The Tumor Immune Microenvironment in Cutaneous Squamous Cell Carcinoma Arising in Organ Transplant Recipients.

Cutaneous squamous cell carcinoma (cSCC) is the most common malignancy in immune-suppressed organ transplant recipients (OTRs). Whilst rates of other malignancies (both cutaneous and non-cutaneous) are elevated in this population, the increase is far less striking. This suggests that cSCC must be a highly immunogenic tumor. The tumor immune microenvironment is altered in cSCC from OTRs. It has reduced anti-tumor properties and instead provides an environment that facilitates tumor growth and survival. Understanding the composition and function of the tumor immune microenvironment in cSCC from OTRs is useful for prognostication and therapeutic decisions.

The Immune Microenvironment in Basal Cell Carcinoma.

The immune system plays a key role in the suppression and progression of basal cell carcinoma (BCC). The primary aetiological factor for BCC development is exposure to ultraviolet radiation (UVR) which, particularly in lighter Fitzpatrick skin types, leads to the accumulation of DNA damage. UVR has roles in the generation of an immunosuppressive environment, facilitating cancer progression. Rates of BCC are elevated in immunosuppressed patients, and BCC may undergo spontaneous immune-mediated regression. Histologic and immunohistochemical profiling of BCCs consistently demonstrates the presence of an immune infiltrate and associated immune proteins. Early studies of immune checkpoint inhibitors reveal promising results in BCC. Therefore, the host immune system and tumor responses to it are important in BCC pathogenesis. Understanding these interactions will be beneficial for disease prognostication and therapeutic decisions.

Downregulation of Cockayne syndrome B protein reduces human 8-oxoguanine DNA glycosylase-1 expression and repair of UV radiation-induced 8-oxo-7,8-dihydro-2'-deoxyguanine.

Human 8-oxoguanine DNA glycosylase-1 (hOGG1) is the key DNA repair enzyme responsible for initiating repair of UV radiation-induced 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG). Previously we have shown that basal cells in human epidermis are particularly sensitive to UVA-mediated DNA damage probably due to low expression of hOGG1. Here we investigate some aspects of the regulatory role of Cockayne syndrome B (CSB) on hOGG1 expression and function. Cockayne syndrome B and hOGG1 genes were knocked down by miRNA technology in the HaCaT human keratinocyte cell line. Loss of the CSB gene decreased hOGG1 mRNA, and loss of hOGG1 increased CSB, indicating that they influence each other's expression. Protein levels were assessed in cells grown into engineered human skin using immunohistochemistry. This confirmed that CSB knockdown with miRNA reduced hOGG1 protein levels, but hOGG1 knockdown did not influence expression of CSB protein. Using comet assay we found that both hOGG1 and CSB knockdown reduced repair of both UVA- and UVB-induced 8-oxo-dG, consistent with CSB downregulation of hOGG1 mRNA and protein. In contrast, CSB but not hOGG1 knockdown reduced repair of UVB- and UVA-induced cyclobutane pyrimidine dimer photolesions. In engineered human skin, repair of UVA-induced 8-oxo-dG was inhibited by both hOGG1 and CSB knockdown, confirming the functional role of both proteins in cells with 3-D cellular contacts. These findings directly indicate that hOGG1 and CSB influence each other's expression. CSB is required for maintaining hOGG1 enzyme levels and function. Cockayne syndrome B could therefore be required for 8-oxo-dG repair due to its regulatory effect on hOGG1 expression. Cockayne syndrome B but not hOGG1 is also required for efficient repair of cyclobutane pyrimidine dimers. Cockayne syndrome B regulation of DNA repair could contribute to the effect of UVA in causing mutations that lead to skin cancer in humans.

Chromatin structure following UV-induced DNA damage-repair or death?

In eukaryotes, DNA is compacted into a complex structure known as chromatin. The unravelling of DNA is a crucial step in DNA repair, replication, transcription and recombination as this allows access to DNA for these processes. Failure to package DNA into the nucleosome, the individual unit of chromatin, can lead to genomic instability, driving a cell into apoptosis, senescence, or cellular proliferation. Ultraviolet (UV) radiation damage causes destabilisation of chromatin integrity. UV irradiation induces DNA damage such as photolesions and subjects the chromatin to substantial rearrangements, causing the arrest of transcription forks and cell cycle arrest. Highly conserved processes known as nucleotide and base excision repair (NER and BER) then begin to repair these lesions. However, if DNA repair fails, the cell may be forced into apoptosis. The modification of various histones as well as nucleosome remodelling via ATP-dependent chromatin remodelling complexes are required not only to repair these UV-induced DNA lesions, but also for apoptosis signalling. Histone modifications and nucleosome remodelling in response to UV also lead to the recruitment of various repair and pro-apoptotic proteins. Thus, the way in which a cell responds to UV irradiation via these modifications is important in determining its fate. Failure of these DNA damage response steps can lead to cellular proliferation and oncogenic development, causing skin cancer, hence these chromatin changes are critical for a proper response to UV-induced injury.

Disruption of E-cadherin by matrix metalloproteinase directly mediates epithelial-mesenchymal transition downstream of transforming growth factor-beta1 in renal tubular epithelial cells.

Epithelial-mesenchymal transition (EMT) plays an important role in organ fibrosis, including that of the kidney. Loss of E-cadherin expression is a hallmark of EMT; however, whether the loss of E-cadherin is a consequence or a cause of EMT remains unknown, especially in the renal system. In this study, we show that transforming growth factor (TGF)-beta1-induced EMT in renal tubular epithelial cells is dependent on proteolysis. Matrix metalloproteinase-mediated E-cadherin disruption led directly to tubular epithelial cell EMT via Slug. TGF-beta1 induced the proteolytic shedding of E-cadherin, which caused the nuclear translocation of beta-catenin, the transcriptional induction of Slug, and the repression of E-cadherin transcription in tubular epithelial cells. These findings reveal a direct role for E-cadherin and for matrix metalloproteinases in causing EMT downstream of TGF-beta1 in fibrotic disease. Specific inhibition rather than activation of matrix metalloproteinases may offer a novel approach for treatment of fibrotic disease.

2-Methoxy-2,4-diphenyl-3(2H)-furanone-labeled gelatin zymography and reverse zymography: a rapid real-time method for quantification of matrix metalloproteinases-2 and -9 and tissue inhibitors of metalloproteinases.

Measurement of matrix metalloproteinases (MMPs) and their specific tissue inhibitors of metalloproteinases (TIMPs) by the techniques of zymography and reverse zymography provide useful information regarding the status of matrix accumulation or breakdown. This report describes the use of 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF), a fluorescent compound which can be used to label gelatin as a substrate for detection of the gelatin degrading MMP-2 and -9 by zymography. In addition, a modification of the zymographic technique by addition of excess MMPs enables the use of the MDPF-labeled gelatin substrate for the identification and quantification of TIMPs by reverse zymography. Both systems are real-time sensitive reliable quantification techniques, easily used for measurement of these MMPs and TIMPs in clinical, biological, and tissue culture samples.