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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-β) 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-β 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-β 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-β, advances in technology, and future perspectives., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

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.

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., (© 2023 John Wiley & Sons Ltd.)

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.

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., (© 2023 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

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-1β, TNF-α, insulin, growth hormone (GH), prostaglandin (PG)), and serotonin (5-HT)), while there are very few factors (e.g., TGF-β1 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.

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-β1) 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-β1-micelles/hydrogel. Moreover, it was observed that TGF-α and TGF-β1 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-β1-micelle/hydrogel-based delivery system provides a suitable alternative for anosmia treatment., (© 2022 American Institute of Chemical Engineers.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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-β 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-β 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-β. Our results indicate that Creb5 is a critical regulator of Prg4/lubricin expression in the articular cartilage.

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.

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., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

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 KRAS G13D 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.

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., (Copyright © 2018 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

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)/β-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 β-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/β-catenin pathways to coordinate the delicate balance between proliferation and differentiation during development.

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., (© 2018 International Society for Neurochemistry.)

Hepatocytes are responsive to mitogenic effects of several ligands acting via EGFR. Studying primary cultures of rat hepatocytes, we found that, as compared to EGF, HB-EGF had a markedly higher affinity of the EGFR, while AR and TGFα had lower affinity. HB-EGF was also more potent compared to the other growth factors regarding phosphorylation of EGFR, Shc, ERK1/2 and Akt. All ligands induced phosphorylation of ErbB2, indicating receptor heterodimerization. TGFα, despite having much lower receptor affinity, was about equally potent and efficacious as HB-EGF as a stimulator of DNA synthesis. In contrast, EGF had relatively high affinity but markedly lower efficacy in stimulation of DNA synthesis. The results suggest that amplifying and/or inhibitory mechanisms may modulate the mitogenic responses downstream of the initial signalling steps, and that this may affect the effects of the EGFR ligands differentially.

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 ., (©2017 American Association for Cancer Research.)

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 H 2 O 2 -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., (Copyright © 2017 American Society for Microbiology.)

Small heat shock proteins (HSPs) regulate a variety of cell functions. Among them, HSP22 and HSP20 are recognized to be ubiquitously expressed in various tissues. With regard to hepatocellular carcinoma (HCC) cells, we previously reported that phosphorylated HSP20 plays a suppressive role in transforming growth factor (TGF)-α-induced cell migration and invasion. In the present study, we investigated whether or not HSP22 is implicated in HCC cell migration. We detected HSP22 protein expression both in human HCC tumor (189.9±68.4ng/mg protein) and the adjacent non-tumor liver tissues (167.9±94.6ng/mg protein). The cases of low-quantity HSP22 protein level group (88.3≧ng/mg protein, the optimum cut-off value of HSP22) were increased in tumor tissues compared with the adjacent non-tumor tissues. The migration of human HCC-derived HuH-7 cells stimulated by TGF-α or hepatocyte growth factor (HGF) was significantly enhanced by the knockdown of HSP22 expression. Down-regulation of HSP22 protein in the cells markedly strengthened the AKT phosphorylation induced by TGF-α or HGF. Inhibitors of the phosphoinositide 3-kinase (PI3K)/AKT pathway, which suppressed the TGF-α-induced migration, significantly reduced the amplification by HSP22 knockdown. PI3K but not AKT was coimmunoprecipitated with HSP22 in HuH-7 cells. In addition, in human HCC tissues, a significantly lower HSP22 protein level in tumor tissues than in adjacent non-tumor tissues was observed more frequently in cases of moderately or poorly differentiated HCC than well-differentiated HCC. Taken together, our results strongly suggest that HSP22 represses HCC progression, especially HCC cell migration, by the down-regulation of the PI3K/AKT signaling pathway., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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-β in PRF on the proliferation and differentiation of osteoblasts. The authors analyzed platelet-derived growth factors and transforming growth factors-β 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.

Pulmonary fibrosis contributes to morbidity and mortality in a range of diseases, and there are no approved therapies for reversing its progression. To understand the mechanisms underlying pulmonary fibrosis and assess potential therapies, mouse models are central to basic and translational research. Unfortunately, metrics commonly used to assess murine pulmonary fibrosis require animals to be grouped and euthanized, increasing experimental difficulty and cost. We examined the ability of magnetic resonance imaging (MRI) to noninvasively assess lung fibrosis progression and resolution in a doxycycline (Dox) regulatable, transgenic mouse model that overexpresses transforming growth factor-α (TGF-α) under control of a lung-epithelial-specific promoter. During 7 wk of Dox treatment, fibrotic lesions were readily observed as high-signal tissue. Mean weighted signal and percent signal volume were found to be the most robust MRI-derived measures of fibrosis, and these metrics correlated significantly with pleural thickness, histology scores, and hydroxyproline content ( R  = 0.75-0.89). When applied longitudinally, percent high signal volume increased by 1.5% wk -1 ( P < 0.001) and mean weighted signal increased at a rate of 0.0065 wk -1 ( P = 0.0062). Following Dox treatment, lesions partially resolved, with percent high signal volume decreasing by -3.2% wk -1 ( P = 0.0034) and weighted mean signal decreasing at -0.015 wk -1 ( P = 0.0028). Additionally, longitudinal MRI revealed dynamic remodeling in a subset of lesions, a previously unobserved behavior in this model. These results demonstrate MRI can noninvasively assess experimental lung fibrosis progression and resolution and provide unique insights into its pathobiology., (Copyright © 2017 the American Physiological Society.)

Cells change their morphology as a response to environmental cues. The quantitative evaluation of single cell spread on extracellular matrices, such as type I collagen, is a key tool in cancer research. Inherent to the manual scoring of cellular spread is inter-observer but also intra-observer variation. To overcome these problems, we have developed the Morphology Analysis Software (MAS). MAS scores phase-contrast images of cells on native type I collagen gels and identifies whether a cell has a spread or round morphology using a combination of four unique parameters: the presence of a cellular extension, the cell area, the cell eccentricity and cell circularity. The MAS software scores are equivalent to the average score of five independent observers but MAS is faster, more objective and standardized. A functional screening assay using six cytokines identified TGFα as a stimulator of HCT8/E11 and SK-BR-3 single cell spreading on top of type I collagen gels. This change in morphology correlates with increased migration potential as evidenced by xCELLigence migration assays and are counteracted by EGFR signaling pathway inhibitors. This underscores the use of morphology classification on a population of unlabeled cells as read-out of an important cancer cell property and the potential for the MAS software in drug screening strategies., (Copyright © 2016. Published by Elsevier Inc.)

The role of the local microenvironment in influencing cell behavior is central to both normal development and cancer formation. Here, we show that sprouty 1 (SPRY1) modulates the microenvironment to enable proper mammary branching morphogenesis. This process occurs through negative regulation of epidermal growth factor receptor (EGFR) signaling in mammary stroma. Loss of SPRY1 resulted in up-regulation of EGFR-extracellular signal-regulated kinase (ERK) signaling in response to amphiregulin and transforming growth factor alpha stimulation. Consequently, stromal paracrine signaling and ECM remodeling is augmented, leading to increased epithelial branching in the mutant gland. By contrast, down-regulation of EGFR-ERK signaling due to gain of Sprouty function in the stroma led to stunted epithelial branching. Taken together, our results show that modulation of stromal paracrine signaling and ECM remodeling by SPRY1 regulates mammary epithelial morphogenesis during postnatal development., Competing Interests: The authors declare no conflict of interest.

The SOCS1 gene coding for suppressor of cytokine signaling 1 is frequently repressed in hepatocellular carcinoma (HCC), and hence SOCS1 is considered a tumor suppressor in the liver. However, the tumor-suppressor mechanisms of SOCS1 are not yet well understood. SOCS1 is known to inhibit pro-inflammatory cytokine production and signaling and to promote activation of the p53 tumor suppressor. However, we observed that SOCS1-deficient mice developed numerous and large liver tumor nodules following treatment with the hepatocarcinogen diethylnitrosamine (DEN) without showing increased interleukin-6 production or activation of p53. On the other hand, the livers of DEN-treated Socs1-null mice showed elevated levels of p21(CIP1/WAF1) protein (p21). Even though p21 generally functions as a tumor suppressor, paradoxically many cancers, including HCC, are known to express elevated levels of p21 that correlate with poor prognosis. We observed elevated p21 expression also in the regenerating livers of SOCS1-deficient mice and in cisplatin-treated Socs1-null hepatocytes, wherein the p21 protein showed increased stability. We show that SOCS1 interacts with p21 and promotes its ubiquitination and proteasomal degradation. Besides, the DEN-treated livers of Socs1-null mice showed increased nuclear and cytosolic p21 staining, and the latter was associated with growth factor-induced, phosphatidylinositol 3-kinase-dependent phosphorylation of p21 in SOCS1-deficient hepatocytes. Cytosolic p21 is often associated with malignancy and chemo-resistance in many cancers. Accordingly, SOCS1-deficient hepatocytes showed increased resistance to apoptosis that was reversed by shRNA-mediated p21 knockdown. In the regenerating livers of Socs1-null mice, increased p21 expression coincided with elevated cyclinD levels. Correspondingly, SOCS1-deficient hepatocytes showed increased proliferation to growth factor stimulation that was reversed by p21 knockdown. Overall, our findings indicate that the tumor-suppressor functions of SOCS1 in the liver could be mediated, at least partly, via regulation of the expression, stability and subcellular distribution of p21 and its paradoxical oncogenic functions, namely, resistance to apoptosis and increased proliferation.

Growth factors of the epidermal growth factor (EGF)/neuregulin family are involved in tumor progression and, accordingly, antibodies that intercept a cognate receptor, epidermal growth factor receptor (EGFR)/ERBB1, or a co-receptor, HER2, have been approved for cancer therapy. Although they might improve safety and delay onset of chemoresistance, no anti-ligand antibodies have been clinically approved. To identify suitable ligands, we surveyed fluids from ovarian and lung cancer patients and found that amphiregulin (AREG) is the most abundant and generalized ligand secreted by advanced tumors. AREG is a low affinity EGFR ligand, which is upregulated following treatment with chemotherapeutic drugs. Because AREG depletion retarded growth of xenografted ovarian tumors in mice, we generated a neutralizing monoclonal anti-AREG antibody. The antibody inhibited growth of ovarian cancer xenografts and strongly enhanced chemotherapy efficacy. Taken together, these results raise the possibility that AREG and other low- or high-affinity binders of EGFR might serve as potential targets for cancer therapy.

Binding of transforming growth factor α (TGF-α) to the epidermal growth factor receptor (EGFR) extracellular domain is encoded through the formation of a unique antiparallel coiled coil within the juxtamembrane segment. This new coiled coil is an "inside-out" version of the coiled coil formed in the presence of epidermal growth factor (EGF). A third, intermediary coiled-coil interface is formed in the juxtamembrane region when EGFR is stimulated with betacellulin. The seven growth factors that activate EGFR in mammalian systems (EGF, TGF-α, epigen, epiregulin, betacellulin, heparin-binding EGF, and amphiregulin) fall into distinct categories in which the structure of the coiled coil induced within the juxtamembrane region correlates with cell state. The observation that coiled-coil state tracks with the downstream signaling profiles for each ligand provides evidence for growth factor functional selectivity by EGFR. Encoding growth factor identity in alternative coiled-coil rotamers provides a simple and elegant method for communicating chemical information across the plasma membrane., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Increasing evidence shows that inflammation may contribute to the pathophysiology of depression. Apigenin, one type of natural flavone, has a number of biological actions including anti-inflammatory effects. Although it has potential antidepressant activity in a chronic mild stress model, the mechanisms of antidepressant effect for apigenin remain unclear. Here, we examined the effects of apigenin on lipopolysaccharide (LPS)-induced depressive-like behavior in male mice. A single administration of LPS (0.5mg/kg, i.p.) increased the immobility time in the tail suspension test (TST) and reduced sucrose preference without changing spontaneous locomotor activity in open field test (OFT). Pre-treatment with apigenin (25, 50mg/kg, i.p.) or fluoxetine (positive control drug, 20mg/kg, i.p.) once daily for 7 consecutive days prevented the abnormal behavior induced by LPS. Apigenin or fluoxetine also effectively attenuated LPS-induced production of pro-inflammatory cytokines IL-1β (interleukin-1β) and TNF-α (tumor necrosis factor-α). Moreover, apigenin or fluoxetine significantly suppressed the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression at both the mRNA and protein level via the modulation of nuclear factor-κB (NF-κB) activation in the prefrontal cortex. Additionally, apigenin (50mg/kg, i.p.) or fluoxetine (20mg/kg, i.p.) effectively reversed the depressive-like behavior induced by TNF-α (0.1fg/site, i.c.v.) without altering the locomotor activity. These results demonstrate that apigenin exhibits antidepressant-like effects in LPS treated mice, partially due to its anti-inflammatory properties., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Accumulating evidence suggests that heat shock proteins (HSPs) are implicated in progression of cancer. HSP22 (HSPB8), a small HSP, is recognized to be ubiquitously expressed in various tissues. However, the expression and the role of HSP22 in ovarian cancer remain to be clarified. In the present study, we investigated the involvement of HSP22 in transforming growth factor (TGF)-α-induced migration of ovarian cancer cells. The expression of HSP22 was detected in a serous ovarian cancer cell line, SKOV3.ip1. The migration was reduced by down-regulation of HSP22 expression. The TGF-α-induced migration was reduced by SB203580 (a p38 MAP kinase inhibitor), SP600125 (a SAPK/JNK inhibitor) and Y27632 (a Rho-kinase inhibitor). However, down-regulation of HSP22 had little effect on the TGF-α-induced phosphorylation of p38 MAP kinase, SAPK/JNK and MYPT, a target protein of Rho-kinase. The HSP22 expression was further analyzed in 20 resected specimens of human ovarian serous carcinoma. The expression of HSP22 was detected in all the twenty tissues (8.24-109.22 pg/mg protein), and the cases with highly expression of HSP22 showed a tendency to acquire the progressive ability. Our results strongly suggest that HSP22 acts as a positive regulator in TGF-α-induced migration of ovarian cancer cells, subsequently directing ovarian cancer toward progression., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The transdifferentiation of pancreatic acinar cells to a ductal phenotype (acinar-to-ductal metaplasia, ADM) occurs after injury or inflammation of the pancreas and is a reversible process. However, in the presence of activating Kras mutations or persistent epidermal growth factor receptor (EGF-R) signalling, cells that underwent ADM can progress to pancreatic intraepithelial neoplasia (PanIN) and eventually pancreatic cancer. In transgenic animal models, ADM and PanINs are initiated by high-affinity ligands for EGF-R or activating Kras mutations, but the underlying signalling mechanisms are not well understood. Here, using a conditional knockout approach, we show that protein kinase D1 (PKD1) is sufficient to drive the reprogramming process to a ductal phenotype and progression to PanINs. Moreover, using 3D explant culture of primary pancreatic acinar cells, we show that PKD1 acts downstream of TGFα and Kras, to mediate formation of ductal structures through activation of the Notch pathway.

Elucidating the mechanisms that underlie metastasis is of paramount importance to understanding tumor progression and to the development of novel therapeutics. Epithelial to Mesenchymal Transition (EMT) plays a vital role in tumor cell dissemination and is regulated by a core cassette of transcription factors. Despite recent advances, the molecular pathways that regulate the EMT program have not yet been fully delineated. We show that Siah ubiquitin ligases regulate Zeb1 protein, a key EMT transcription factor. The induction of EMT in breast cancer cells leads to the down-regulation of Siah, while the loss of Siah induces a mesenchymal phenotype, concurrent with an up-regulation of Zeb1. Overexpression of Siah in vitro mediates Zeb1 degradation, which can be blocked with a Siah peptide inhibitor. Thus, this work demonstrates that Siah is a novel regulator of EMT. This work is the first to identify a mechanism of post-translational regulation of the key Epithelial to Mesenchymal Transition transcription factor Zeb1.

Purpose: Targeted inhibition of EGFR with the mAbs cetuximab or panitumumab is a valuable treatment for RAS wild-type colorectal cancers. The efficacy of EGFR blockade is limited by the emergence of acquired resistance often attributed to secondary KRAS mutations. Remarkably, tumor biopsies from resistant patients show that only a fraction of the resilient cells carry KRAS mutations. We hypothesized that a paracrine cross-talk driven by the resistant subpopulation may provide in trans protection of surrounding sensitive cells., Experimental Design: Conditioned medium assays and three-dimensional cocultures were used to assess paracrine networks between cetuximab-sensitive and -resistant cells. Production of EGFR ligands by cells sensitive to cetuximab and panitumumab was measured. The ability of recombinant EGFR ligands to protect sensitive cells from cetuximab was assessed. Biochemical activation of the EGFR signaling pathway was measured by Western blotting., Results: Colorectal cancer cells sensitive to EGFR blockade can successfully grow despite cetuximab treatment when in the company of their resistant derivatives. Media conditioned by resistant cells protect sensitive parental cells from cetuximab. EGFR blockade triggers increased secretion of TGFα and amphiregulin. Increased secretion of ligands by resistant cells can sustain EGFR/ERK signaling in sensitive cells., Conclusions: Colorectal cancer cells that develop resistance to cetuximab and panitumumab secrete TGFα and amphiregulin, which protect the surrounding cells from EGFR blockade. This paracrine protective mechanism might be therapeutically exploitable., (©2014 American Association for Cancer Research.)

Bone marrow-derived mesenchymal stem cells (MSCs) contribute to breast cancer progression by releasing soluble factors that sustain tumor progression. MSCs express functional epidermal growth factor receptor (EGFR) and breast cancer cells secrete EGFR-ligands including transforming growth factor-α (TGFα). Using RNA-sequencing, we analysed the whole transcriptome of MSCs stimulated with TGFα. We identified 1,640 highly differentially regulated genes: 967 genes up-regulated with Fold Induction (FI) ≥ 1.50 and 673 genes down-regulated with FI ≤ 0.50. When highly regulated genes were categorized according to GO molecular function classification and KEGG pathways analysis, a large number of genes coding for potentially secreted proteins or surface receptors resulted enriched following TGFα treatment, including VEGFA, IL6, EREG, HB-EGF, LIF, NGF, NRG1, CCL19, CCL2, CCL25 and CXCL3. Secretion of corresponding proteins was confirmed for selected factors. Finally, we identified 4,377 and 4,262 alternatively spliced genes in untreated and TGFα-treated MSCs, respectively. Among these, an unannotated splice variant of VEGFA coding for a secreted VEGF protein of 172 amino acids (VEGFA(172)), was found only in MSCs stimulated with TGFα. These findings suggest that EGFR activation in MSCs leads to a significant change in the expression of a wide array of genes coding for secreted proteins that can significantly enhance tumor progression.

Aberrant epidermal growth factor receptor (EGFR) activation is associated with ovarian cancer progression. In this study, we report that the EGFR ligand amphiregulin (AREG) stimulates cell invasion and down-regulates E-cadherin expression in two human ovarian cancer cell lines, SKOV3 and OVCAR5. In addition, AREG increases the expression of transcriptional repressors of E-cadherin including SNAIL, SLUG and ZEB1. siRNA targeting SNAIL or SLUG abolishes AREG-induced cell invasion. Moreover, ERK1/2 and AKT pathways are involved in AREG-induced E-cadherin down-regulation and cell invasion. Finally, we show that three EGFR ligands, AREG, epidermal growth factor (EGF) and transforming growth factor-α (TGF-α), exhibit comparable effects in down-regulating E-cadherin and promoting cell invasion. This study demonstrates that AREG induces ovarian cancer cell invasion by down-regulating E-cadherin expression., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

In a fraction of patients surgically treated for cleft lip/palate, excessive scarring disturbs maxillary growth and dento-alveolar development. Since certain genes are involved in craniofacial morphogenesis as well as tissue repair, a primary defect causing cleft lip/palate could lead to altered wound healing. We performed in vitro wound healing assays with primary lip fibroblasts from 16 cleft lip/palate patients. Nine foreskin fibroblast strains were included for comparison. Cells were grown to confluency and scratch wounds were applied; wound closure was monitored morphometrically over time. Wound closure rate showed highly significant differences between fibroblast strains. Statistically, fibroblast strains from the 25 individuals could be divided into three migratory groups, namely "fast", "intermediate", and "slow". Most cleft lip/palate fibroblasts were distributed between the "fast" (5 strains) and the "intermediate" group (10 strains). These phenotypes were stable over different cell passages from the same individual. Expression of genes involved in cleft lip/palate and wound repair was determined by quantitative PCR. Transforming growth factor-α mRNA was significantly up-regulated in the "fast" group. 5 ng/ml transforming growth factor-α added to the culture medium increased the wound closure rate of cleft lip/palate strains from the "intermediate" migratory group to the level of the "fast", but had no effect on the latter group. Conversely, antibody to transforming growth factor-α or a specific inhibitor of its receptor most effectively reduced the wound closure rate of "fast" cleft lip/palate strains. Thus, fibroblasts from a distinct subgroup of cleft lip/palate patients exhibit an increased migration rate into wounds in vitro, which is linked to higher transforming growth factor-α expression and attenuated by interfering with its signaling.

Transforming growth factor-α (TGF-α) is upregulated in advanced stages of prostate cancer and strongly correlated with metastasis. However, the effect of TGF-α on epithelial-mesenchymal transition (EMT) in prostate cancer and the underlying mechanisms remain unclear. Recently, microRNAs have emerged as new regulators of EMT. This study found that treatment of DU145 cells with TGF-α suppressed the expression of epithelial marker E-cadherin and increased the expression of mesenchymal marker Vimentin as well as changed the cell morphology from cobblestone shape to spindle shape. The level of miR-124 was downregulated by TGF-α in several different cancer cell lines. Enforced expression of miR-124 abolished TGF-α-induced EMT. Slug was proven to be a target of miR-124 and mediated the inhibitory effect of miR-124 on TGF-α-induced EMT. Furthermore, overexpression of miR-124 reduced the migratory and invasive capacity of TGF-α-treated DU145 cells. In conclusion, our findings suggest that miR-124 inhibits TGF-α-induced EMT in DU145 cells by targeting Slug. Thus, miR-124 may be a potential target for prostate cancer therapeutic intervention.

Alectinib is a new generation ALK inhibitor with activity against the gatekeeper L1196M mutation that showed remarkable activity in a phase I/II study with echinoderm microtubule associated protein-like 4 (EML4)--anaplastic lymphoma kinase (ALK) non-small cell lung cancer (NSCLC) patients. However, alectinib resistance may eventually develop. Here, we found that EGFR ligands and HGF, a ligand of the MET receptor, activate EGFR and MET, respectively, as alternative pathways, and thereby induce resistance to alectinib. Additionally, the heat shock protein 90 (Hsp90) inhibitor suppressed protein expression of ALK, MET, EGFR, and AKT, and thereby induced apoptosis in EML4-ALK NSCLC cells, even in the presence of EGFR ligands or HGF. These results suggest that Hsp90 inhibitors may overcome ligand-triggered resistance to new generation ALK inhibitors and may result in more successful treatment of NSCLC patients with EML4-ALK.

A number of growth factors and signaling pathways regulate matrix deposition and fibroblast proliferation in the lung. The epidermal growth factor receptor (EGFR) family of receptors and the transforming growth factor-β (TGF-β) family are active in diverse biological processes and are central mediators in the initiation and maintenance of fibrosis in many diseases. Transforming growth factor-α (TGF-α) is a ligand for the EGFR, and doxycycline (Dox)-inducible transgenic mice conditionally expressing TGF-α specifically in the lung epithelium develop progressive fibrosis accompanied with cachexia, changes in lung mechanics, and marked pleural thickening. Although recent studies demonstrate that EGFR activation modulates the fibroproliferative effects involved in the pathogenesis of TGF-β induced pulmonary fibrosis, in converse, the direct role of EGFR induction of the TGF-β pathway in the lung is unknown. The αvβ6 integrin is an important in vivo activator of TGF-β activation in the lung. Immunohistochemical analysis of αvβ6 protein expression and bronchoalveolar analysis of TGF-β pathway signaling indicates activation of the αvβ6/TGF-β pathway only at later time points after lung fibrosis was already established in the TGF-α model. To determine the contribution of the αvβ6/TGF-β pathway on the progression of established fibrotic disease, TGF-α transgenic mice were administered Dox for 4 wk, which leads to extensive fibrosis; these mice were then treated with a function-blocking anti-αvβ6 antibody with continued administration of Dox for an additional 4 wk. Compared with TGF-α transgenic mice treated with control antibody, αvβ6 inhibition significantly attenuated pleural thickening and altered the decline in lung mechanics. To test the effects of genetic loss of the β6 integrin, TGF-α transgenic mice were mated with β6-null mice and the degree of fibrosis was compared in adult mice following 8 wk of Dox administration. Genetic ablation of the β6 integrin attenuated histological and physiological changes in the lungs of TGF-α transgenic mice although a significant degree of fibrosis still developed. In summary, inhibition of the β6 integrin led to a modest, albeit significant, effect on pleural thickening and lung function decline observed with TGF-α-induced pulmonary fibrosis. These data support activation of the αvβ6/TGF-β pathway as a secondary effect contributing to TGF-α-induced pleural fibrosis and suggest a complex contribution of multiple mediators to the maintenance of progressive fibrosis in the lung.

Our previous data demonstrated that type II cGMP‑dependent protein kinase (PKG II) inhibited epidermal growth factor (EGF)-induced MAPK/ERK/JNK‑mediated signal transduction through inhibiting the phosphorylation/activation of the epidermal growth factor receptor (EGFR). Since the EGFR also binds with several other ligands as well as EGF, the present study was designed to investigate whether PKG II inhibited transforming growth factor-α (TGF-α), betacellulin (BTC) and epiregulin (EPR) induced phosphorylation/activation of the EGFR and consequent MAPK/ERK‑mediated signaling. The human gastric cancer cell line AGS, was infected with adenoviral constructs encoding cDNA of PKG II (Ad-PKG II) to increase the expression of PKG II and was treated with 8-pCPT-cGMP to activate the kinase. Western blotting was applied to detect the phosphorylation of EGFR and MAPK/ERK. The results demonstrated that treatment with EGF (100 ng/ml, 5 min), TGF-α (100 ng/ml, 5 min), BTC (100 ng/ml, 5 min) and EPR (100 ng/ml, 5 min) increased the tyrosine (tyr) 1068 phosphorylation of the EGFR and the threonine (thr) 202/tyr 204 phosphorylation of MAPK/ERK. Infecting the cells with Ad-PKG II and stimulating the kinase with 8-pCPT-cGMP efficiently inhibited the phosphorylation of the EGFR and MAPK/ERK induced by EGF, TGF-α, BTC and EPR. The results indicated that PKG II also inhibits the activation of the EGFR caused by diverse ligands of the receptor.

The complement system is activated in response to tissue injury. During wound healing, complement activation seems beneficial in acute wounds but may be detrimental in chronic wounds. We found that the epidermal expression of many complement components was only increased to a minor extent in skin wounds in vivo and in cultured keratinocytes after exposure to supernatant from stimulated mononuclear cells. In contrast, the epidermal expression of complement components was downregulated in ex vivo injured skin lacking the stimulation from infiltrating inflammatory cells but with intact injury-induced epidermal growth factor receptor (EGFR)-mediated growth factor response. In cultured primary keratinocytes, stimulation with the potent EGFR ligand, TGF-α, yielded a significant downregulation of complement component expression. Indeed, EGFR inhibition significantly enhanced the induction of complement components in keratinocytes and epidermis following stimulation with proinflammatory cytokines. Importantly, EGFR inhibition of cultured keratinocytes either alone or in combination with proinflammatory stimulus promoted activation of the complement system after incubation with serum. In keratinocytes treated solely with the EGFR inhibitor, complement activation was dependent on serum-derived C1q, whereas in keratinocytes stimulated with a combination of proinflammatory cytokines and EGFR inhibition, complement activation was found even with C1q-depleted serum. In contrast to human keratinocytes, EGFR inhibition did not enhance complement component expression or cause complement activation in murine keratinocytes. These data demonstrate an important role for EGFR in regulating the expression of complement components and complement activation in human epidermis and keratinocytes and, to our knowledge, identify for the first time a pathway important for the epidermal regulation of complement activation.

Based on gene expression patterns, breast cancers can be divided into subtypes that closely resemble various developmental stages of normal mammary epithelial cells (MECs). Thus, understanding molecular mechanisms of MEC development is expected to provide critical insights into initiation and progression of breast cancer. Epidermal growth factor receptor (EGFR) and its ligands play essential roles in normal and pathological mammary gland. Signals through EGFR is required for normal mammary gland development. Ligands for EGFR are over-expressed in a significant proportion of breast cancers, and elevated expression of EGFR is associated with poorer clinical outcome. In the present study, we examined the effect of signals through EGFR on MEC differentiation using the human telomerase reverse transcriptase (hTERT)-immortalized human stem/progenitor MECs which express cytokeratin 5 but lack cytokeratin 19 (K5(+)K19(-) hMECs). As reported previously, these cells can be induced to differentiate into luminal and myoepithelial cells under appropriate culture conditions. K5(+)K19(-) hMECs acquired distinct cell fates in response to EGFR ligands epidermal growth factor (EGF), amphiregulin (AREG) and transforming growth factor alpha (TGFα) in differentiation-promoting MEGM medium. Specifically, presence of EGF during in vitro differentiation supported development into both luminal and myoepithelial lineages, whereas cells differentiated only towards luminal lineage when EGF was replaced with AREG. In contrast, substitution with TGFα led to differentiation only into myoepithelial lineage. Chemical inhibition of the MEK-Erk pathway, but not the phosphatidylinositol 3-kinase (PI3K)-AKT pathway, interfered with K5(+)K19(-) hMEC differentiation. The present data validate the utility of the K5(+)K19(-) hMEC cells for modeling key features of human MEC differentiation. This system should be useful in studying molecular/biochemical mechanisms of human MEC differentiation.

Objective: To observe the effects of hepatocyte growth factor (HGF) derived from tumor microenvironment and/or afatinib on the growth of human lung adenocarcinoma H1975 cells and explore the potential mechanisms by which HGF induces primary resistance to afatinib., Methods: The effects of HGF, TGF-α and afatinib on the growth of H1975 cells were evaluated by MTT assay. The HGF concentrations of normal human fetal lung fibroblasts MRC-5 cells and human lung adenocarcinoma H1975 cells co-cultured or separately cultured were determined by ELISA assay. Western blot was used to detect the expressions of EGFR and Met signal pathway-related proteins and epithelial-mesenchymal transition (EMT) markers in H1975 cells treated with HGF and/or afatinib., Results: The MTT assay showed that H1975 cells were hyposensitive to afatinib in the presence of HGF. The ELISA assay showed that HGF production by H1975 cells was less than 0.1 ng/2.0×10(6) cells, but HGF production by MRC-5 cells was (151.37 ± 2.07)ng/2.0×10(6) cells incubated for 48 h. When H1975 cells and MRC-5 cells were co-cultured for 72 h, the concentration of HGF in the culture supernatant was (61.13 ± 16.21)ng/ml. In the presence of HGF, the expression of p-Met, p-Akt and p-ERK proteins in the H1975 cells was markedly up-regulated. afatinib inhibited p-EGFR, but did not affect the expression of p-Met, p-Akt and p-ERK proteins. In the presence of afatinib, HGF up-regulated the expression of vimentin and down-regulated the expression of E-cadherin., Conclusions: HGF secreted by stromal cells in the tumor micro-environment may confer resistance to afatinib in H1975 cells by activation of the Met/PI3K/Akt and Met/MAPK/ERK signaling pathways, and is involved in the epithelial-mesenchymal transition process.

Keratinocyte migration during re-epithelization is crucial in wound healing under biochemical and biomechanical microenvironment. However, little is known about the underlying mechanisms whereby mechanical tension and cocultured fibroblasts or keratinocytes modulate the migration of keratinocytes or fibroblasts. Here we applied a tensile device together with a modified transwell assay to determine the lateral and transmembrane migration dynamics of human HaCaT keratinocytes or HF fibroblasts. A novel pattern of asymmetric migration was observed for keratinocytes when they were cocultured with non-contact fibroblasts, i.e., the accumulative distance of HaCaT cells was significantly higher when moving away from HF cells or migrating from down to up cross the membrane than that when moving close to HF cells or when migrating from up to down, whereas HF migration was symmetric. This asymmetric migration was mainly regulated by EGF derived from fibroblasts, but not transforming growth factor α or β1 production. Mechanical stretch subjected to fibroblasts fostered keratinocyte asymmetric migration by increasing EGF secretion, while no role of mechanical stretch was found for EGF secretion by keratinocytes. These results provided a new insight into understanding the regulating mechanisms of two- or three-dimensional migration of keratinocytes or fibroblasts along or across dermis and epidermis under biomechanical microenvironment.

The biological functions of recombinant human follicle-stimulating hormone (FSH) and transforming growth factor-α (TGF-α) on in vitro maturation of porcine oocytes were investigated. Cumulus-oocyte complexes were matured in defined porcine oocyte medium containing 0-0.1 IU/ml FSH in the presence or absence of 10 ng/ml TGF-α. The percentage of oocytes reaching metaphase II was significantly higher with the addition of 0.01-0.1 IU/ml FSH compared with no addition, and was further enhanced in the presence of TGF-α. The rates of sperm penetration and blastocyst formation were significantly higher with the addition of 0.05-0.1 IU/ml FSH compared with no addition after in vitro fertilization and embryo culture. There was no beneficial effect of FSH and TGF-α on nuclear maturation of denuded oocytes. The specific EGF receptor inhibitor, AG1478, completely inhibited TGF-α-induced meiotic resumption, but did not completely prevent the stimulatory effect of FSH. Addition of both FSH and TGF-α significantly enhanced cumulus expansion compared with no addition. When cumulus expansion-related genes (HAS2, HAPLN1, and VCAN) mRNA expression in COCs was measured during in vitro maturaiton, addition of both of FSH and TGF-α upregulated the expression of HAS2 mRNA after 20 hr culture and HAPLN1 mRNA after 44 hr culture compared with no addition. Expression of VCAN mRNA was significantly higher in the presence of FSH compared with addition of TGF-α alone. These results suggest that FSH and TGF-α synergistically enhance porcine oocyte maturation via cumulus cells, and act through different signaling pathways., (Copyright © 2013 Wiley Periodicals, Inc.)

In the hen ovary, selection of a follicle into the preovulatory hierarchy occurs from a small cohort of prehierarchal (6-8 mm) follicles. Prior to follicle selection the granulosa layer remains in an undifferentiated state despite elevated follicle-stimulating hormone receptor (FSHR) expression. The present studies describe a role for bone morphogenetic protein 4 (BMP4) in supporting FSHR mRNA expression in granulosa cells from prehierarchal follicles and promoting differentiation at follicle selection. Culture of undifferentiated granulosa cells in culture medium alone resulted in a significant decline in levels of FSHR mRNA (by ~80% compared to freshly collected cells). By comparison, granulosa cultured with BMP4 (10-100 ng/ml) maintained FSHR and expression at approximately in vivo levels. Because both granulosa and theca tissues from prehierarchal follicles express BMP4, it is suggested that BMP4 acts in a paracrine and/or autocrine fashion to support elevated FSHR expression prior to follicle selection. Granulosa cells cultured with BMP4 for 24 h also initiated FSH-induced cAMP production and indirectly initiated anti-Mullerian hormone (AMH), CYP11A, and STAR expression plus progesterone production. However, pretreatment with the BMP antagonist NOGGIN or the mitogen-activated protein kinase (MAPK) agonist transforming growth factor alpha attenuated or blocked each action promoted by BMP4. We conclude that prior to and immediately after selection, BMP4 serves to support FSHR expression within the granulosa layer, yet prior to selection, multiple factors (including inhibitory MAPK signaling, AMH, and BMP antagonists) can modulate FSHR expression and suppress FSH-mediated cell signaling to prevent granulosa cell differentiation prior to follicle selection.

The inhibition of the Ras/mitogen-activated protein kinase (Ras/MAPK) pathway through the suppression of mutated Ras or MAPK/extracellular signal-regulated kinase 1/2 (MEK1/2) has been shown to sensitize tumor cells to ionizing radiation (IR). The molecular mechanisms of this sensitization however, are not yet fully understood. In this study, we investigated the role of transforming growth factor-α (TGF-α) in the radiosensitizing effects of selumetinib, a selective inhibitor of MEK1/2. The expression of epidermal growth factor receptor (EGFR) ligands was assessed by ELISA in both Ras wild-type and Ras mutant cells that were exposed to radiation with or without selumetinib. The effects of selumetinib on the TGF-α/EGFR signaling cascade in response to radiation were examined by western blot analysis, clonogenic assay and by determing the yield of mitotic catastrophe. The treatment of cells with selumetinib reduced the basal and IR-induced secretion of TGF-α in both Ras wild-type and Ras mutant cell lines in vitro and in vivo. The reduction of TGF-α secretion was accompanied with a reduction in phosphorylated tumor necrosis factor-α converting enzyme (TACE) in the cells treated with selumetinib with or without IR. The treatment of cells with selumetinib with or without IR inhibited the phosphorylation of EGFR and checkpoint kinase 2 (Chk2), and reduced the expression of survivin. Supplementation with exogenous TGF-α partially rescued the selumetinib-treated cells from IR-induced cell death, restored EGFR and Chk2 phosphorylation and increased survivin expression. These data suggest that the inhibition of MEK1/2 with selumetinib may provide a mechanism to sensitize tumor cells to IR in a fashion that prevents the activation of the TGF-α autocrine loop following IR.

The receptor tyrosine kinase (RTK) Met is known to be over-expressed in canine osteosarcoma (OSA). In human cancers, the RTKs Met, epidermal growth factor receptor (EGFR) and Ron are frequently co-expressed and engage in heterodimerization, altering signal transduction and promoting resistance to targeted therapeutics. We found that EGFR and Ron are expressed in canine OSA cell lines and primary tissues, EGFR and Ron are frequently phosphorylated in OSA tumour samples, and Met is co-associated with EGFR and Ron in canine OSA cell lines. Transforming growth factor alpha (TGFα) and hepatocyte growth factor (HGF) stimulation induced amplification of ERK1/2 and STAT3 phosphorylation in OSA cells and Met was phosphorylated following TGFα stimulation providing evidence for receptor cross-talk. Lastly, treatment of OSA cells with combined gefitinib and crizotinib inhibited cell proliferation in an additive manner. Together, these data support the notion that Met, EGFR and Ron interact in OSA cells and as such, may represent viable targets for therapeutic intervention., (© 2011 John Wiley & Sons Ltd.)

Tympanic membrane perforations are common and represent a management challenge to clinicians. Current treatments for chronic perforations involve a graft surgery and require general anaesthesia, including associated costs and morbidities. Bioactive molecules (e.g. growth factors, cytokines) play an important role in promoting TM wound healing following perforation and the use of growth factors as a topical treatment for tympanic membrane perforations has been suggested as an alternative to surgery. However, the choice of bioactive molecules best suited to promote wound healing has yet to be identified. We investigated the effects of hyaluronic acid, vitronectin, TGF-α, IL-24 and their combinations on migration, proliferation and adhesion of cultured human tympanic membrane-derived keratinocytes (hTM), in addition to their possible mechanisms of action. We found that TGF-α, TGF-α/HA and TGF-α/IL-24 promoted wound healing by significantly increasing both migration and proliferation. TGF-α and/or HA treated cells showed comparable cell-cell adhesion whilst maintaining an epithelial cell phenotype. With the use of receptor binding inhibitors for ErbB1 (AG1478) and CD44 (BRIC235), we revealed that the activation of ErbB1 is required for TGF-α/HA-mediated migration and proliferation. These results suggest factors that may be incorporated into a tissue-engineered membrane or directly as topical treatment for tympanic membrane perforations and hence reduce the need for a surgery., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Foregut neuroendocrine tumors [NETs] usually pursuit a benign course, but some show aggressive behavior. The treatment of patients with advanced NETs is marginally effective and new approaches are needed. In other tumors, transactivation of the EGF receptor (EGFR) by growth factors, gastrointestinal (GI) hormones and lipids can stimulate growth, which has led to new treatments. Recent studies show a direct correlation between NET malignancy and EGFR expression, EGFR inhibition decreases basal NET growth and an autocrine growth effect exerted by GI hormones, for some NETs. To determine if GI hormones can stimulate NET growth by inducing transactivation of EGFR, we examined the ability of EGF, TGFα and various GI hormones to stimulate growth of the human foregut carcinoid,BON, the somatostatinoma QGP-1 and the rat islet tumor,Rin-14B-cell lines. The EGFR tyrosine-kinase inhibitor, AG1478 strongly inhibited EGF and the GI hormones stimulated cell growth, both in BON and QGP-1 cells. In all the three neuroendocrine cell lines studied, we found EGF, TGFα and the other growth-stimulating GI hormones increased Tyr(1068) EGFR phosphorylation. In BON cells, both the GI hormones neurotensin and a bombesin analogue caused a time- and dose-dependent increase in EGFR phosphorylation, which was strongly inhibited by AG1478. Moreover, we found this stimulated phosphorylation was dependent on Src kinases, PKCs, matrix metalloproteinase activation and the generation of reactive oxygen species. These results raise the possibility that disruption of this signaling cascade by either EGFR inhibition alone or combined with receptor antagonists may be a novel therapeutic approach for treatment of foregut NETs/PETs., (Published by Elsevier B.V.)