The purpose of this study was to validate an immunohistochemical assay for XPG protein using a commercially available antibody for use in FFPE human tissues and to analyse its expression in human normal and cancer samples. The optimal pretreatment procedure to maximize staining was determined by testing 9 different antigen retrieval techniques. The optimal primary antibody titer was selected to maximize signal to noise through analysis of a breast cancer tissue while assay precision was determined in 2 breast cancer, 1 ovarian cancer, and 1 sarcoma tissues on 5 different days. These experiments established that the best IHC assay was: pretreament of samples with High Tide for 40 minutes at 95°C in a water-bath and staining using a 2.5 μg/mL antibody dilution. The optimized assay was then validated by staining 2 tumor cell lines. Once validated, the differential expression of XPG was determined in 28 normal tissues and 20 tumor tissues consisting of 10 breast, 5 ovarian, and 5 sarcoma cancer samples.
XPG is one of multiple proteins that are members of the NER system in mammalian cells [6
]. The NER pathway is involved in DNA repair and allows tumor cells to survive DNA damage caused by ultraviolet light or different genotoxins such as anticancer therapeutic agents [21
]. Briefly, after DNA damage recognition, the sequential action of NER helicases and endonucleases open the double helix and cleave the damaged strand few bases away from the lesion. This is followed by the removal of the DNA segment containing the lesion, DNA gap filling using the intact strand as template and restoration of the chromatin structure [1
]. During this process, XPG functions as a structure-specific endonuclease that cleaves DNA bubbles and flaps near the junctions of single-stranded and double-stranded DNA with specific polarity [10
]. XPG endonuclease activity has no preference for the sequencing of DNA damage. At the site of injury, NER complex proteins create a DNA bubble of an approximate length of 25 nucleotides. XPG cuts the DNA damage between 0-2 nucleotides below 3’ of the double-fork single-stranded chain. The cells that do not express ERCC5/XPG are inefficient in repairing damage to DNA.
XPG also binds strongly to various structured DNAs that it does not cleave, implying separate biological functions for its binding and incision activities [13
]. For example, XPG has a non-enzymatic scaffolding role in several steps of NER, including coordination of incision with the resynthesis step [28
]. In addition, XPG forms a complex with the transcription and repair factor TFIIH being this important for stable association of the CAK kinase subunit with TFIIH [12
]. Thus, XPG alleles with C-terminal mutations or truncations are unable to bind TFIIH, resulting in impaired activated RNA polymerase II-mediated transcription [26
]. More recently, it was shown that XPG has an intrinsic single-strand annealing activity that is independent of its nuclease activity but requires intact N- and C-terminal domains [29
]. This activity is performed in cooperation with WRN protein through a direct physical interaction between both proteins and is observed during the late S-phase of the cell cycle [13
]. Finally, XPG has a role in the early steps of base excision repair (BER) of oxidative DNA damage through direct interaction and stimulation of different DNA glycosylases [13
As the incision activity of XPG is essential for removing bulky DNA adducts by NER, point mutations that inactivate the XPG endonuclease function cause the cancer-prone, sun-sensitive disorder xeroderma pigmentosum (XP) in XP-G patients [14
] University Medical Centre (CMU. Moreover, patients with rare truncating mutations in XPG have the combined diseases of XP with Cockayne syndrome (XP-G/CS) [32
]. XP-G/CS presents as severe primarily postnatal neurological and developmental dysfunction with mental retardation, wasting, greatly accelerated symptoms of segmental aging and death in early childhood. Mutations and polymorphisms of the ERCC5 gene were also associated to an increase risk of different cancers types such as endometrial, melanoma, prostate, bladder, gastric, cervix or lung cancer [15
]. Polymorphisms of ERCC5 gene or differential expression levels of its mRNA were correlated with patient prognosis in different tumor pathologies. For example, the Asp1104His or His46His polymorphisms were reported to be an independent prognostic factor in ovarian cancer, sarcoma and cutaneous melanoma, and non-small cell lung cancer (NSCLC), respectively [19
]. On the other hand, high expression of XPG mRNA was associated as an independent prognostic factor in NSCLC and sarcoma patients and to significantly correlate with increased response to chemotherapeutic agents such as oxaliplatin, fluoropyrimidin or trabectedin [42
]. Thus, XPG seems to be an important molecular factor involved in tumorigenesis and prognosis of cancer patients. However, all these studies were performed using DNA sequencing or real-time PCR techniques that are quite cumbersome to be used in a routine pathology laboratory.
In the present work, we have validated an IHC assay for detection of expression levels of XPG protein in FFPE human tissue samples. We have also tested this IHC assay in different normal and tumor human tissues. XPG expression was firstly determined on a microarray containing 28 normal tissue cores. Positive staining was observed in 60% of the normal samples. The highest staining was detected in adrenal gland, breast, colon, heart, kidney, thyroid and tongue. A weaker staining was observed in cerebellum, cerebrum, salivary gland, skin, spleen, stomach, testis, thymus, tonsil and uterus. Finally, cervix, esophagus, liver, lung, skeletal muscle, smooth muscle, ovary, pancreas, parathyroid, pituitary, and prostate samples were considered as negative. Subcellular localization was predominantly nuclear, although cytoplasmic staining was also observed in 5 specimens (adrenal gland, heart, kidney, cerebellum and cerebrum). Interestingly, a weak to moderate staining intensity was also correlated with a cytoplasmic staining in adrenal gland, heart and kidney samples while both central nervous system samples presented cytoplasmic staining even in the case of presenting a weak nuclear staining. Another interesting observation concerned the staining of macrophages present on these normal tissues. In fact, these inflammatory cells showed a weak nuclear staining in positive and negative samples. However, while in positive samples (colon, tongue, salivary gland, thymus and tonsil) it was mainly nuclear, in negative tissues (lung and ovary), it was cytoplasmic. Other interesting observations were the expression of XPG in the Hassall’s corpuscles of the thymus or smooth muscle cells in esophagus and uterus.
In tumors, positive staining was observed in 90% of breast cancer samples and in all ovarian cancer and sarcomas samples. XPG expression in breast and ovary cancer samples was expected as breast and uterus normal samples already expressed weak to moderate levels of the protein. Subcellular localization was predominantly nuclear. Of note, in all positive samples (19/20 analyzed samples), inflammatory cells showed 2+, or even, 3+ nuclear staining. These contrasted with that observed in normal tissues where macrophage nuclear staining was seen only in 17% (5/28) of analyzed tissues. The biological significance of this XPG overexpression in tumor-associated macrophages (TAMs) is not well understood. One possibility is that this over-expression is related to chronic hypoxia or inflammation in tissue microenvironments that will favour higher levels of DNA lesions that need to be repaired. A second possibility would be that TAMs would need high levels of XPG to support high levels of transcription in order to maintain elevated inflammatory cytokine secretions as observed in tumor microenvironments. Other non-tumor cells showing a positive staining were endothelial cells, smooth muscle and fibroblasts.
In summary, we have validated an immunohistochemical assay for XPG protein detection using a commercially available antibody for use in formalin-fixed, FFPE human tissues. To our knowledge, this is the first time that XPG expression was evaluated at the protein level in human samples. The use of this validated methodology would help to interpret the role of XPG in tumorogenesis and its use as a possible prognostic or predictive factor.