High expression of keratin 6C is associated with poor prognosis and accelerates cancer proliferation and migration by modulating epithelial-mesenchymal transition in lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD) is a more frequent subtype of lung cancer and most cases are discovered in the late stages. The proliferation and metastasis of LUAD are pivotal for disease progression. Despite unremitting deeper understanding of LUAD biology, the mechanisms involved in the proliferation and metastasis of LUAD remain unclear. The objective of our article was to inquiry the expression and the function of keratin 6C (KRT6C) in LUAD cells. METHODS: First, the expression level and prognostic value of KRT6C in LUAD tissues were analyzed on the basis of the data acquired from TCGA database. Through qRT-PCR, the expression level of KRT6C on LUAD cell lines (A549, H1299, PC-9) and human normal lung cell line MRC-5 was tested. After that, CCK8 and colony formation assays was utilized to detect cell proliferation. In addition, to explore the influence of KRT6C on LUAD migration and invasion ability, scratch wound healing and transwell assays were utilized. Through western blotting, the protein expression levels of KRT6C, PCNA, E-cadherin, N-cadherin, Snail and Vimentin were detected. RESULTS: The outcomes revealed that KRT6C was highly expressed in LUAD tissues and cell lines. Besides, elevated level of KRT6C was related to worse prognosis in LUAD patients. Ablation of KRT6C restrained proliferation, migration and invasion of A549 cells. KRT6C deficiency augmented the expression of E-cadherin as well as reduced the expression of N-cadherin, Snail and Vimentin. CONCLUSION: Above all, these consequences indicated that depletion of KRT6C suppressed A549 cell proliferation, migration and invasion, which might be achieved by regulating EMT. In general, KRT6C is identified as a potential therapeutic target for LUAD.

Statins downregulate K6a promoter activity: a possible therapeutic avenue for pachyonychia congenita.

Pachyonychia congenita (PC) is a keratinizing disorder predominantly caused by mutations in keratin 6a (K6a) (∼50% of cases) or K6b, K16, or K17. One means of treating PC is identification of small-molecule inhibitors of PC-related keratins. Here, we cloned the human K6a promoter, and using a cell-based reporter gene assay, a chemical library was screened for K6a inhibitors. One compound, compactin, the precursor of all cholesterol-lowering statins, was of particular interest. We found that, surprisingly, simvastatin and other statins inhibit K6a promoter activity and K6a protein expression. Further investigation showed that this effect works through cholesterol/mevalonate pathway inhibition rather than an off-target effect. Inhibition of both basal and IFN-γ-inducible K6a expression by statins was demonstrated. Both these K6a inhibitory effects were found to be mediated by Stat1 transcription factor, but only the IFN-γ-inducible promoter activity was controlled via the Stat/JAK pathway. The repressive effect of statins was found to be mediated by the isoprenoid pathway downstream of mevalonate (the intermediate following 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase) but upstream of cholesterol, specifically the geranylgeranylation pathway. These data set the scene for further unraveling signaling pathways that control the K6a promoter, as well as facilitating clinical trials for statins in PC patients.

Use of self-delivery siRNAs to inhibit gene expression in an organotypic pachyonychia congenita model.

Although RNA interference offers therapeutic potential for treating skin disorders, delivery hurdles have hampered clinical translation. We have recently demonstrated that high pressure, resulting from intradermal injection of large liquid volumes, facilitated nucleic acid uptake by keratinocytes in mouse skin. Furthermore, similar intradermal injections of small interfering RNA (siRNA; TD101) into pachyonychia congenita (PC) patient foot lesions resulted in improvement. Unfortunately, the intense pain associated with hypodermic needle administration to PC lesions precludes this as a viable delivery option for this disorder. To investigate siRNA uptake by keratinocytes, an organotypic epidermal model, in which pre-existing endogenous gene or reporter gene expression can be readily monitored, was used to evaluate the effectiveness of "self-delivery" siRNA (i.e., siRNA chemically modified to enhance cellular uptake). In this model system, self-delivery siRNA treatment resulted in reduction of pre-existing fluorescent reporter gene expression under conditions in which unmodified controls had little or no effect. Additionally, treatment of PC epidermal equivalents with self-delivery "TD101" siRNA resulted in marked reduction of mutant keratin 6a mRNA with little or no effect on wild-type expression. These results indicate that chemical modification of siRNA may overcome certain limitations to transdermal delivery (specifically keratinocyte uptake) and may have clinical utility for inhibition of gene expression in the skin.

Therapeutic interference: a step closer for pachyonychia congenita?

The identification of mutations in keratin genes as the cause of several inherited skin disorders raised the possibility that molecular-based therapies might be developed to treat these conditions. In this issue, Smith et al. (2007) have identified small interfering RNAs that specifically and potently silence keratin 6a expression. These molecules have great promise as therapeutic agents for the treatment of pachyonychia congenita.

Development of therapeutic siRNAs for pachyonychia congenita.

Pachyonychia congenita (PC) is an autosomal-dominant keratin disorder where the most painful, debilitating aspect is plantar keratoderma. PC is caused by mutations in one of four keratin genes; however, most patients carry K6a mutations. Knockout mouse studies suggest that ablation of one of the several K6 genes can be tolerated owing to compensatory expression of the others. Here, we have developed potent RNA interference against K6a as a paradigm for treating a localized dominant skin disorder. Four small interfering RNAs (siRNAs) were designed against unique sequences in the K6a 3'-untranslated region. We demonstrated near-complete ablation of endogenous K6a protein expression in two keratinocyte cell lines, HaCaT and NEB-1, by transient transfection of each of the four K6a siRNAs. The siRNAs were effective at very low, picomolar concentrations. One potent lead K6a inhibitor, which was highly specific for K6a, was tested in a mouse model where reporter gene constructs were injected intradermally into mouse paw and luciferase activity was used as an in vivo readout. Imaging in live mice using the Xenogen IVIS system demonstrated that the K6a-specific siRNA strongly inhibited bicistronic K6a-luciferase gene expression in vivo. These data suggest that siRNAs can specifically and very potently target mutated genes in the skin and support development of these inhibitors as potential therapeutics.