Lack of collagen α6(IV) chain in mice does not cause severe-to-profound hearing loss or cochlear malformation, a distinct phenotype from nonsyndromic hearing loss with COL4A6 missense mutation.

Congenital hearing loss affects 1 in every 1000 births, with genetic mutations contributing to more than 50% of all cases. X-linked nonsyndromic hereditary hearing loss is associated with six loci (DFNX1-6) and five genes. Recently, the missense mutation (c.1771G>A, p.Gly591Ser) in COL4A6, encoding the basement membrane (BM) collagen α6(IV) chain, was shown to be associated with X-linked congenital nonsyndromic hearing loss with cochlear malformation. However, the mechanism by which the COL4A6 mutation impacts hereditary hearing loss has not yet been elucidated. Herein, we investigated Col4a6 knockout (KO) effects on hearing function and cochlear formation in mice. Immunohistochemistry showed that the collagen α6(IV) chain was distributed throughout the mouse cochlea within subepithelial BMs underlying the interdental cells, inner sulcus cells, basilar membrane, outer sulcus cells, root cells, Reissner's membrane, and perivascular BMs in the spiral limbus, spiral ligament, and stria vascularis. However, the click-evoked auditory brainstem response analysis did not show significant changes in the hearing threshold of Col4a6 KO mice compared with wild-type (WT) mice with the same genetic background. In addition, the cochlear structures of Col4a6 KO mice did not exhibit morphological alterations, according to the results of high-resolution micro-computed tomography and histology. Hence, loss of Col4a6 gene expression in mice showed normal click ABR thresholds and normal cochlear formation, which differs from humans with the COL4A6 missense mutation c.1771G>A, p.Gly591Ser. Therefore, the deleterious effects in the auditory system caused by the missense mutation in COL4A6 are likely due to the dominant-negative effects of the α6(IV) chain and/or α5α6α5(IV) heterotrimer with an aberrant structure that would not occur in cases with loss of gene expression.

Nanog is a promising chemoresistant stemness marker and therapeutic target by iron chelators for esophageal cancer.

Esophageal cancer is a disease showing poor prognosis. Although combination chemotherapy using cisplatin (CDDP) and 5-fluorouracil is standard for unresectable esophageal cancer, the response rate is 35%. Cancer stem cells (CSCs) and inflammation are reportedly responsible for the poor prognosis of esophageal cancer. However, comprehensive analyses have not been conducted and proposals for progress remain lacking. Iron is known to be a key factor in the stemness of CSCs. Our study focused on the therapeutic potential of iron control using iron chelators for CSCs in esophageal cancer. Among 134 immunohistochemically analyzed cases, Nanog expression was high in 98 cases and low in 36 cases. High Nanog expression correlated with low overall and disease-free survivals. The iron chelators deferasirox (DFX) and SP10 suppressed the proliferation and expression of stemness markers in TE8 and OE33 cells. DFX and SP10 did not induce compensatory interleukin (IL)-6 secretion, although CDDP did result in high induction. Moreover, BBI608 and SSZ, as other CSC-targeting drugs, could not suppress the expression of stemness markers. Overall, Nanog expression appears related to poor prognosis in esophageal cancer patients, and inhibition of stemness and compensatory IL-6 secretion by iron chelators may offer a novel therapeutic strategy for esophageal cancer.

Pathological application of carbocyanine dye-based multicolour imaging of vasculature and associated structures.

Simultaneous visualisation of vasculature and surrounding tissue structures is essential for a better understanding of vascular pathologies. In this work, we describe a histochemical strategy for three-dimensional, multicolour imaging of vasculature and associated structures, using a carbocyanine dye-based technique, vessel painting. We developed a series of applications to allow the combination of vessel painting with other histochemical methods, including immunostaining and tissue clearing for confocal and two-photon microscopies. We also introduced a two-photon microscopy setup that incorporates an aberration correction system to correct aberrations caused by the mismatch of refractive indices between samples and immersion mediums, for higher-quality images of intact tissue structures. Finally, we demonstrate the practical utility of our approach by visualising fine pathological alterations to the renal glomeruli of IgA nephropathy model mice in unprecedented detail. The technical advancements should enhance the versatility of vessel painting, offering rapid and cost-effective methods for vascular pathologies.

Development of an exon skipping therapy for X-linked Alport syndrome with truncating variants in COL4A5.

Currently, there are no treatments for Alport syndrome, which is the second most commonly inherited kidney disease. Here we report the development of an exon-skipping therapy using an antisense-oligonucleotide (ASO) for severe male X-linked Alport syndrome (XLAS). We targeted truncating variants in exon 21 of the COL4A5 gene and conducted a type IV collagen α3/α4/α5 chain triple helix formation assay, and in vitro and in vivo treatment efficacy evaluation. We show that exon skipping enabled trimer formation, leading to remarkable clinical and pathological improvements including expression of the α5 chain on glomerular and the tubular basement membrane. In addition, the survival period was clearly prolonged in the ASO treated mice group. This data suggests that exon skipping may represent a promising therapeutic approach for treating severe male XLAS cases.

Collagen XVIII Deposition in the Basement Membrane Zone beneath the Newly Forming Epidermis during Wound Healing in Mice.

The basement membrane (BM) is composed of various extracellular molecules and regulates tissue regeneration and maintenance. Here, we demonstrate that collagen XVIII was spatiotemporally expressed in the BM during skin wound healing in a mouse excisional wound-splinting model. Re-epithelialization was detected at days 3 and 6 post-wounding. The ultrastructure of epidermal BM was discontinuous at day 3, whereas on day 6 a continuous BM was observed in the region proximal to the wound edge. Immunohistochemistry demonstrated that collagen XVIII was deposited in the BM zone beneath newly forming epidermis in day 3 and 6 wounds. Laminin-332, known to be the earliest BM component appearing in wounds, was colocalized with collagen XVIII in the epidermal BM zone at days 3 and 6. The deposition of α1(IV) collagen and nidogen-1 in the epidermal BM zone occurred later than that of collagen XVIII. We also observed the short isoform of collagen XVIII in the epidermal BM zone at day 3 post-wounding. Collectively, our results suggested that collagen XVIII plays a role in the formation of the dermal-epidermal junction during re-epithelialization, and that it is the short isoform that is involved in the early phase of re-epithelialization.

A Novel Combination Cancer Therapy with Iron Chelator Targeting Cancer Stem Cells via Suppressing Stemness.

Excess iron causes cancer and is thought to be related to carcinogenesis and cancer progression including stemness, but the details remain unclear. Here, we hypothesized that stemness in cancer is related to iron metabolism and that regulating iron metabolism in cancer stem cells (CSCs) may be a novel therapy. In this study, we used murine induced pluripotent stem cells that expressed specific stem cell genes such as Nanog, Oct3/4, Sox2, Klf4, and c-Myc, and two human cancer cell lines with similar stem cell gene expression. Deferasirox, an orally available iron chelator, suppressed expression of stemness markers and spherogenesis of cells with high stemness status in vitro. Combination therapy had a marked antitumor effect compared with deferasirox or cisplatin alone. Iron metabolism appears important for maintenance of stemness in CSCs. An iron chelator combined with chemotherapy may be a novel approach via suppressing stemness for CSC targeted therapy.

Biallelic COLGALT1 variants are associated with cerebral small vessel disease.

OBJECTIVE: Approximately 5% of cerebral small vessel diseases are hereditary, which include COL4A1/COL4A2-related disorders. COL4A1/COL4A2 encode type IV collagen α1/2 chains in the basement membranes of cerebral vessels. COL4A1/COL4A2 mutations impair the secretion of collagen to the extracellular matrix, thereby resulting in vessel fragility. The diagnostic yield for COL4A1/COL4A2 variants is around 20 to 30%, suggesting other mutated genes might be associated with this disease. This study aimed to identify novel genes that cause COL4A1/COL4A2-related disorders. METHODS: Whole exome sequencing was performed in 2 families with suspected COL4A1/COL4A2-related disorders. We validated the role of COLGALT1 variants by constructing a 3-dimensional structural model, evaluating collagen ß (1-O) galactosyltransferase 1 (ColGalT1) protein expression and ColGalT activity by Western blotting and collagen galactosyltransferase assays, and performing in vitro RNA interference and rescue experiments. RESULTS: Exome sequencing demonstrated biallelic variants in COLGALT1 encoding ColGalT1, which was involved in the post-translational modification of type IV collagen in 2 unrelated patients: c.452 T > G (p.Leu151Arg) and c.1096delG (p.Glu366Argfs*15) in Patient 1, and c.460G > C (p.Ala154Pro) and c.1129G > C (p.Gly377Arg) in Patient 2. Three-dimensional model analysis suggested that p.Leu151Arg and p.Ala154Pro destabilized protein folding, which impaired enzymatic activity. ColGalT1 protein expression and ColGalT activity in Patient 1 were undetectable. RNA interference studies demonstrated that reduced ColGalT1 altered COL4A1 secretion, and rescue experiments showed that mutant COLGALT1 insufficiently restored COL4A1 production in cells compared with wild type. INTERPRETATION: Biallelic COLGALT1 variants cause cerebral small vessel abnormalities through a common molecular pathogenesis with COL4A1/COL4A2-related disorders. Ann Neurol 2018;84:843-853.

Hair follicle epidermal stem cells define a niche for tactile sensation.

The heterogeneity and compartmentalization of stem cells is a common principle in many epithelia, and is known to function in epithelial maintenance, but its other physiological roles remain elusive. Here we show transcriptional and anatomical contributions of compartmentalized epidermal stem cells in tactile sensory unit formation in the mouse hair follicle. Epidermal stem cells in the follicle upper-bulge, where mechanosensory lanceolate complexes innervate, express a unique set of extracellular matrix (ECM) and neurogenesis-related genes. These epidermal stem cells deposit an ECM protein called EGFL6 into the collar matrix, a novel ECM that tightly ensheathes lanceolate complexes. EGFL6 is required for the proper patterning, touch responses, and αv integrin-enrichment of lanceolate complexes. By maintaining a quiescent original epidermal stem cell niche, the old bulge, epidermal stem cells provide anatomically stable follicle-lanceolate complex interfaces, irrespective of the stage of follicle regeneration cycle. Thus, compartmentalized epidermal stem cells provide a niche linking the hair follicle and the nervous system throughout the hair cycle.

A novel, nontoxic iron chelator, super-polyphenol, effectively induces apoptosis in human cancer cell lines.

Iron chelation therapy is the main treatment for iron overload disease. Iron chelators were recently reported to be useful for cancer therapy; however, they cause side effects that make them difficult to use in some cancer patients. Thus, a novel oral iron chelator, super-polyphenol (SP), was developed for cancer therapy to decrease the side effects. SP is either water soluble or insoluble, and has different isoforms according to the number of side chains. Of these isoforms, water-soluble SP6 and SP10 appear to be the best candidates, as they have the strongest chelating abilities. In this study, we focused on the usefulness and safety of SP6 and SP10 as anti-cancer drugs, and examined their anti-cancer effects and toxicity. The results showed that SP6 and SP10 inhibited cancer cell proliferation by inducing apoptosis in HCT116, HSC-2, A549, and MCF-7 cancer cells. SP10 also inhibited tumor growth in an HCT116 xenograft model. SP6 and SP10 had no acute toxicities. An intravenous injection test revealed that SP6 and SP10 had better safety profiles than the iron chelator deferoxamine. In conclusion, SP is a novel oral iron chelator with anti-cancer effects and few adverse side effects. This is the first report of SP in the literature.

Regulation of keratin 5/14 intermediate filaments by CDK1, Aurora-B, and Rho-kinase.

We previously reported that vimentin, GFAP, and desmin (type III intermediate filament [IF] proteins) are mitotically phosphorylated by CDK1, Aurora-B, and Rho-kinase. This phosphorylation is critical for efficient separation of these IFs and completion of cytokinesis. Keratin 5 (K5) and K14 form a heterodimer, which constitutes IF network in basal layer cells of stratified squamous epithelia. Here, we report that the solubility of K5/K14 increased in mitosis. The in vitro assays revealed that three mitotic kinases phosphorylate K5 more than K14. We then identified Thr23/Thr144, Ser30, and Thr159 on murine K5 as major phosphorylation sites for CDK1, Aurora-B, and Rho-kinase, respectively. Using site- and phosphorylation-state-specific antibodies, we demonstrated that K5-Thr23 was phosphorylated in entire cytoplasm from prometaphase to metaphase, whereas K5-Ser30 phosphorylation occurred specifically at the cleavage furrow from anaphase to telophase. Efficient K5/K14-IF separation was impaired by K5 mutations at the sites phosphorylated by these mitotic kinases. K5-Thr23 phosphorylation was widely detected in dividing K5-positive cells of murine individuals. These results suggested that mitotic reorganization of K5/K14-IF network is governed largely through K5 phosphorylation by CDK1, Aurora-B, and Rho-kinase.

Iron depletion is a novel therapeutic strategy to target cancer stem cells.

Adequate iron levels are essential for human health. However, iron overload can act as catalyst for the formation of free radicals, which may cause cancer. Cancer stem cells (CSCs), which maintain the hallmark stem cell characteristics of self-renewal and differentiation capacity, have been proposed as a driving force of tumorigenesis and metastases. In the present study, we investigated the role of iron in the proliferation and stemness of CSCs, using the miPS-LLCcm cell model. Although the anti-cancer agents fluorouracil and cisplatin suppressed the proliferation of miPS-LLCcm cells, these drugs did not alter the expression of stemness markers, including Nanog, SOX2, c-Myc, Oct3/4 and Klf4. In contrast, iron depletion by the iron chelators deferasirox and deferoxamine suppressed the proliferation of miPS-LLCcm cells and the expression of stemness markers. In an allograft model, deferasirox inhibited the growth of miPS-LLCcm implants, which was associated with decreased expression of Nanog and Sox2. Altogether, iron appears to be crucial for the proliferation and maintenance of stemness of CSCs, and iron depletion may be a novel therapeutic strategy to target CSCs.

The specific localization of advanced glycation end-products (AGEs) in rat pancreatic islets.

Advanced glycation end-products (AGEs) are produced by non-enzymatic glycation between protein and reducing sugar such as glucose. Although glyceraldehyde-derived AGEs (Glycer-AGEs), one of the AGEs subspecies, have been reported to be involved in the pathogenesis of various age-relating diseases such as diabetes mellitus or arteriosclerosis, little is known about the pathological and physiological mechanism of AGEs in vivo. In present study, we produced 4 kinds of polyclonal antibodies against AGEs subspecies and investigated the localization of AGEs-modified proteins in rat peripheral tissues, making use of these antibodies. We found that Glycer-AGEs and methylglyoxal-derived AGEs (MGO-AGEs) were present in pancreatic islets of healthy rats, distinguished clearly into the pancreatic α and ß cells, respectively. Although streptozotocin-induced diabetic rats suffered from remarkable impairment of pancreatic islets, the localization and deposit levels of the Glycer- and MGO-AGEs were not altered in the remaining α and ß cells. Remarkably, the MGO-AGEs in pancreatic ß cells were localized into the insulin-secretory granules. These results suggest that the cell-specific localization of AGEs-modified proteins are presence generally in healthy peripheral tissues, involved in physiological intracellular roles, such as a post-translational modulator contributing to the secretory and/or maturational functions of insulin.

Monoclonal Suncus Antibodies: Generation of Fusion Partners to Produce Suncus-Suncus Hybridomas.

We used suncus (Suncus murinus; house musk shrew) to generate partner cells for cell fusion to produce suncus monoclonal antibodies. Suncus are insectivores that are genetically distant to rodents, and recognize antigens and epitopes that are not immunogenic in mice and rats, which are the animals most commonly used in basic life science research and from which monoclonal antibodies are usually produced. To date, monoclonal antibodies from suncus have not been generated due to the lack of a plasmacytoma fusion partner. To obtain suncus plasmacytoma cell lines suitable as a cell fusion partner, we injected suncus at both sides of the tail base with antigen emulsion, collected the lymph nodes and spleens, and cultured the cells to obtain immortalized lymphoid cell lines visually resembling mouse SP2/0-Ag14 myeloma cells. Three suncus immunized with the antigen provided 4 cell lines of suncus plasmacytoma, but they did not secrete immunoglobulins. Antibody-producing hybrid cells were generated from these cell lines using a cell fusion technique. Using one of the cell lines as a fusion partner, we obtained six lines of immunoglobulin-producing hybrid cells which secreted an unidentified monoclonal IgG. When these 6 lines were used as new fusion partners, we obtained several hybrid cell lines which secreted immunogen-specific monoclonal antibodies. These hybrid cells can be cloned and cryopreserved. We also obtained another good fusion partner which initially secreted antibody but later stopped doing so. These suncus-suncus hybrid cell lines will be useful for the production of suncus monoclonal antibodies.

Iron depletion-induced downregulation of N-cadherin expression inhibits invasive malignant phenotypes in human esophageal cancer.

Esophageal carcinomas often have a poor prognosis due to early lymph node metastasis. Epithelial-mesenchymal transition (EMT) is strongly associated with the acquisition of cancer metastasis and invasion. However, there is no established treatment to eliminate the EMT of cancer cells. Iron is an essential element for both normal and cancer cells in humans. Recently, iron depletion has been discovered to suppress tumor growth. Therefore, we hypothesized that decreased iron conditions would regulate EMT phenotypes, as well as suppressing tumor growth. The human TE esophageal cancer cell lines and OE19 were used in our study. Decreased iron conditions were made using an iron-depletion diet in mice and the iron chelator deferasirox for cell studies. Migration and invasion abilities of cells were measured using migration, invasion, and sphere-formation assays. Esophageal subcutaneous tumor growth was suppressed in decreased iron conditions. In vitro study showed that decreased iron conditions inhibited esophageal cancer cell proliferation as well as migration and invasion abilities, with downregulation of N-cadherin expression. Also, migration and invasion abilities were suppressed by inhibiting expression of N-cadherin. In conclusion, decreased iron conditions revealed a profound anticancer effect by the suppression of tumor growth and the inhibition of migration and invasion abilities via N-cadherin.

Desmin phosphorylation by Cdk1 is required for efficient separation of desmin intermediate filaments in mitosis and detected in murine embryonic/newborn muscle and human rhabdomyosarcoma tissues.

Desmin is a type III intermediate filament (IF) component protein expressed specifically in muscular cells. Desmin is phosphorylated by Aurora-B and Rho-kinase specifically at the cleavage furrow from anaphase to telophase. The disturbance of this phosphorylation results in the formation of unusual long bridge-like IF structures (IF-bridge) between two post-mitotic (daughter) cells. Here, we report that desmin also serves as an excellent substrate for the other type of mitotic kinase, Cdk1. Desmin phosphorylation by Cdk1 loses its ability to form IFs in vitro. We have identified Ser6, Ser27, and Ser31 on murine desmin as phosphorylation sites for Cdk1. Using a site- and phosphorylation-state-specific antibody for Ser31 on desmin, we have demonstrated that Cdk1 phosphorylates desmin in entire cytoplasm from prometaphase to metaphase. Desmin mutations at Cdk1 sites exhibit IF-bridge phenotype, the frequency of which is significantly increased by the addition of Aurora-B and Rho-kinase site mutations to Cdk1 site mutations. In addition, Cdk1-induced desmin phosphorylation is detected in mitotic muscular cells of murine embryonic/newborn muscles and human rhabdomyosarcoma specimens. Therefore, Cdk1-induced desmin phosphorylation is required for efficient separation of desmin-IFs and generally detected in muscular mitotic cells in vivo.

Iron depletion enhances the effect of sorafenib in hepatocarcinoma.

ABSTACT Human hepatocellular carcinoma (HCC) is known to have a poor prognosis. Sorafenib, a molecular targeted drug, is most commonly used for HCC treatment. However, its effect on HCC is limited in clinical use and therefore new strategies regarding sorafenib treatment are required. Iron overload is known to be associated with progression of chronic hepatitis and increased risk of HCC. We previously reported that iron depletion inhibited cancer cell proliferation and conversely induced angiogenesis. Indeed iron depletion therapy including iron chelator needs to be combined with anti-angiogenic drug for its anti-cancer effect. Since sorafenib has an anti-angiogenic effect by its inhibitory targeting VEGFR, we hypothesized that sorafenib could complement the anti-cancer effect of iron depletion. We retrospectively analyzed the relationship between the efficacy of sorafenib and serum iron-related markers in clinical HCC patients. In clinical cases, overall survival was prolonged in total iron binding capacity (TIBC) high- and ferritin low-patients. This result suggested that the low iron-pooled patients, who could have a potential of more angiogenic properties in/around HCC tumors, could be adequate for sorafenib treatment. We determined the effect of sorafenib (Nexavar®) and/or deferasirox (EXJADE®) on cancer cell viability, and on cell signaling of human hepatocarcinoma HepG2 and HLE cells. Both iron depletion by deferasirox and sorafenib revealed insufficient cytotoxic effect by each monotherapy, however, on the basis of increased angiogenesis by iron depletion, the addition of deferasirox enhanced anti-proliferative effect of sorafenib. Deferasirox was confirmed to increase vascular endothelial growth factor (VEGF) secretion into cellular supernatants by ELISA analysis. In in vivo study sorafenib combined with deferasirox also enhanced sorafenib-induced apoptosis. These results suggested that sorafenib combined with deferasirox could be a novel combination chemotherapy for HCC.

Oxidative stress balance is dysregulated and represents an additional target for treating cholangiocarcinoma.

BACKGROUND: Pancreatico-biliary malignancies exhibit similar characteristics, including obesity-related features and poor prognosis, and require new treatment strategies. Oxidative stress is known to induce DNA damage and carcinogenesis, and its reduction is viewed as being favorable. However, it also has anti-infection and anti-cancer functions that need to be maintained. To reveal the effect of oxidative stress on cancer progression, we evaluated oxidative stress and anti-oxidative balance in pancreatic cancer (PC) and cholangiocarcinoma (CC) patients, as well as the effect of add-on antioxidant treatment to chemotherapy in a mouse cholangiocarcinoma model. METHODS: We recruited 84 CC and 80 PC patients who were admitted to our hospital. Serum levels of reactive oxygen metabolites (ROM) and the anti-oxidative OXY-adsorbent test were determined and the balance of these tests was defined as an oxidative index. A diabetic mouse-based cholangiocarcinoma model was utilized to evaluate the effects of add-on antioxidant therapy on cholangiocarcinoma chemotherapy. RESULTS: Serum ROM was higher and anti-oxidant OXY was lower in CC patients with poor outcomes. These parameters were not significantly different in PC patients. In mice, vitamin E administration induced antioxidant hemeoxygenase (HO)-1 protein expression in cancer tissue, while the number of stem-like cells increased. l-carnitine administration improved intestinal microbiome and biliary acid balance, upregulated the hepatic mitochondrial membrane uptake related gene Cpt1 in non-cancerous tissue, and did not alter stem-like cell numbers. CONCLUSION: Oxidative stress balance was dysregulated in cholangiocarcinoma with poor outcome. The mitochondrial function-supporting agent l-carnitine is a good candidate to control oxidative stress conditions.

Ectodomain Shedding of Lymphatic Vessel Endothelial Hyaluronan Receptor 1 (LYVE-1) Is Induced by Vascular Endothelial Growth Factor A (VEGF-A).

Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), a type I transmembrane glycoprotein, is known as one of the most specific lymphatic vessel markers in the skin. In this study, we found that the ectodomain of LYVE-1 undergoes proteolytic cleavage, and this process produces soluble LYVE-1. We further identified the cleavage site for ectodomain shedding and generated an uncleavable mutant of LYVE-1. In lymphatic endothelial cells, ectodomain shedding of LYVE-1 was induced by vascular endothelial growth factor (VEGF)-A, an important factor for angiogenesis and lymphangiogenesis under pathological conditions. VEGF-A-induced LYVE-1 ectodomain shedding was mediated via the extracellular signal-regulated kinase (ERK) and a disintegrin and metalloproteinase (ADAM) 17. Wild-type LYVE-1, but not uncleavable LYVE-1, promoted migration of lymphatic endothelial cells in response to VEGF-A. Immunostaining analyses in human psoriasis skin lesions and VEGF-A transgenic mouse skin suggested that the ectodomain shedding of LYVE-1 occurred in lymphatic vessels undergoing chronic inflammation. These results indicate that the ectodomain shedding of LYVE-1 might be involved in promoting pathological lymphangiogenesis.

Inhibition of the growth factor MDK/midkine by a novel small molecule compound to treat non-small cell lung cancer.

Midkine (MDK) is a heparin-binding growth factor that is highly expressed in many malignant tumors, including lung cancers. MDK activates the PI3K pathway and induces anti-apoptotic activity, in turn enhancing the survival of tumors. Therefore, the inhibition of MDK is considered a potential strategy for cancer therapy. In the present study, we demonstrate a novel small molecule compound (iMDK) that targets MDK. iMDK inhibited the cell growth of MDK-positive H441 lung adenocarcinoma cells that harbor an oncogenic KRAS mutation and H520 squamous cell lung cancer cells, both of which are types of untreatable lung cancer. However, iMDK did not reduce the cell viability of MDK-negative A549 lung adenocarcinoma cells or normal human lung fibroblast (NHLF) cells indicating its specificity. iMDK suppressed the endogenous expression of MDK but not that of other growth factors such as PTN or VEGF. iMDK suppressed the growth of H441 cells by inhibiting the PI3K pathway and inducing apoptosis. Systemic administration of iMDK significantly inhibited tumor growth in a xenograft mouse model in vivo. Inhibition of MDK with iMDK provides a potential therapeutic approach for the treatment of lung cancers that are driven by MDK.

Antiproliferative effect of a novel mTOR inhibitor temsirolimus contributes to the prolonged survival of orthotopic esophageal cancer-bearing mice.

Esophageal squamous cell carcinoma (ESCC) remains one of the most aggressive cancers with poor prognosis regardless of a several reports that indicate a better therapeutic efficacy using some new chemotherapeutic agents. Recent drug development has contributed to an improved specificity to suppress mTOR activity by which many types of malignancies can be explosively progressed. Temsirolimus (CCI-779, TricelTM) is one of recently synthesized analogs of rapamycin and has provided better outcomes for patients with renal cell carcinoma. In this study, we experimentally evaluated an efficacy of targeting mTOR by temsirolimus for ESCC treatment, with an assessment of its survival advantage using an advanced ESCC animal model. First, we confirmed that the expression of phosphorylated mTOR was increased in 46 of 58 clinical ESCC tumor tissues (79.3%) and appeared to get strengthened with tumor progression. All of ESCC cell lines used in this study revealed an increase of mTOR phosphorylation, accompanied with the upregulation of hypoxia inducible factor-I α (HIF-1α), one of the critical effectors regulated by mTOR. Temsirolimus treatment apparently suppressed the activation of mTOR and its downstream effectors, resulting in the reduced ability of ESCC cell proliferation. Finally, the weekly administration of temsirolimus significantly diminished the size of subcutaneous tumors (vehicle, 3261.6 ± 722.0; temsirolimus, 599.2 ± 122.9; p = 0.007) in nude mice and effectively prolonged orthotopic esophageal cancer-bearing mice (median survival periods: control, 31 d; temsirolimus, 43 d; p = 0.0024). These data suggests that targeting mTOR by temsirolimus may become a therapeutic alternative for esophageal cancer, with a contribution to a better outcome.