Modern Pathology (2014) 27, 1203–1211 & 2014 USCAP, Inc. All rights reserved 0893-3952/14 $32.00 1203 DLC1 expression is reduced in human cutaneous melanoma and correlates with patient survival Cecilia Sjoestroem1, Shahram Khosravi1, Yabin Cheng1, Gholamreza Safaee Ardekani1, Magdalena Martinka2 and Gang Li1 1Department of Dermatology and Skin Science, Vancouver Coastal Health Research Institute, University of British Columbia, Vancouver, BC, Canada and 2Department of Pathology, Vancouver General Hospital, Vancouver, BC, Canada Deleted in Liver Cancer-1 (DLC1) is a Rho-GTPase-activating protein known to be downregulated and function as a tumor suppressor in numerous solid and hematological cancers. Its expression status in melanoma is currently unknown however, prompting us to examine this. Using immunohistochemistry and tissue microarrays containing a large set of melanocytic lesions (n ¼ 539), we examined the expression profile of DLC1 in melanoma progression, as well as the association between DLC1 and patient survival. We detected both cytoplasmic and nuclear DLC1 expression, and found that whereas cytoplasmic DLC1 was significantly downregulated in metastatic melanoma compared with nevi and primary melanoma, nuclear DLC1 expression was significantly down in primary melanoma compared with nevi, and then further down in metastatic melanoma. Loss of cytoplasmic DLC1 was significantly associated with poorer overall and disease-specific 5-year survival rates of all melanoma (Po0.001 and P ¼ 0.001, respectively) and metastatic melanoma patients (P ¼ 0.020 and 0.008, respectively), and similar results were seen for nuclear DLC1 (Po0.001 for both overall and disease-specific survival for all melanoma patients, and P ¼ 0.004 for metastatic melanoma patients). Next, we examined the correlation between cytoplasmic and nuclear DLC1 and found that concomitant loss of both forms was associated with the worst outcome for metastatic melanoma patients (P ¼ 0.013 and P ¼ 0.008 for overall and disease-specific 5-year survival, respectively). Finally, multivariate Cox regression analysis determined that strong cytoplasmic and nuclear DLC1 expression was a favorable independent prognostic factor for all melanoma (HR, 0.61; 95% CI, 0.42–0.88; P ¼ 0.008) and metastatic melanoma patients (HR, 0.42; 95% CI, 0.23–0.77; P ¼ 0.005). Although more research still needs to be done on the topic, these preliminary results support the hypothesis that DLC1 is a tumor suppressor in melanoma. Modern Pathology (2014) 27, 1203–1211; doi:10.1038/modpathol.2013.223; published online 21 February 2014 Keywords: DLC1; immunohistochemistry; melanoma; metastasis; rhogap; tissue microarray; tumor suppressor The incidence of cutaneous melanoma has been accounts for less than 5% of all skin cancers, it is steadily increasing in the non-Hispanic White responsible for over 80% of all skin cancer-related population worldwide since the 1950s.1 In the deaths.2 These numbers are greatly attributable to United States, incidence rates have increased by the high metastatic potential of melanoma, and are an average 2.8% per annum since 1992 in non- reflected by a 5-year survival rate of only 5–16% for Hispanic Whites, currently making it the fifth and patients with distant metastases compared with a seventh most commonly diagnosed cancer in men rate well above 90% for patients with early-stage, and women, respectively.2 Although melanoma localized melanoma.2–5 Rho-GTPases belong to the small GTPase family of proteins, and are commonly implicated in the Correspondence: C Sjoestroem, MSc, Department of Dermatology progression and metastasis of a number of cancers, and Skin Science, Vancouver Coastal Health Research Institute, including melanoma, where specifically RhoA, University of British Columbia, Vancouver, BC, Canada. RhoC, and Cdc42 have all been positively associated E-mail: [email protected] 6,7 Received 20 May 2013; revised 14 October 2013; accepted 15 with a metastatic phenotype. Rho-GTPase activity October 2013; published online 21 February 2014 is dependent on the binding of either GTP or GDP www.modernpathology.org DLC1 expression in melanoma 1204 C Sjoestroem et al and is controlled by three groups of proteins: Materials and methods GTPase-activating proteins (GAPs), guanine exchange factors (GEFs), and guanine nucleotide dissociation Tissue Microarray Construction inhibitors. Whereas GEFs catalyze the nucleotide Seven hundred and forty-eight formalin-fixed, par- exchange, GAPs conversely catalyze the hydrolyza- affin-embedded tissues were acquired from Vancou- tion of GTP to GDP, rendering the Rho-GTPases ver General Hospital, Department of Pathology, 7–9 inactive. between 1992 and 2009 in agreement with the One RhoGAP protein extensively researched Declaration of Helsinki guidelines, as approved by over the past decade is Deleted in Liver Cancer-1 the Clinical Research Ethics Board of the University (DLC1), a protein, as the name suggests, originally of British Columbia, Vancouver, BC, Canada. Tissues found to be deleted or downregulated in about 50% with lost cores or insufficient tumor cells were 10 of primary hepatocellular carcinomas. DLC1 was excluded from the study, leaving 539 tissues avail- mapped to chromosome 8p21.3–22, a region able for evaluation. The tissue microarrays were frequently deleted in many solid tumors, including assembled as previously described.32,33 liver cancer, but has later also been found to be silenced by other, mainly epigenetic, mecha- nisms.10–12 Since its initial discovery, several Immunohistochemistry of Tissue Microarrays studies have reported DLC1 downregulation Deparaffinization of the tissue microarray slides was in a number of malignancies including breast, accomplished by heating the slides at 55 1C for renal, lung, nasopharyngeal, esophageal, cervical, 20 min followed by three 5-min washes with xylene. prostate, colorectal, oral squamous cell, and gastric Next, the slides were rehydrated by successive 5- carcinomas, as well as in multiple myelomas and min washes in 100, 95 and 80% ethanol, and lymphomas,13–23 and these studies together distilled water. Antigen retrieval was achieved by with recent in vitro and in vivo studies have heating the samples at 95 1C for 30 min in 10 mM confirmed the role of DLC1 as a bona fide tumor sodium citrate (pH 6.0), and endogenous peroxidase suppressor.24–26 activity was blocked by incubation of the slides in In addition to containing a RhoGAP domain, 3% hydrogen peroxide for 30 min. Next, the tissues responsible for catalyzing the hydrolysis of GTP were blocked with Dako antibody diluent (Dako bound to RhoA, RhoB, RhoC, and Cdc42, DLC1 Diagnostics, Glostrup, Denmark) for 30 min, fol- also contains a steroidogenic acute regulatory- lowed by incubation with a primary monoclonal related lipid transfer domain, a sterile alpha mouse anti-DLC1 antibody (1:50 dilution, Santa motif domain, and a focal adhesion-targeting (FAT) Cruz Biotechnology, Santa Cruz, CA, USA) over- domain.27,28 The FAT domain is of particular night at 4 1C. The samples were then incubated with interest as it has been shown to enhance the tumor a universal biotinylated secondary antibody and suppressor activities of DLC1.29 It is well established streptavidin-HRP (Dako Diagnostics) for 30 min that DLC1 is recruited to the focal adhesions via each, and developed using 3,30-diaminobenzidine interactions between its FAT domain and the substrate (Vector Laboratories, Burlington, ON, SH2 domains of the tensin proteins, which have Canada), followed by hematoxylin counterstaining. moreover been found to differentially regulate DLC1 activity in vitro.30,31 Our group has recently found that one tensin family member, Cten, is Evaluation of Immunostaining upregulated in the progression from melanocytic nevi to primary tumors,32 and display oncogenic DLC1 staining was evaluated and scored based on properties in vitro (unpublished data). This, intensity of staining (0–3) and percentage of DLC1- along with the numerous studies showing DLC1 expressing cells (1 (0–25%); 2 (26–50%); 3 (51– downregulation in cancer, prompted us to examine 75%); and 4 (76–100%)) by two independent the expression profile of DLC1 in melanoma, as a first observers. The degree of staining was calculated by step to characterize its role in melanoma progression multiplying the intensity score with the percentage and metastasis. of staining, and was accordingly classified as Using immunohistochemical staining and negative (0); weak (1–3); moderate (4–6); and strong tissue microarrays we examined the expression (8–12). In the event of two duplicate cores having status of DLC1 in melanoma, as well as the different staining, the higher of the two scores was correlation between DLC1 and patient survival. used for the subsequent analysis. Our results show that DLC1 exists both as a cytoplasmic and a nuclear protein in mela- Statistical Analysis nocytic lesions, and indicate that downregulation of either cytoplasmic or nuclear DLC1 is asso- Differences in demographic and clinicopathological ciated with a significantly worse 5-year patient characteristics, and DLC1 expression between sub- survival for metastatic melanoma patients, with groups were evaluated by w2-tests. Kaplan–Meier concurrent loss of both being associated with the analyses and log-rank tests were used to evaluate the worst outcome. correlation between DLC1 expression and patient Modern Pathology (2014) 27, 1203–1211 DLC1 expression in melanoma C Sjoestroem et al 1205 survival. Univariate