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Orthodontic therapy applies forces to teeth, causing an inflammatory reaction in the periodontal ligament. This is repaired by remodeling of the periodontium, allowing tooth displacement. Although orthodontic therapy is mostly initiated during childhood and adolescence, the number of adults seeking this treatment is increasing as our society's esthetic awareness rises. However, adults may already have periodontal tissue abnormalities, rendering orthodontic treatment inefficient because a healthy periodontium is essential for success. Numerous risk factors have been linked to periodontal lesions, with orthodontic tooth movement possibly playing a minimal influence. Although such tissue damages are mostly of esthetic rather than functional concern for patients, restoration frequently requires invasive procedures. Autologous cells for the treatment of periodontal complications have grown in popularity as a less intrusive alternative. The present review analyzed the literature on the use of mesenchymal stem cells and oral tissue-derived fibroblasts for the healing of periodontal defects that may be related to orthodontic tooth movement. Furthermore, the advantages and challenges of the two cell types have been examined. Although the number of clinical studies is currently limited, our study demonstrates that oral fibroblasts have the potential to be the next emergent frontrunners for tissue engineering in the periodontium.
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AIMS: To test whether titanium surface roughness disparity might be used to specifically guide the behavior of gingiva fibroblasts and keratinocytes, thereby improving the quality of soft tissue (ST) integration around abutments. METHODS: Titanium discs resembling the roughness of enamel (M) or cementum (MA) were created with normal or increased hydrophilicity and used as substrates for human fibroblasts and keratinocytes. Adhesion and proliferation assays were performed to assess cell-type specific responses upon encountering the different surfaces. Additionally, immunofluorescence and qPCR analyses were performed to study more in depth the behavior of fibroblasts and keratinocytes on MA and M surfaces, respectively. RESULTS: While enamel-like M surfaces supported adhesion, growth and a normal differentiation potential of keratinocytes, cementum-emulating MA surfaces specifically impaired the growth of keratinocytes. Vice versa, MA surfaces sustained regular adhesion and proliferation of fibroblasts. Yet, a more intimate adhesion between fibroblasts and titanium was achieved by an increased hydrophilicity of MA surfaces, which was associated with an increased expression of elastin. CONCLUSION: The optimal titanium implant abutment might be achieved by a bimodal roughness design, mimicking the roughness of enamel (M) and cementum with increased hydrophilicity (hMA), respectively. These surfaces can selectively elicit cell responses favoring proper ST barrier by impairing epithelial downgrowth and promoting firm adhesion of fibroblasts.
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OBJECTIVES: Cell models have shown great promise as tools for research, potentially providing intriguing alternatives to animal models. However, the original tissue characteristics must be maintained in culture, a fact that is often assumed, but seldom assessed. We aimed to follow the retention of the original tissue identities of cleft lip-derived skin and mucosa keratinocytes in vitro. METHODS: Cleft lip-derived keratinocytes were isolated from discarded tissue along the cleft margins during cheiloplasty. Cell identities were assessed by immunohistochemistry and quantitative real-time PCR for tissue-specific markers and compared with native lip tissue. Moreover, keratinocytes were regularly analyzed for the retention of the original tissue characteristics by the aforementioned methods as well as by differentiation assays. RESULTS: The various anatomical zones of the human lip could be distinguished using a panel of differentiation and functional-based markers. Using these markers, retention of the original tissue identities could be followed and confirmed in the corresponding primary keratinocytes in culture. CONCLUSIONS: Our findings promote patient-derived cells retaining their original identities as astonishing and clinically relevant in vitro tools. Such cells allow a better molecular understanding of various lip-associated pathologies as well as their modeling in vitro, including but not restricted to orofacial clefts.
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Interferon Regulatory Factor 6 (IRF6) and Grainyhead Like Transcription Factor 3 (GRHL3) are transcription factors that orchestrate gene regulatory networks required for the balance between keratinocyte differentiation and proliferation. Absence of either protein results in the lack of a normal stratified epidermis with keratinocytes failing to stop proliferating and to terminally differentiate. Numerous pathological variants within IRF6 and GRHL3 have been identified in orofacial cleft-affected individuals and expression of the two transcription factors has been found to be often dysregulated in cancers. However, whether orofacial cleft-associated IRF6 and GRHL3 variants in patients might also affect their cancer risk later in life, is not clear yet. The fact that the role of IRF6 and GRHL3 in cancer remains controversial makes this question even more challenging. Some studies identified IRF6 and GRHL3 as oncogenes, while others could attribute tumor suppressive functions to them. Trying to solve this apparent conundrum, we herein aimed to characterize IRF6 and GRHL3 function in various types of carcinomas. We screened multiple cancer and normal cell lines for their expression, and subsequently proceeded with functional assays in cancer cell lines. Our data uncovered consistent downregulation of IRF6 and GRHL3 in all types of carcinomas analyzed. Reduced levels of IRF6 and GRHL3 were found to be associated with several tumorigenic properties, such as enhanced cell proliferation, epithelial mesenchymal transition, migration and reduced differentiation capacity. Based on our findings, IRF6 and GRHL3 can be considered as tumor suppressor genes in various carcinomas, which makes them potential common etiological factors for cancer and CLP in a fraction of CLP-affected patients.
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BACKGROUND: Regularly discarded lip tissue obtained from corrective surgeries to close the cleft lip represents an easily accessible and rich source for the isolation of primary fibroblasts. Primary fibroblasts have been described to show compelling similarities to mesenchymal stem cells (MSCs). Hence, cleft lip and palate (CLP) lip-derived fibroblasts could be thought as an intriguing cell source for personalized regenerative therapies in CLP-affected patients. METHODS: Initially, we thoroughly characterized the fibroblastic nature of the lip-derived mesenchymal outgrowths by molecular and functional assays. Next, we compared their phenotype and genotype to that of bone marrow-mesenchymal stem cells (BM-MSCs) and of human lung-derived fibroblasts WI38, by assessing their morphology, surface marker expression, trilineage differentiation potential, colony-forming (CFU) capacity, and immunomodulation property. Finally, to better decipher the heterogeneity of our CLP cultures, we performed a single cell clonal analysis and tested expanded clones for surface marker expression, as well as osteogenic and CFU potential. RESULTS: We identified intriguingly similar phenotypic and genotypic properties between CLP lip fibroblasts and BM-MSCs, which makes them distinct from WI38. Furthermore, our own data in combination with the complex anatomy of the lip tissue indicated heterogeneity in our CLP cultures. Using a clonal analysis, we discovered single cell-derived clones with increased levels of the MSC markers CD106 and CD146 and clones with variabilities in their commitment to differentiate into bone-forming cells and in their potential to form single cell-derived colonies. However, we were not able to gain clones possessing superior MSC-like capacities when compared to the heterogeneous parental CLP population. Additionally, all clones could still generate contractile forces and retained robust levels of the fibroblast specific marker FSP1, which was not detectable in BM-MSCs. CONCLUSIONS: Our results suggest that we isolate heterogeneous populations of fibroblasts from discarded CLP lip tissue, which show a prominently multipotent character in their entirety avoiding the need for elaborate subpopulation selections in vitro. These findings suggest that CLP lip fibroblasts might be a novel potential cell source for personalized regenerative medicine of clinical benefit for CLP patients.
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Labio Leporino , Fisura del Paladar , Células Madre Mesenquimatosas , Células de la Médula Ósea , Diferenciación Celular , Células Cultivadas , Labio Leporino/genética , Labio Leporino/metabolismo , Fisura del Paladar/genética , Fibroblastos , Humanos , Células Madre Mesenquimatosas/metabolismoRESUMEN
For their full manifestation, tumors require support from the surrounding tumor microenvironment (TME), which includes a specific extracellular matrix (ECM), vasculature, and a variety of non-malignant host cells. Together, these components form a tumor-permissive niche that significantly differs from physiological conditions. While the TME helps to promote tumor progression, its special composition also provides potential targets for anti-cancer therapy. Targeting tumor-specific ECM molecules and stromal cells or disrupting aberrant mesenchyme-cancer communications might normalize the TME and improve cancer treatment outcome. The tenascins are a family of large, multifunctional extracellular glycoproteins consisting of four members. Although each have been described to be expressed in the ECM surrounding cancer cells, tenascin-C and tenascin-W are currently the most promising candidates for exploitability and clinical use as they are highly expressed in various tumor stroma with relatively low abundance in healthy tissues. Here, we review what is known about expression of all four tenascin family members in tumors, followed by a more thorough discussion on tenascin-C and tenascin-W focusing on their oncogenic functions and their potential as diagnostic and/or targetable molecules for anti-cancer treatment purposes.
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Variants within the gene encoding for the transcription factor Interferon Regulatory Factor 6 (IRF6) are associated with syndromic and non-syndromic Cleft Lip/Palate (CLP) cases. IRF6 plays a vital role in the regulation of the proliferation/differentiation balance in keratinocytes and is involved in wound healing and migration. Since a fraction of CLP patients undergoing corrective cleft surgery experience wound healing complications, IRF6 represents an interesting candidate gene linking the two processes. However, Irf6 function has been mainly studied in mice and knowledge on IRF6 in human cells remains sparse. Here, we aimed to elucidate the role of IRF6 in human postnatal skin- and oral mucosa-derived keratinocytes. To do so, we applied CRISPR/Cas9 to ablate IRF6 in two TERT-immortalized keratinocyte cultures, which we used as model cell lines. We show that IRF6 controls the appearance of single cells and colonies, with the latter being less cohesive in its absence. Consequently, IRF6 knockout keratinocytes often moved as single cells instead of a collective epithelial sheet migration but maintained their epithelial character. Lack of IRF6 triggered severe keratinocyte differentiation defects, which were already apparent in the stratum spinosum and extended to the stratum corneum in 3D organotypic skin cultures, while it did not alter their growth rate. Finally, proteomics revealed that most of the differentially expressed proteins in the absence of IRF6 could be associated with differentiation, cell-cell adhesion as well as immune response. Our data expand the knowledge on IRF6 in human postnatal keratinocytes, which will help to better understand IRF6-related pathologies.
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The prevalence of congenital anomalies in newborns is estimated to be as high as 6%, many of which involving the cranio-/orofacial region. Such malformations, including several syndromes, are usually identified prenatally, at birth, or rarely later in life. The lack of clinically relevant human cell models of these often very rare conditions, the societal pressure to avoid the use of animal models and the fact that the biological mechanisms between rodents and human are not necessarily identical, makes studying cranio-/orofacial anomalies challenging. To overcome these limitations, we are developing a living cell repository of healthy and diseased cells derived from the cranio-/orofacial region. Ultimately, we aim to make patient-derived cells, which retain the molecular and genetic characteristics of the original anomaly or disease in vitro, available for the scientific community. We report our efforts in establishing a human living cell bank derived from the cranio-/orofacial region of otherwise discarded tissue samples, detail our strategy, processes and quality checks. Such specific cell models have a great potential for discovery and translational research and might lead to a better understanding and management of craniofacial anomalies for the benefit of all affected individuals.
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Galectin-9 is one of the key proteins employed by a variety of human malignancies to suppress anti-cancer activities of cytotoxic lymphoid cells and thus escape immune surveillance. Human cancer cells in most cases express higher levels of galectin-9 compared to non-transformed cells. However, the biochemical mechanisms underlying this phenomenon remain unclear. Here we report for the first time that in human cancer as well as embryonic cells, the transcription factors hypoxia-inducible factor 1 (HIF-1) and activator protein 1 (AP-1) are involved in upregulation of transforming growth factor beta 1 (TGF-ß1) expression, leading to activation of the transcription factor Smad3 through autocrine action. This process triggers upregulation of galectin-9 expression in both malignant (mainly in breast and colorectal cancer as well as acute myeloid leukaemia (AML)) and embryonic cells. The effect, however, was not observed in mature non-transformed human cells. TGF-ß1-activated Smad3 therefore displays differential behaviour in human cancer and embryonic vs non-malignant cells. This study uncovered a self-supporting biochemical mechanism underlying high levels of galectin-9 expression operated by the human cancer and embryonic cells employed in our investigations. Our results suggest the possibility of using the TGF-ß1 signalling pathway as a potential highly efficient target for cancer immunotherapy.
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Galectinas/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Comunicación Autocrina , Galectinas/genética , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HaCaT , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Células MCF-7 , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , Transducción de Señal , Proteína smad3/genética , Proteína smad3/metabolismo , Células THP-1 , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Factor de Crecimiento Transformador beta1/genética , Escape del Tumor , Hipoxia Tumoral , Microambiente TumoralRESUMEN
Van der Woude syndrome (VWS) is a genetic syndrome that leads to typical phenotypic traits, including lower lip pits and cleft lip/palate (CLP). The majority of VWS-affected patients harbor a pathogenic variant in the gene encoding for the transcription factor interferon regulatory factor 6 (IRF6), a crucial regulator of orofacial development, epidermal differentiation and tissue repair. However, most of the underlying mechanisms leading from pathogenic IRF6 gene variants to phenotypes observed in VWS remain poorly understood and elusive. The availability of one VWS individual within our cohort of CLP patients allowed us to identify a novel VWS-causing IRF6 variant and to functionally characterize it. Using VWS patient-derived keratinocytes, we reveal that most of the mutated IRF6_VWS transcripts are subject to a non-sense-mediated mRNA decay mechanism, resulting in IRF6 haploinsufficiency. While moderate levels of IRF6_VWS remain detectable in the VWS keratinocytes, our data illustrate that the IRF6_VWS protein, which lacks part of its protein-binding domain and its whole C-terminus, is noticeably less stable than its wild-type counterpart. Still, it maintains transcription factor function. As we report and characterize a so far undescribed VWS-causing IRF6 variant, our results shed light on the physiological as well as pathological role of IRF6 in keratinocytes. This acquired knowledge is essential for a better understanding of the molecular mechanisms leading to VWS and CLP.
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Of the four tenascins found in bony fish and tetrapods, tenascin-W is the least understood. It was first discovered in the zebrafish and later in mouse, where it was mistakenly named tenascin-N. Tenascin-W is expressed primarily in developing and mature bone, in a subset of stem cell niches, and in the stroma of many solid tumors. Phylogenetic studies show that it is the most recent tenascin to evolve, appearing first in bony fishes. Its expression in bone and the timing of its evolutionary appearance should direct future studies to its role in bone formation, in stem cell niches, and in the treatment and detection of cancer.
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Evolución Molecular , Proteínas de Neoplasias , Neoplasias , Tenascina , Proteínas de Pez Cebra , Pez Cebra , Animales , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Neoplasias/genética , Neoplasias/inmunología , Tenascina/genética , Tenascina/inmunología , Pez Cebra/genética , Pez Cebra/inmunología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/inmunologíaRESUMEN
Extrahepatic cancers of the biliary system are typically asymptomatic until after metastasis, which contributes to their poor prognosis. Here we examined intrahepatic cholangiocarcinomas (n = 8), carcinomas of perihilar bile ducts (n = 7), carcinomas of the gallbladder (n = 11) and hepatic metastasis from carcinomas of the gallbladder (n = 4) for the expression of the extracellular matrix glycoproteins tenascin-C and tenascin-W. Anti-tenascin-C and anti-tenascin-W immunoreactivity was found in all biliary tract tumors examined. Unlike tenascin-C, tenascin-W was not detected in normal hepatobiliary tissue. Tenascin-W was also expressed by the cholangiocarcinoma-derived cell line Huh-28. However, co-culture of Huh-28 cells with immortalized bone marrow-derived stromal cells was necessary for the formation and organization of tenascin-W fibrils in vitro. Our results indicate that tenascin-W may be a novel marker of hepatobiliary tumor stroma, and its absence from many normal tissues suggests that it may be a potential target for biotherapies.
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Neoplasias del Sistema Biliar/inmunología , Biomarcadores de Tumor/inmunología , Proteínas de Neoplasias/inmunología , Tenascina/inmunología , Neoplasias del Sistema Biliar/patología , Línea Celular Tumoral , Humanos , Células del Estroma/inmunología , Células del Estroma/patologíaRESUMEN
Tenascins are a family of multifunctional glycoproteins found in the extracellular matrix of chordates. Two of the tenascins, tenascin-C and tenascin-W, form hexabrachions. In this review, we describe the discovery and domain architecture of tenascin-W, its evolution and patterns of expression during embryogenesis and in tumors, and its effects on cells in culture. In avian and mammalian embryos tenascin-W is primarily expressed at sites of osteogenesis, and in the adult tenascin-W is abundant in certain stem cell niches. In primary cultures of osteoblasts tenascin-W promotes cell migration, the formation of mineralized foci and increases alkaline phosphatase activity. Tenascin-W is also prominent in many solid tumors, yet it is missing from the extracellular matrix of most adult tissues. This makes it a potential candidate for use as a marker of tumor stroma and a target for anti-cancer therapies.
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To gain more understanding of the complex molecular processes underlying cleft lip/palate (CLP), we established a unique human cell bank, consisting of keratinocytes and corresponding fibroblasts from individual CLP patients as a new study tool. After their careful characterization, we used such patient-derived cell cultures as well as control keratinocytes for in vitro differentiation and proliferation assays. Foreskin-derived control cells as a group showed significant higher induction of the late differentiation markers Loricrin and Filaggrin than the group of CLP patients-derived keratinocytes. Additionally, we detected great variations between individual CLP keratinocyte cell cultures in regard to their potential to terminally differentiate as assessed by the induction of Loricrin and Filaggrin. Primary patient cell cultures that did not properly differentiate, exhibited high proliferation rates. Moreover, we could correlate the expression levels of transcription factor IRF6 to the ability of individual cell cultures to terminally differentiate. Using clinically relevant, patient-derived cells, our results suggest that some of the genetic predispositions causing CLP might also lead to deficiencies in keratinocyte differentiation manifested in in vitro assays.
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Muscle wasting in old age or cancer may result from failed myofiber regeneration and/or accelerated atrophy. This study aimed to determine from transcriptomic analysis of human muscle the integrity of the cellular stress response system in relation to satellite cell differentiation or apoptosis in patients with cancer (weight-stable (CWS) or weight-losing (CWL)) or healthy elderly (HE) when compared with healthy middle-aged controls (HMA). 28 patients with cancer (CWS: 18 and CWL: 10), HE: 21 and HMA: 20 underwent biopsy of quadriceps muscle. The expression of transcription factors for muscle regeneration (Pax3, Pax7 and MyoD) was increased in CWS and HE compared with HMA (p≤0.001). In contrast, the expression of the late myogenic differentiation marker MyoG was reduced in CWS and CWL but increased in HE (p≤0.0001). Bax was significantly increased in CWS, CWL and HE (p≤0.0001). Expression of the oxidative defense genes SOD2, GCLM, and Nrf2 was decreased in CWS and CWL but increased in HE (p≤0.0001). There is evidence for blockade of satellite cell maturation, upregulation of apoptosis and reduced oxidative defense in the muscle of cancer patients. In the healthy elderly the potential for differentiation and oxidative defense is maintained.
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Caquexia/metabolismo , Desarrollo de Músculos/fisiología , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Neoplasias/metabolismo , Células Satélite del Músculo Esquelético/metabolismo , Anciano , Anciano de 80 o más Años , Caquexia/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Músculo Esquelético/patología , Atrofia Muscular/patología , Neoplasias/patología , Células Satélite del Músculo Esquelético/patologíaRESUMEN
Krüppel-like factor 4 (KLF4), a transcription factor involved in both tumor suppression and oncogenesis in various human tumors, is subject to alternative splicing that produces KLF4α. KLF4α is primarily expressed in the cytoplasm because it lacks exon 3 of KLF4, which contains the nuclear localization signal. The role of KLF4 in breast cancer remains unclear and nothing is known yet about the expression and function of the isoform KLF4α. Here, we show that KLF4α is expressed in normal and tumoral tissue of the breast and provide evidence that the KLF4α/KLF4(full-length) (FL) ratio is increased in tumors compared to corresponding normal tissue. Forced increase of the KLF4α/KLF4(FL) ratio in the metastatic breast cancer cell line MDA-MB-231 decreases the levels of E-Cadherin, p21Cip1, and p27Kip1, three known KLF4(FL) target genes, and stimulates cell proliferation. We suggest that cytoplasmic KLF4α binds to KLF4(FL) and retains it in the cytoplasm thereby antagonizing the gene regulatory activities of KLF4(FL) in the nucleus. Our results establish KLF4α as a KLF4 isoform that opposes the function of KLF4(FL) and as an important factor in the complex and unresolved role of KLF4(FL) in breast carcinogenesis.
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Neoplasias de la Mama/patología , Proliferación Celular/fisiología , Factores de Transcripción de Tipo Kruppel/metabolismo , Isoformas de Proteínas/metabolismo , Empalme Alternativo , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Transformación Celular Neoplásica/metabolismo , Femenino , Humanos , Factor 4 Similar a KruppelRESUMEN
BACKGROUND: Cachexia affects the majority of patients with advanced cancer and is associated with a reduction in treatment tolerance, response to therapy, and duration of survival. One impediment towards the effective treatment of cachexia is a validated classification system. METHODS: 41 patients with resectable upper gastrointestinal (GI) or pancreatic cancer underwent characterisation for cachexia based on weight-loss (WL) and/or low muscularity (LM). Four diagnostic criteria were used >5%WL, >10%WL, LM, and LM+>2%WL. All patients underwent biopsy of the rectus muscle. Analysis included immunohistochemistry for fibre size and type, protein and nucleic acid concentration, Western blots for markers of autophagy, SMAD signalling, and inflammation. FINDINGS: Compared with non-cachectic cancer patients, patients with LM or LM+>2%WL, mean muscle fibre diameter was reduced by about 25% (pâ=â0.02 and pâ=â0.001 respectively). No significant difference in fibre diameter was observed if patients had WL alone. Regardless of classification, there was no difference in fibre number or proportion of fibre type across all myosin heavy chain isoforms. Mean muscle protein content was reduced and the ratio of RNA/DNA decreased in patients with either >5%WL or LM+>2%WL. Compared with non-cachectic patients, SMAD3 protein levels were increased in patients with >5%WL (pâ=â0.022) and with >10%WL, beclin (pâ=â0.05) and ATG5 (pâ=â0.01) protein levels were increased. There were no differences in phospho-NFkB or phospho-STAT3 levels across any of the groups. CONCLUSION: Muscle fibre size, biochemical composition and pathway phenotype can vary according to whether the diagnostic criteria for cachexia are based on weight loss alone, a measure of low muscularity alone or a combination of the two. For intervention trials where the primary end-point is a change in muscle mass or function, use of combined diagnostic criteria may allow identification of a more homogeneous patient cohort, reduce the sample size required and enhance the time scale within which trials can be conducted.
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Caquexia/diagnóstico , Caquexia/etiología , Músculo Esquelético/patología , Neoplasias/complicaciones , Fenotipo , Anciano , Autofagia , Biomarcadores , Índice de Masa Corporal , Femenino , Humanos , Inflamación/metabolismo , Inflamación/patología , Masculino , Persona de Mediana Edad , Músculo Esquelético/metabolismo , Transducción de Señal , Proteínas Smad/metabolismo , Pérdida de PesoRESUMEN
Overexpression of the basement membrane protein Laminin γ2 (Lamγ2) is a feature of many epidermal and oral dysplasias and all invasive squamous cell carcinomas (SCCs). This abnormality has potential value as an immunohistochemical biomarker of premalignancy but its mechanism has remained unknown. We recently reported that Lamγ2 overexpression in culture is the result of deregulated translation controls and depends on the MAPK-RSK signaling cascade. Here we identify eIF4B as the RSK downstream effector responsible for elevated Lamγ2 as well as MYC protein in neoplastic epithelial cells. Premalignant dysplastic keratinocytes, SCC cells, and keratinocytes expressing the E6 oncoprotein of human papillomavirus (HPV) type 16 displayed MAPK-RSK and mTOR-S6K1 activation and overexpressed Lamγ2 and MYC in culture. Immunohistochemical staining of oral dysplasias and SCCs for distinct, RSK- and S6K1-specific S6 phosphorylation events revealed that their respective upstream pathways become hyperactive at the same time during neoplastic progression. However, pharmacologic kinase inhibitor studies in culture revealed that Lamγ2 and MYC overexpression depends on MAPK-RSK activity, independent of PI3K-mTOR-S6K1. eIF4B knockdown reduced Lamγ2 and MYC protein expression, consistent with the known requirement for eIF4B to translate mRNAs with long, complex 5' untranslated regions (5'-UTRs). Accordingly, expression of a luciferase reporter construct preceded by the Lamγ2 5'-UTR proved to be RSK-dependent and mTOR-independent. These results demonstrate that RSK activation of eIF4B is causally linked to elevated Lamγ2 and MYC protein levels during neoplastic progression to invasive SCC. These findings have potential clinical significance for identifying premalignant lesions and for developing targeted drugs to treat SCC.
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Carcinoma de Células Escamosas/metabolismo , Laminina/metabolismo , Sistema de Señalización de MAP Quinasas , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Factores Eucarióticos de Iniciación/genética , Factores Eucarióticos de Iniciación/metabolismo , Factores Eucarióticos de Iniciación/fisiología , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Inmunohistoquímica , Queratinocitos/metabolismo , Laminina/genética , Invasividad Neoplásica/genética , Procesos Neoplásicos , ARN Mensajero/metabolismoRESUMEN
As life expectancy lengthens, cases of non-viral-associated vulvar squamous cell carcinoma and its precursor lesion, so-called differentiated vulvar intraepithelial neoplasia (VIN), continue to increase in frequency. Differentiated VIN often is difficult to recognize and failure to detect it before invasion results in morbidity and mortality. Thus, identification of a reliable biomarker for this type of lesion would be of great clinical benefit. Our recent studies have identified activation (ser235/236 phosphorylation) of ribosomal protein S6 (p-S6) in basal epithelial cells as an event that precedes and accompanies laminin γ(2) overexpression in most preinvasive oral dysplasias. To test this as a potential biomarker of vulvar dysplasia, we immunostained seven differentiated VINs and nine papillomavirus-related 'classic' VINs, most of which were associated with carcinoma, for p-S6. All carcinomas, all differentiated VINs, and most classic VINs contained regions of p-S6 staining in the basal layer, whereas basal and parabasal cells of normal vulvar epithelium and hyperplastic and inflamed lesions lacking cellular atypia were p-S6 negative. Laminin γ(2) was expressed in a subset of VINs, always occurring within basal p-S6 positive regions, as we had found previously for oral dysplasias. Lichen sclerosus is considered a potential precursor of vulvar carcinoma. Two lichen sclerosus lesions of patients with a concurrent carcinoma and one of six lichen sclerosus lesions without atypia or known concurrent carcinoma were basal p-S6 positive. In summary, there is a distinct difference in p-S6 basal cell layer staining between benign and neoplastic vulvar squamous epithelium, with consistent staining of differentiated VIN and of some lichen sclerosus lesions. These results support further studies to assess the potential of p-S6 as a biomarker to identify vulvar lesions at risk of progressing to invasive cancer.
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Biomarcadores de Tumor/análisis , Carcinoma in Situ/química , Carcinoma/química , Células Epiteliales/química , Inmunohistoquímica , Lesiones Precancerosas/química , Proteína S6 Ribosómica/análisis , Neoplasias de la Vulva/química , Anciano , Anciano de 80 o más Años , Carcinoma/patología , Carcinoma in Situ/patología , Progresión de la Enfermedad , Células Epiteliales/patología , Femenino , Humanos , Laminina/análisis , Persona de Mediana Edad , Invasividad Neoplásica , Fosforilación , Lesiones Precancerosas/patología , Valor Predictivo de las Pruebas , Factores de Riesgo , Liquen Escleroso Vulvar/metabolismo , Liquen Escleroso Vulvar/patología , Neoplasias de la Vulva/patologíaRESUMEN
BACKGROUND: Tenascins are large glycoproteins found in the extracellular matrix of many embryonic and adult tissues. Tenascin-C is a well-studied biomarker known for its high overexpression in the stroma of most solid cancers. Tenascin-W, the least studied member of the family, is highly expressed in the stroma of colon and breast tumors and in gliomas, but not in the corresponding normal tissues. Other solid tumors have not been analyzed. The present study was undertaken to determine whether tenascin-W could serve as a cancer-specific extracellular matrix protein in a broad range of solid tumors. METHODS: We analyzed the expression of tenascin-W and tenascin-C by immunoblotting and by immunohistochemistry on multiple frozen tissue microarrays of carcinomas of the pancreas, kidney and lung as well as melanomas and compared them to healthy tissues. RESULTS: From all healthy adult organs tested, only liver and spleen showed detectable levels of tenascin-W, suggesting that tenascin-W is absent from most human adult organs under normal, non-pathological conditions. In contrast, tenascin-W was detectable in the majority of melanomas and their metastases, as well as in pancreas, kidney, and lung carcinomas. Comparing lung tumor samples and matching control tissues for each patient revealed a clear overexpression of tenascin-W in tumor tissues. Although the number of samples examined is too small to draw statistically significant conclusions, there seems to be a tendency for increased tenascin-W expression in higher grade tumors. Interestingly, in most tumor types, tenascin-W is also expressed in close proximity to blood vessels, as shown by CD31 co-staining of the samples. CONCLUSIONS: The present study extends the tumor biomarker potential of tenascin-W to a broad range of solid tumors and shows its accessibility from the blood stream for potential therapeutic strategies.