in TGF-beta superfamily members, such as bone morphogenetic proteins (BMP) and anti-Mullerian hormone (AMH). Here, effects of TGFα on bovine GC proliferation, intracellular signaling, and cytokine-induced apoptosis were evaluated. Briefly, all small antral follicles (3-5 mm) from slaughterhouse specimens of bovine ovary pairs were aspirated and the cells were plated in T25 flasks containing DMEM/F12 medium, 10% FBS, and antibiotic-antimycotic, and incubated at 37 °C in 5% CO2 for 3 to 4 d. Once confluent, the cells were sub-cultured for experiments (in 96-, 12-, or 6-well plates) in serum-free conditions (DMEM/F12 medium with ITS). Exposure of the bGC to TGFα (10 or 100 ng/mL) for 24 h stimulated cell proliferation compared to control (P < 0.05; n = 7 ovary pairs). Proliferation was accompanied by a concomitant increase in mitogen-activated protein kinase (MAPK) signaling within 2 h of treatment, as evidenced by phosphorylated ERK1/2 expression (P < 0.05, n = 3 ovary pairs). These effects were entirely negated, however, by the MAPK inhibitor, U0126 (10uM, P < 0.05). Additionally, prior exposure of the bGC to TGFα (100 ng/mL) failed to prevent Fas Ligand (100 ng/mL)-induced apoptosis, as measured by caspase 3/7 activity (P < 0.05, n = 7 ovary pairs). Collectively, the results indicate TGFα stimulates proliferation of bGC from small antral follicles via a MAPK/ERK-mediated mechanism, but this action alone fails to prevent apoptosis, suggesting that TGFα may be incapable of promoting their persistence in follicles during the process of follicular selection/dominance.

A variety of hormones regulate ovarian function in the cow, thus influencing fertility. One such hormone, transforming growth factor-alpha, TGFα, is expressed by the oocyte (egg) of the bovine ovary; yet little other information about the actions of this molecule on ovarian cells is available. In this study, we determined that although TGFα directly stimulates growth and proliferation of cells of the bovine ovary, and does so via specific signaling mechanisms, it fails to prevent immune-mediated programmed cell death. The latter observation diminishes the importance of TGFα relative to other oocyte-derived hormones in terms of ovarian function and overall animal fertility.

Bisacodyl Suppresses TGF-α-Induced MUC5AC Production in NCI-H292 Cells.

MUC5AC overproduction is commonly observed in chronic inflammatory lung diseases and worsens these conditions. Therefore, drugs that inhibit MUC5AC production are urgently needed. To identify novel drugs directly inhibiting MUC5AC production, 640 already approved drugs were screened. We found that the laxative bisacodyl suppressed transforming growth factor (TGF)-α-induced MUC5AC production in a concentration-dependent manner. Additionally, bisacodyl also suppressed TGF-α-induced MUC5AC mRNA expression in the same concentration range. These results suggested that bisacodyl could be a new drug for treating mucin overproduction.

GLP-1 reduces the migration of hepatocellular carcinoma cells via suppression of the stress-activated protein kinase/c-Jun N-terminal kinase pathway.

Incretins, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), are hormones secreted from small intestine accompanied with oral intake. We previously showed that transforming growth factor (TGF)-α stimulates the migration of hepatocellular carcinoma (HCC) cells via mitogen-activated protein (MAP) kinases, AKT and Rho-kinase. However, it remains to be elucidated whether incretins affect HCC cell functions. In the present study, therefore, we investigated whether incretins affect the migration of HCC cells using human HCC-derived HuH7 cells. GLP-1, but not GIP, reduced both TGF-α- and hepatocyte growth factor (HGF)-induced cell migration. IBMX, an inhibitor of cyclic nucleotide phosphodiesterase, enhanced the suppressive effect of GLP-1. GLP-1 attenuated the phosphorylation of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) by TGF-α and HGF. Our results strongly suggest that GLP-1 suppresses TGF-α- and HGF-induced migration of HCC cells through inhibiting the SAPK/JNK signaling pathway, and that the inhibition by GLP-1 is due to cAMP production.

Creb5 establishes the competence for Prg4 expression in articular cartilage.

A hallmark of cells comprising the superficial zone of articular cartilage is their expression of lubricin, encoded by the Prg4 gene, that lubricates the joint and protects against the development of arthritis. Here, we identify Creb5 as a transcription factor that is specifically expressed in superficial zone articular chondrocytes and is required for TGF-ß and EGFR signaling to induce Prg4 expression. Notably, forced expression of Creb5 in chondrocytes derived from the deep zone of the articular cartilage confers the competence for TGF-ß and EGFR signals to induce Prg4 expression. Chromatin-IP and ATAC-Seq analyses have revealed that Creb5 directly binds to two Prg4 promoter-proximal regulatory elements, that display an open chromatin conformation specifically in superficial zone articular chondrocytes; and which work in combination with a more distal regulatory element to drive induction of Prg4 by TGF-ß. Our results indicate that Creb5 is a critical regulator of Prg4/lubricin expression in the articular cartilage.

Quercetin suppresses the migration of hepatocellular carcinoma cells stimulated by hepatocyte growth factor or transforming growth factor-α: Attenuation of AKT signaling pathway.

Flavonol, which is found abundantly in plants such as fruits and vegetables, belongs to the family of flavonoid, natural polyphenols. Quercetin, one of the flavonol, reportedly has anti-cancer effects and prevents the proliferation of various cancer cells, including hepatocellular carcinoma (HCC). However, the effects of quercetin on HCC cells migration have not yet been clarified. We have previously shown that the migration of human HCC-derived HuH7 cells induced by hepatocyte growth factor (HGF) or transforming growth factor-α (TGF-α) is mediated through p38 MAPK and AKT. In this study, we investigated whether quercetin affects the HGF- or TGF-α-induced migration of HuH7 cells. Quercetin significantly suppressed both HGF- and TGF-α-induced migration of HuH7 cells in a dose-dependent manner. In addition, myricetin, another flavonol, also showed significant inhibition of the cell migration. Each HGF- and TGF-α-induced autophosphorylation of receptors were not affected by quercetin or myricetin. Quercetin did not suppress HGF- or TGF-α-induced p38 MAPK phosphorylation. On the contrary, quercetin and myricetin inhibited the growth factors-induced phosphorylation of AKT. Our results strongly suggest that quercetin suppresses the growth factor-induced migration of HCC cells by inhibiting the signaling pathway of AKT but not p38 MAPK.

Lipocalin-2 Regulates Epidermal Growth Factor Receptor Intracellular Trafficking.

Epidermal growth factor receptor (EGFR) activation and lipocalin-2 (Lcn2) expression are frequently observed in the same pathological contexts, such as cancers or chronic kidney disease (CKD). However, the significance of this association is unknown. Here, we describe the role of Lcn2 in regulating EGFR trafficking. We show that Lcn2 increases EGFR cell surface abundance and is required for transforming growth factor α (TGF-α)-induced EGFR recycling to the plasma membrane and sustained activation. Lcn2 binds to the intracellular domain of EGFR in late endosomal compartments and inhibits its lysosomal degradation. Consistently, Lcn2 enhances EGFR-induced cell migration after TGF-α stimulation. In vivo, Lcn2 gene inactivation prevents EGFR recycling to the plasma membrane in an experimental model of CKD. Remarkably, this is associated with a dramatic decrease of renal lesions. Together, our data identify Lcn2 as a key mediator of EGFR trafficking processes. Hence, therapeutic inhibition of Lcn2 may counteract the deleterious effect of EGFR activation.

Transcriptional and metabolic rewiring of colorectal cancer cells expressing the oncogenic KRASG13D mutation.

BACKGROUND: Activating mutations in KRAS frequently occur in colorectal cancer (CRC) patients, leading to resistance to EGFR-targeted therapies. METHODS: To better understand the cellular reprogramming which occurs in mutant KRAS cells, we have undertaken a systems-level analysis of four CRC cell lines which express either wild type (wt) KRAS or the oncogenic KRASG13D allele (mtKRAS). RESULTS: RNAseq revealed that genes involved in ribosome biogenesis, mRNA translation and metabolism were significantly upregulated in mtKRAS cells. Consistent with the transcriptional data, protein synthesis and cell proliferation were significantly higher in the mtKRAS cells. Targeted metabolomics analysis also confirmed the metabolic reprogramming in mtKRAS cells. Interestingly, mtKRAS cells were highly transcriptionally responsive to EGFR activation by TGFα stimulation, which was associated with an unexpected downregulation of genes involved in a range of anabolic processes. While TGFα treatment strongly activated protein synthesis in wtKRAS cells, protein synthesis was not activated above basal levels in the TGFα-treated mtKRAS cells. This was likely due to the defective activation of the mTORC1 and other pathways by TGFα in mtKRAS cells, which was associated with impaired activation of PKB signalling and a transient induction of AMPK signalling. CONCLUSIONS: We have found that mtKRAS cells are substantially rewired at the transcriptional, translational and metabolic levels and that this rewiring may reveal new vulnerabilities in oncogenic KRAS CRC cells that could be exploited in future.

Growth-inhibitory effects of TGFαL3-SEB chimeric protein on colon cancer cell line.

BACKGROUND: TGFαL3-SEB chimeric protein is a synthetic protein, which is produced by combining the third loop (L3) of transforming growth factor-α (TGF-α) with staphylococcal enterotoxin type B. To the best of our knowledge, anti-cancer activity of this chimeric protein against colon cancer that overexpresses epidermal growth factor receptor (EGFR) has not yet been studied. Thus, in the present study, the anti-tumor effects of TGFαL3-SEB chimeric protein on HT-29 colon cancer cells were evaluated. MATERIALS AND METHODS: The TGFαL3-SEB chimeric protein was previously designed and cloned in Escherichia coli (E. coli) [1,2]. The level of expression and the purity of this novel protein were examined for further analysis. For this purpose, the cells were treated with different concentrations (25, 50 and 75 µg/ml) of TGFαL3-SEB and then the proliferation was detected using the MTT assay. The apoptosis-inducing potential of TGFαL3-SEB in HT-29 and HEK-293 cells was evaluated by flow cytometry using Annexin V/PI double staining method; in addition, bax/bcl2 mRNA ratio, caspase-3 and caspase-9 activity were also assessed. RESULTS: In the present study, TGFαL3-SEB chimeric protein was produced in E. coli. After effective purification, its growth inhibitory effect was evaluated. Our results indicated that the incubation of HT-29 colon cancer cell with 25, 50 and 75 µg/ml of TGFαL3-SEB for 24 h leads to significant reduction of proliferation in a dose-dependent manner (P < 0.05). Further analysis indicated that exposure of EGFR expressing HT-29 cells to TGFαL3-SEB leads to significant increase of the caspase-3 and caspase-9 activity in a concentration-dependent manner (P < 0.05). Bax/bcl-2 ratio also confirmed that TGFαL3-SEB has the pro-apoptotic effect. Flow cytometry analysis of TGFαL3-SEB treated cells showed that in addition to apoptotic cells, necrotic cells were also increased significantly at the concentration of 25, 50 and 75 µg/ml (P < 0.05). CONCLUSION: In conclusion, our results demonstrated that TGFαL3-SEB chimeric protein induced cell death through both mechanisms of apoptosis and necrosis in HT-29 colon cancer cells. This paper has highlighted that TGFαL3-SEB has the potential to target EGFR expressing cancer cell.

Suppression of Wnt/ß-catenin signaling by EGF receptor is required for hair follicle development.

Mice that lack the epidermal growth factor receptor (EGFR) fail to develop a hair coat, but the mechanism responsible for this deficit is not completely understood. Here, we show that EGFR plays a critical role to attenuate wingless-type MMTV integration site family member (Wnt)/ß-catenin signaling during postnatal hair follicle development. Genetic ablation of EGFR in mice resulted in increased mitotic activity in matrix cells, apoptosis in hair follicles, and impaired differentiation of epithelial lineages that form hair. EGFR is activated in wild-type hair follicle stem cells marked with SOX9 or NFATc1 and is essential to restrain proliferation and support stem cell numbers and their quiescence. We observed elevated levels of Wnt4, 6, 7b, 10a, 10b, and 16 transcripts and hyperactivation of the ß-catenin pathway in EGFR knockout follicles. Using primary keratinocytes, we linked ligand-induced EGFR activation to suppression of nascent mRNA synthesis of Wnt genes. Overexpression of the Wnt antagonist sFRP1 in mice lacking EGFR demonstrated that elevated Wnts are a major cause for the hair follicle defects. Colocalization of transforming growth factor α and Wnts regulated by EGFR in stem cells and progeny indicates that EGFR autocrine loops control Wnts. Our findings define a novel mechanism that integrates EGFR and Wnt/ß-catenin pathways to coordinate the delicate balance between proliferation and differentiation during development.

Epidermal growth factor receptor and ligand family expression and activity in glioblastoma.

Epidermal growth factor family of receptor tyrosine kinases (ERBB) family cell surface receptors, including epidermal growth factor receptor (EGFR/ERBB1), are phosphorylated upon binding by various EGF family ligands and signal via multiple kinase pathways. EGFR signaling is enhanced because of mutational activation of EGFR in almost half of glioblastomas, the most common malignant primary brain tumor. Therapeutic targeting of EGFR in glioblastoma has remained largely unsuccessful. Here, we profiled nine long-term (LTC) and five glioma-initiating (GIC) cell lines for expression and activation of ERBB family receptors and expression of their ligands. Receptors and ligands were abundantly expressed, with patterns overall similar to glioblastoma expression profiles in vivo as deposited in The Cancer Genome Atlas database. No differences between LTC and GIC emerged. Irrespective of ligand or receptor expression, neither an EGFR antibody, erbitux, nor an EGFR tyrosine kinase inhibitor, gefitinib, were particularly active against LTC or GIC at clinically relevant concentrations. Self-renewal capacity of GIC was severely compromised by epidermal growth factor (EGF) withdrawal, but rescued by transforming growth factor alpha (TGF-α), although not by neuregulin-1 (NRG-1). Subcellular fractionation indicated high levels of nuclear phosphorylated EGFR in all LTC and GIC. In LN-229 cells, pERBB2 and pERBB3 were also detected in the nucleus. Nuclear pERBB2 was less sensitive, whereas pERBB3 was induced, in response to gefitinib. This study provides an extensive characterization of human glioma cell models, including stem-like models, with regard to ERBB receptor/ligand expression and signaling. Redundant signaling involving multiple ERBB family ligands and receptors may contribute to the challenges of developing more effective EGFR-targeted therapies for glioblastoma.

EGFR Downregulation after Anti-EGFR Therapy Predicts the Antitumor Effect in Colorectal Cancer.

Anti-EGFR mAb is reported to induce EGFR internalization in colorectal cancer cells. However, the biological relevance of EGFR internalization with anti-EGFR mAb is unknown. Therefore, the relevance of EGFR downregulation with anti-EGFR mAb to antitumor activity in colorectal cancer cells was investigated. Quantification of EGFR on the cell surface before cetuximab treatment was assessed by flow cytometry, and its growth-inhibitory effects were measured by Trypan blue exclusion, in 10 RAS, BRAF wild-type colorectal cancer cell lines, but there was no significant correlation between EGFR number and its growth-inhibitory effect. However, a significant correlation existed between the percentage decrease in the number of EGFRs after cetuximab treatment and its growth-inhibitory effect in those cell lines. Treatment with TGFα, a ligand for EGFR, induced EGFR internalization in colorectal cancer cells, but most EGFRs subsequently recycled to the cell surface, consistent with previous studies. While cetuximab treatment induced EGFR internalization, most receptors subsequently translocated into the late endosome, leading to lysosomal degradation, as revealed by immunoblotting and double immunofluorescence. Cetuximab-sensitive colorectal cancer cells showed greater EGFR internalization, stronger cell growth inhibition, and more augmented apoptotic signals than nonsensitive cells. IHC for EGFR, performed using an EGFR pharmDx Kit (mouse anti-human EGFR mAb clone 2-18C9), in clinical specimens before and after anti-EGFR mAb therapy in 13 colorectal cancer patients showed a significant correlation between the response to anti-EGFR mAb and decreased staining after therapy.Implications: This report clearly demonstrates that anti-EGFR mAb facilitates internalization and subsequent degradation of EGFRs in lysosomes, which is an important determinant of the efficacy of anti-EGFR mAb treatment for colorectal cancer. Mol Cancer Res; 15(10); 1445-54. ©2017 AACR.

PRAS40 Connects Microenvironmental Stress Signaling to Exosome-Mediated Secretion.

Secreted exosomes carrying lipids, proteins, and nucleic acids conduct cell-cell communications within the microenvironment of both physiological and pathological conditions. Exosome secretion is triggered by extracellular or intracellular stress signals. Little is known, however, about the signal transduction between stress cues and exosome secretion. To identify the linker protein, we took advantage of a unique finding in human keratinocytes. In these cells, although transforming growth factor alpha (TGF-α) and epidermal growth factor (EGF) share the same EGF receptor and previously indistinguishable intracellular signaling networks, only TGF-α stimulation causes exosome-mediated secretion. However, deduction of EGF-activated pathways from TGFα-activated pathways in the same cells allowed us to identify the proline-rich Akt substrate of 40 kDa (PRAS40) as the unique downstream effector of TGF-α but not EGF signaling via threonine 308-phosphorylated Akt. PRAS40 knockdown (KD) or PRAS40 dominant-negative (DN) mutant overexpression blocks not only TGF-α- but also hypoxia- and H2O2-induced exosome secretion in a variety of normal and tumor cells. Site-directed mutagenesis and gene rescue studies show that Akt-mediated activation of PRAS40 via threonine 246 phosphorylation is both necessary and sufficient to cause exosome secretion without affecting the endoplasmic reticulum/Golgi pathway. Identification of PRAS40 as a linker protein paves the way for understanding how stress regulates exosome secretion under pathophysiological conditions.

Effects of Growth Factors From Platelet-Rich Fibrin on the Bone Regeneration.

Platelet-rich fibrin (PRF) has simple manufacturing process without risk of immunologic rejection or infection by additional materials. This study was designed to investigate the effects of platelet-derived growth factors and transforming growth factors-ß in PRF on the proliferation and differentiation of osteoblasts. The authors analyzed platelet-derived growth factors and transforming growth factors-ß in normal human serum (NHS) and PRF by enzyme-linked immunosorbent assay quantitatively and those in PRF group were significantly increased when compared with NHS. After harvesting osteoblasts from iliac bone, the authors added 10% fetal bovine serum, 10% NHS, and 3% PRF to osteoblasts, respectively and incubated for 24, 48, 72 hours. For analysis of osteoblast proliferation and differentiation, the authors measured DNA synthesis by [3H]-thymidine isotope activity, protein synthesis by sulforhoamine B assay and those were increased according to elapsed time in all groups, but they were significantly increased in 3% PRF. The authors measured alkaline phosphatase activity of osteoblasts, it was increased according to elapsed to time in all groups, but significantly increased in 72 hours. In conclusion, application of PRF to bone defect sites may have an enhanced effect of bone regeneration with low risk of complications, and relatively simple manufacturing process.