Expression pattern of class I histone deacetylases in vulvar intraepithelial neoplasia and vulvar cancer: a tissue microarray study

Invasive vulvar squamous cell carcinomas (VSCC) represent the fourth most common type of malignant tumor of the female genital tract in the United States, with an estimated 3, 580 new cases and 900 deaths in 2009 [1]. Recently, a significant increase of precancerous lesions and invasive vulvar carcinomas has been observed in industrialized countries. The incidence of invasive and vulvar intraepithelial neoplasia (VIN) has risen 2.4% per year in the U.S. from 1992 to 1998 [2]. A Scandinavian study describes an increase in VIN incidence more than 4 times from 1973 to 2000 and of 20% for invasive vulvar cancer [3]. The described increase in incidence is seen primarily in younger women, whereas in elderly women, the incidence rates of vulvar cancer have remained relatively stable over the past few decades.

VIN is most commonly treated by local excision, laser evaporation, or a combination of both methods, to preserve vulvar function and morphology. The preferred treatment modality for VSCC is surgery whenever feasible. Small tumors are treated by wide local excision, with or without partial or radical vulvectomy, combined with lymph node staging via sentinel lymph node biopsy or inguinofemoral lymphadenectomy if lymph node metastases are present. For patients with extensively involved inguinofemoral lymph nodes, radiotherapy of the pelvis is advantageous. For patients with recurrent or metastatic disease, irradiation and chemotherapy offer some benefit; however, response rates are regarded as low [4, 5]. Targeted therapies for VSCC have not yet been established in clinical practice [6], but given the low benefit of conventional chemotherapies, novel systemic treatment modalities are urgently needed for these patients.

Epigenetics characterize the hereditary changes in the pattern of gene expression that are not due to changes in DNA sequence. Genetics and epigenetics interact at all stages of cancer development. Epigenetic alterations in mammalian genomes fall into two main categories: DNA methylation and histone modification. Histones are strongly alkaline proteins that are able to package the DNA and condense it into structural units called nucleosomes. Acetylation and deacetylation of histones are performed by histone acetyltransferase (HATs) and histone deacetylases (HDACs), respectively. HDACs increase the affinity of histone complexes to DNA. The chromatin is thereby more condensed and transcriptionally repressed [7–9].

Additionally HDACs can modify proteins other than histones, such as transcription factors (e.g. p53, E2F, pRb). Acetylation can also affect protein stability and protein-protein interactions. Therefore, HDACs are emerging as important regulators of cell growth, differentiation, and apoptosis [10, 11]. There are at least eighteen deacetylase enzymes known in human cells, categorized into four classes: class I (HDAC 1, 2, 3, 8), class II (HDAC 4, 5, 6, 7, 9, 10), class III (SIRT 1-7) and class IV (HDAC 11). HDAC1 and HDAC2 are one of the best-characterized HDACs. However, the isoenzyme-specific biological functions of HDACs are still mostly unknown [8]. It has been postulated that dysregulated function of HDACs leads to cancer formation and development [11]. Altered HDAC expression is observed in a variety of cancer types, such as prostate adenocarcinoma [12], gastric carcinoma [13], colorectal carcinoma [14], cervical dysplasia and endometrial stromal sarcoma [15]. In vulvar intraepithelial neoplasia and vulvar cancer, no data on HDAC expression has been published.

The aim of this study was to analyze the expression of the class I HDACs 1, 2 and 3 by immunohistochemistry in a series of VIN and VSCC samples using the tissue microarray technique and to correlate the finding with the clinicopathological features of the patients.

This study shows that class I HDACs are highly expressed in the majority of VIN and VSCC; however, the expression patterns differ between VIN and VSCC. High HDAC-2 protein expression is found more often in VIN, and high HDAC-3 protein expression is found more often in VSCC. These two types of HDACs are neither associated with patient age nor with level of p16 expression. Therefore, the observed differences are not explained by the difference of the average age or frequency of p16 positivity in VIN and VSCC. The immunohistochemical staining showed high intensity in the majority of tissue samples, and negative results were not found. HDAC 3 was the most intensely expressed isoform of all three class I HDACs.

Based on the expression pattern of histone deacetylating proteins, we hypothesized that epigenetic regulation plays a major role in the pathogenesis of invasive vulvar cancer, as has been demonstrated for several other malignancies [12–15]. The transformation of non-invasive to invasive vulvar neoplasia may be promoted by epigenetic regulation. To our knowledge, this report is the first on class I HDAC expression in vulvar cancer or vulvar intraepithelial neoplasia.

P16 has been proposed as a surrogate marker for HPV associated neoplasia [21–23]. We found no difference between class I HDAC expression in p16-negative or p16-positive tumor tissue. Therefore, the regulation of gene expression by HDACs seems to be independent of HPV infection.

One mechanism by which HDACs appear to stimulate tumor cell growth is through the repression of the tumor suppressor gene, p21 [26]. In this study, high expression of HDAC 1 in VIN and high expression of HDAC 2 in VSCC were associated with increased cell proliferation, as defined by Ki-67. This result supports previous findings in other tumor types, such as prostate cancer and colorectal cancer [27, 28]. With the exception of the significant correlation of HADC 1 and 2 with the proliferation marker Ki-67, no relation was found between HDAC expression and clinicopathological features. Still, the utility of these results must be considered carefully because of the lack of a full clinical data set.

The epigenetic regulation of HDACs has recently been explored as a therapeutic target by the discovery and development of HDAC-inhibitors (HDACi). In-vitro data suggest that HDACi induce cell cycle arrest, differentiation, and apoptosis [15, 29–32]. The antitumor effects of HDACi emphasize the important role of HDACs in cancer development. However, HDACi affects the activity of several enzymes, and it is difficult to identify the particular functions of different HDAC isoforms involved in cancerogenesis. Several HDACi are currently under clinical investigation involving various hematological malignancies and solid tumors, of which vorinostat has already been approved for the systemic treatment of cutaneous T cell lymphoma [33]. Particularly, in oral squamous cell cancer, there are different phase I and II trials using HDAC-i as a monotherapy or in combination with other agents. This treatment method is of interest regarding VSCC [34–37].

In summary, HDACs, the targets of HDACi, are highly expressed in the majority of VIN and VSCC and show a different expression pattern among these two tissue types irrespective of the HPV-related etiology. Investigations of HDAC-i for the topical or systemic treatment of VIN and VSCC are warranted.