Levels of the EMT-related protein Snail/Slug are not correlated with p53/p63 in cutaneous squamous cell carcinoma.

BACKGROUND: The contribution of the E-cadherin transcriptional repressors Snail and Slug to invasion and metastasis has strengthened the evidence for the importance of epithelial-mesenchymal transition (EMT) in carcinoma progression. However, to the best of our knowledge, no study has described the immunohistochemical staining of the EMT-related proteins Snail/Slug in skin tumors and the correlation between Snail/Slug and tumor suppressor p53/p63. METHODS: We performed immunohistological staining of Snail, Slug, E-cadherin, p53 and p63 in 20 archived specimens each of seborrheic keratosis (SK), actinic keratosis (AK) and squamous cell carcinoma in situ (SCCIS), and 53 specimens of cutaneous squamous cell carcinomas (SCC). Fifteen normal skin (NS) specimens served as controls. RESULTS: Significant negative correlations were observed between Snail and E-cadherin expression and between Slug and E-cadherin expression (Snail: R(2) = 0.5432, p < 0.01; Slug: R(2) = 0.4666, p < 0.01). CONCLUSIONS: The staining intensities of Snail and Slug are associated with decreased E-cadherin staining in SCC and this may promote EMT. However, the staining intensities of p53 and p63 are not significantly correlated with the loss of E-cadherin.

Expression of CD10 predicts tumor progression and unfavorable prognosis in malignant melanoma.

BACKGROUND: CD10 expression in malignant melanoma (MM) has been reported to increase according to tumor progression and metastasis; however, its association with patient outcome has not been clarified. OBJECTIVE: We examined the immunohistochemical expression of CD10 in MM to determine whether or not it could serve as a marker for tumor progression and prognosis. METHODS: A total of 64 formalin-fixed, paraffin-embedded samples of primary MM were immunostained for CD10. Similarly, 40 samples of melanocytic nevus and 20 of metastatic MM were analyzed for comparison. The following clinicopathologic variables were evaluated: age, gender, histologic type, tumor site, Breslow thickness, Clark level, the presence or absence of ulceration and tumor-infiltrating lymphocytes, and survival. Statistical analyses were performed to assess for associations. Several parameters were analyzed for survival using the Kaplan-Meier method and Cox proportional hazards model. RESULTS: Immunohistochemical analysis revealed that 34 of 64 cases (53%) of primary MM expressed CD10, compared with 15 of 20 cases (75%) of metastatic MM and only 4 of 40 cases (10%) of nevus. There was a significant positive relationship between CD10 expression and Breslow thickness, Clark level, and ulceration. Univariate analysis revealed 4 significant factors for shorter survival periods: CD10 expression, high Breslow thickness, high Clark level, and the presence of ulceration (P < .01 each). In multivariate analysis, CD10 expression was revealed to be a statistically significant and independent prognostic factor. LIMITATIONS: The major limitation was the small sample size. CONCLUSION: CD10 expression may serve as a progression marker and can predict unfavorable prognosis in patients with MM.

Loss of Wnt-5α is associated with an invasive phenotype of extramammary Paget's disease.

BACKGROUND: The Wnt (wingless-type MMTV integration site) gene family encodes secretory signaling molecules that play a diverse biological role in the regulation of normal and pathological processes, including cell growth, differentiation and oncogenesis. However, the role of Wnt genes in the development of extramammary Paget's disease remains unknown. OBJECTIVE: To investigate the expression of Wnt-1, Wnt-5α and their downstream genes, ß-catenin and c-Myc, in extramammary Paget's disease. METHODS: Paraffin-embedded specimens of extramammary Paget's disease (33 specimens from 22 patients), including 7 specimens with dermal invasion and 4 with lymph node metastasis, were examined immunohistochemically for Wnt-1, Wnt-5α, ß-catenin and c-Myc. Seven normal genital skin specimens served as controls. RESULTS: The expression levels of Wnt-1 and ß-catenin in extramammary Paget's disease were significantly correlated with each other; however, their expression levels in the invasive extramammary Paget's disease were similar to those of wholly intraepithelial extramammary Paget's disease. Nuclear expression of c-Myc was significantly higher in the invasive extramammary Paget's disease in comparison with intraepithelial extramammary Paget's disease. Interestingly, the expression of Wnt-5α in invasive extramammary Paget's disease was significantly downregulated compared to wholly intraepithelial extramammary Paget's disease. CONCLUSION: The Wnt-1/ß-catenin pathway may not play an important role in the progression of extramammary Paget's disease. The loss of Wnt-5α, however, may play a role in the invasiveness of extramammary Paget's disease.

CD10 expressed by fibroblasts and melanoma cells degrades endothelin-1 secreted by human keratinocytes.

Endothelin-1 (ET-1) is a potent multifunctional peptide linked to wound healing, pigmentation, carcinogenesis, and fibrosclerotic processes in the skin. Whereas ET-1 was thought to be digested by receptor-mediated endocytosis, it is also reported to be biochemically degraded by the neutral endopeptidase CD10 using kidney homogenates. Although keratinocytes (KC) and fibroblasts (Fb) are sources of both ET-1 and CD10, respectively, there is no report investigating the direct association between CD10 expression and its function in relation to ET-1 degradation in the skin. CD10 expression in melanoma cells is associated with clinical prognosis, suggesting an important role in the invasive and metastatic potential of melanoma cells. Here, cultured KC produced much higher amounts of ET-1 than did cultured Fb or melanoma cells. In contrast, KC and A375 melanoma cells did not express CD10, while Fb, SK-MEL-28 and G361 melanoma cells constitutively expressed CD10. KC-derived ET-1 was down-modulated by both CD10-positive Fb and CD10-positive melanoma cells, and the inhibition was partially reversed under substitution conditions using CD10-knockdown Fb or CD10-knockdown melanoma cells. This indicates that CD10 on cultured Fb and melanoma cells is biochemically active in the degradation or down-modulation of ET-1 secreted from KC. These findings may lead to better understanding of skin homeostasis and of the malignant potential of melanoma.

Hematopoietic progenitor kinase 1, mitogen-activated protein/extracellular signal-related protein kinase kinase kinase 1, and phosphomitogen-activated protein kinase kinase 4 are overexpressed in extramammary Paget disease.

The c-Jun amino-terminal kinase (JNK) pathway seems to play important roles in the pathogenesis of several tumors, but its significance in extramammary Paget disease (EMPD) has not been investigated yet. The purpose of the study was to investigate the potential contribution of the JNK-associated molecules, such as hematopoietic progenitor kinase 1 (HPK1), mitogen-activated protein/extracellular signal-related protein kinase kinase kinase1 (MEKK1), transforming growth factor-ß activated kinase 1 (TAK1), and phosphomitogen-activated protein kinase kinase 4 (p-MKK4) to the development of EMPD. Thirty-five paraffin-embedded EMPD specimens were subjected to immunohistochemical staining for HPK1, MEKK1, TAK1, and p-MKK4. All the 35 EMPD, including 13 dermal invasive EMPD and 2 lymph node metastasis, showed cytoplasmic overexpression of HPK1, MEKK1, and p-MKK4. The expression (%positive cells) of HPK1, MEKK1, and p-MKK4 in EMPD (92.3% ± 8.6%, 92.9% ± 8.6%, and 92.7% ± 7.4%, respectively) were significantly higher than in normal eccrine sweat gland cells (51.6% ± 10.4%, 44.7% ± 11.7%, 0% ± 0%). In addition, the expression of HPK1-, MEKK1-, and p-MKK4 in invasive EMPD was significantly higher than in noninvasive EMPD. Meanwhile, the expression of TAK1 was basically low and no significantly different between EMPD and normal controls. In conclusion, these results indicate that JNK pathway may play a role in the pathogenesis of EMPD.

CD10-bearing fibroblast inhibits matrigel invasive potency of interleukin-1α-producing squamous cell carcinoma by diminishing substance P levels in the tumor microenvironment.

CD10 is a neutral endopeptidase, which cleaves various peptide substrates including substance P. CD10 expression has been detected in peritumoral fibroblasts (Fb) within the invasive area of various cancers such as squamous cell carcinoma (SCC). However, the biological significance of CD10-bearing Fb remains largely unknown. We examined dynamic interactions of Fb with tumorigenic A431 SCC cells or non-tumorigenic HaCaT squamous cells. The SCC and HaCaT cells did not synthesize CD10, while Fb constitutively expressed CD10. When co-cultured, SCC markedly upregulated fibroblastic CD10 expression compared with HaCaT, which was mainly attributable to SCC-derived interleukin-1α (IL-1α). Both SCC and Fb autonomously secreted substance P, which eventually enhanced the invasive capacity of SCC in a matrigel invasion assay by upregulating matrix metalloproteinase (MMP)-1 and MMP-2, but not MMP-9. Transfection of siRNA for CD10 successfully knocked down the CD10 expression in Fb (CD10ND-Fb). In the presence of CD10ND-Fb, substance P levels in supernatants as well as MMP production and the invasive potency of SCC were significantly augmented compared with control scramble RNA-transfected Fb. We also transfected CD10 vector to Fb and found that the matrigel invasive ability of SCC cells was downregulated co-cultured with CD10 vector-transfected Fb rather than empty vector-transfected Fb. In conclusion, the CD10-bearing Fb generated by SCC-derived IL-1 inhibited the invasive capacity of SCC by diminishing the microenvironmental concentration of substance P.

Nerve growth factor, brain-derived neurotrophic factor and their high-affinity receptors are overexpressed in extramammary Paget's disease.

BACKGROUND: Neurotrophin (NT) systems appear to play important roles in the pathogenesis of several tumors, but their expression in extramammary Paget's disease (EPD) has not been investigated. METHODS: Thirty-four paraffin-embedded EPD specimens (32 primary EPD and 2 metastatic to lymph nodes) were subject to immunohistochemical staining for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT3, NT4, their high-affinity receptors (TrkA, TrkB and TrkC) and the common low-affinity receptor, p75 NT receptor (p75). RESULTS: All 34 EPD specimens, including 2 metastatic to lymph nodes, showed cytoplasmic overexpression of NGF, BDNF, TrkA and TrkB. The expression (% positive cells) of NGF, BDNF, NT3, NT4, TrkA and TrkB (81.6 ± 14.9, 86.0 ± 10.4, 89.6 ± 14.9, 87.8 ± 17.9, 83 ± 14.4 and 86.2 ± 11.7%) in EPD was significantly higher than in normal skin (21.6 ± 6.5, 27.6 ± 4.5, 19.7 ± 10.1, 8.2 ± 10.0, 25.0 ± 5.3 and 25.4 ± 6.4%), and the expression of these factors in invasive EPD was significantly higher than in noninvasive EPD. Interestingly, Paget cells were negative for p75 and TrkC in all the 34 EPD specimens. CONCLUSIONS: These results suggest that overexpression of NGF, BDNF and their high-affinity receptors (TrkA and TrkB) might play a role in the pathogenesis of EPD.

Aberrant expression of tenascin-c and neuronatin in malignant peripheral nerve sheath tumors.

Development of neurofibroma (NF) and its malignant counterpart, malignant peripheral nerve sheath tumor (MPNST), is a hallmark of type I neurofibromatosis (NF1). Newly identified glycoprotein neuronatin (Nnat) is predominantly expressed in the fetal central and peripheral nervous systems and is gradually diminished according to the neural maturation. However, its expression in NFs and MPNSTs is unknown. Since an overexpression of tenascin-C (Tn-C), an extracellular matrix component, has been observed in neural malignancies, we investigated the immunohistological expressions of Nnat and Tn-C in NFs and MPNSTs, and compared their expression with that of the proliferation marker Ki-67 to possibly distinguish MPNSTs from ordinal NFs. Standard immunohistological procedure was performed for Nnat, Tn-C and Ki-67 in 9 sporadic NFs, 15 diffuse NFs (NF1), 15 plexiform NFs (NF1) and 6 MPNSTs (NF1), as well as 5 normal skins. All of the MPNSTs showed positive staining for Nnat, Tn-C and Ki-67, in sharp contrast to completely negative staining in all sporadic or NF-1-derived NFs. The aberrant expression of Nnat and Tn-C was a useful marker for distinguishing MPNSTs from benign NFs.

Design of tumor-homing and pH-responsive polypeptide-doxorubicin nanoparticles with enhanced anticancer efficacy and reduced side effects.

Tumor-homing and pH-responsive polypeptide-drug nanoparticles for targeted cancer therapy are precisely designed by site-specific drug conjugation to a bioactive and well-defined elastin-like polypeptide through an acid-labile linker. In a murine cancer model, these nanoparticles show significantly better anti-tumor efficacy and less systemic toxicity than not only free drugs, but also polypeptide-drug nanoparticles without the tumor-homing function.

A herpes simplex virus 2 (HSV-2) glycoprotein D-expressing nonreplicating dominant-negative HSV-2 virus vaccine is superior to a gD2 subunit vaccine against HSV-2 genital infection in guinea pigs.

We recently constructed a novel non-replicating dominant-negative HSV-2 recombinant viral vaccine (CJ2-gD2) capable of expressing various HSV-2 antigens that are dominant targets of HSV-2-specific CD8 T-cell response. Importantly, CJ2-gD2 expresses gD2, the HSV-2 major antigen glycoprotein D, as efficiently as wild-type HSV-2 infection and can lead to a nearly 500-fold reduction in wild-type HSV-2 viral replication in cells co-infected with CJ2-gD2 and wild-type HSV-2. In this report, we show that CJ2-gD2 elicits a strong antibody response to various HSV-2 antigens and is highly effective in the prevention of primary and recurrent HSV-2 genital infection and disease in the immunized guinea pigs. The direct comparison study between CJ2-gD2 and a gD2 subunit vaccine (gD2-alum/MPL) with a formulation akin to a vaccine tested in phase III clinical trials shows that CJ2-gD2 is 8 times more effective than the gD2-alum/MPL subunit vaccine in eliciting an anti-HSV-2 specific neutralizing antibody response and offers significantly superior protection against primary and recurrent HSV-2 genital infections. Importantly, no challenge wild-type HSV-2 viral DNA was detectable in dorsal root ganglia DNA isolated from CJ2-gD2-immunized guinea pigs on day 60 post-challenge. CJ2-gD2 should be an excellent HSV-2 vaccine candidate for protection against HSV-2 genital infection and disease in humans.

Arginine metabolism in soft tissue sarcoma.

BACKGROUND: L-Arginine (L-Arg) is a conditionally essential amino acid for humans, which is the substrate for both arginase (ARG) and the inducible form of nitric oxide synthase (iNOS) enzymes. Whether L-Arg metabolism has detrimental or beneficial influence on the tumor growth depends on local up regulation of the NOS or ARG pathways at the tumor site. OBJECTIVE: To evaluate the expression profile of ARG and iNOS in various histological subtypes of soft tissue sarcomas (STSs). METHODS: A series of 81 adult STSs were tested for ARG1, ARG2 and iNOS expression by immunohistochemical analysis. RESULTS: ARG1, ARG2 and iNOS expression was found in tumor cells of all cases of STSs except dermatofibrosarcoma protuberans (DFSP) in a cytoplasmic pattern. However, there was no significant correlation found between ARG, iNOS expression and histopathological parameters. Conversely, the majority of DFSP were devoid of ARG and iNOS expression, while only two cases showed focal and weak expression. CONCLUSIONS: Overexpression of L-Arg-metabolizing enzymes ARG and iNOS in tumor cells of all of the STS cases except DFSP may have a role in mediating the biological processes which characterize STSs. New knowledge of the regulation of arginine metabolism in tumor tissues is key to designing sound therapeutic means to effectively prevent tumorigenesis. Further studies are needed to clarify the absence of ARG and iNOS staining in DFSP.

Cathepsin K-upregulation in fibroblasts promotes matrigel invasive ability of squamous cell carcinoma cells via tumor-derived IL-1α.

BACKGROUND: Cathepsin K (CTSK), a cysteine protease with strong collagenolytic properties, is involved in extracellular matrix turnover. In the previous studies, CTSK expression was detected in peritumoral fibroblasts (Fbs) around squamous cell carcinoma (SCC), but not in those surrounding benign epidermal tumors. However, the mechanism governing CTSK expression in epidermal tumors remains unclear. OBJECTIVE: To study the regulatory mechanisms of fibroblastic CTSK expression in the SCC-stromal interaction. METHODS: We examined dynamic interactions of Fbs with tumorigenic SCC cells (A431 and A253) or normal human keratinocytes. RESULTS: SCC cells and normal keratinocytes did not synthesize CTSK, while Fbs constitutively expressed CTSK. When cocultured, SCC cells upregulated fibroblastic CTSK expression more potently than did normal keratinocytes, which was mainly attributable to SCC-derived IL-1α. Coculturing Fbs with SCC cells significantly augmented the matrigel invasive ability of SCC cells, which was downregulated when cocultured with CTSK knockdown Fbs or in the presence of neutralizing anti-IL-1α antibody. CONCLUSION: The CTSK-upregulated Fbs generated by SCC-derived IL-1α may play a crucial role in the progression and invasion of SCC.

CD10-bearing fibroblasts may inhibit skin inflammation by down-modulating substance P.

Substance P (SP) is a multipotent neuropeptide that affects the proliferation, activation and motility of keratinocytes and fibroblasts (Fbs). SP in pulmonary and synovial cells is degraded by CD10, a 90- to 110-kDa cell surface zinc-dependent metalloprotease. However, the expression and function of CD10 in human dermal Fbs have not yet been investigated in vivo and in vitro specifically with reference to SP. Our immunohistologic study revealed moderate to strong fibroblastic CD10 expression in the majority of psoriasis vulgaris (16/16), chronic eczema (15/16), lichen planus (18/20) and atopic dermatitis (4/5). Keratinocytes showed no CD10 expression in vivo and in vitro. Cultured Fbs constitutively expressed CD10 and SP. CD10 expression was augmented by external interleukin (IL)-1ß and IL-22, but not by IL-8 and IL-17A in Fbs. SP production was enhanced in CD10 knockdown-Fbs (CD10ND-Fbs) compared with control-Fbs. In the presence of IL-1ß or IL-22, the enhancement of SP production was more prominent in CD10ND-Fbs than in control-Fbs, suggesting the down-modulating activity of CD10 on SP in cytokine-mediated inflammation. In conclusion, fibroblastic CD10 expression may down-regulate skin inflammation by degrading SP or reducing its level in the dermal microenvironment.

Clinicopathologic and prognostic significance of SATB1 in cutaneous malignant melanoma.

BACKGROUND: Special AT-rich sequence-binding protein-1 (SATB1), a new type of gene regulator, has been reported to be expressed in several human cancers and may have malignant potential. However, no data on SATB1 expression and its relationship to tumor progression in cutaneous malignant melanoma (CMM) has yet been reported. OBJECTIVE: We examined the immunohistochemical expression of SATB1 in CMM to determine whether it could serve as a prognostic marker. METHODS: A total of 97 samples of primary CMM and controls were immunostained for SATB1. The following clinicopathologic variables were evaluated: age, gender, subtype, SATB1 expression, Breslow thickness, Clark level, presence of ulceration, lymph node metastasis, distant metastasis, and survival. Statistical analyses were performed to assess for associations. Several parameters were analyzed for survival using the Kaplan-Meier method and Cox proportional-hazards model. RESULTS: Forty cases (85.1%) of CMM showed positive staining for SATB1 by immunohistochemistry. The intensity of SATB1 staining was significantly higher in CMM than in nevus NV and normal skin (NS) (P < 0.01). High SATB1 expression was significantly correlated with Breslow thickness, Clark level, mortality, presence of ulceration, and lymph node metastasis (P < 0.01). Moreover, Kaplan-Meier analysis revealed that SATB1 overexpression was significantly associated with worse survival (P < 0.01). Further univariate analysis and multivariate regression analysis indicated that SATB1 expression was an independent prognostic marker for CMM (P = 0.03). CONCLUSIONS: The overexpression of SATB1 correlated with metastatic potential of CMM and is a novel independent prognostic marker for predicting outcome.