Induction of ZEB proteins by inactivation of RB protein is key determinant of mesenchymal phenotype of breast cancer.

We previously showed that depletion of the retinoblastoma protein (RB) induces down-regulation of the adhesion molecule E-cadherin and thereby triggers the epithelial-mesenchymal transition. To further characterize the effect of RB inactivation on the phenotype of cancer cells, we have now examined RB expression in human breast cancer cell lines and clinical specimens. We found that RB-inactive cells exhibit a mesenchymal-like morphology and are highly invasive. We also found that ZEB proteins, transcriptional repressors of the E-cadherin gene, are markedly up-regulated in these cells in a manner sensitive to the miR-200 family of microRNAs. Moreover, depletion of ZEB in RB-inactive cells suppressed cell invasiveness and proliferation and induced epithelial marker expression. These results implicate ZEB in induction of the epithelial-mesenchymal transition, as well as in maintenance of the mesenchymal phenotype in RB-inactive cells. We also developed a screening program for inhibitors of ZEB1 expression and thereby identified several cyclin-dependent kinase inhibitors that blocked both ZEB1 expression and RB phosphorylation. Together, our findings suggest that RB inactivation contributes to tumor progression not only through loss of cell cycle control but also through up-regulation of ZEB expression and induction of an invasive phenotype.

Loss of p16 expression is associated with the stem cell characteristics of surface markers and therapeutic resistance in estrogen receptor-negative breast cancer.

Triple-negative breast cancer [TNBC, which is negative for the estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2] is a high-risk form of the disease without a specific therapy. DNA microarray and immunohistochemical analyses have shown that most TNBCs fall within the basal-like histological subset of breast cancers, which frequently exhibit inactivation of the retinoblastoma tumor suppressor (Rb) and upregulation of the cyclin-dependent kinase inhibitor p16(INK4a) (p16). However, downregulation of p16 expression has been observed in some basal-like breast cancer cell lines, suggesting that such cells can be divided into two groups according to Rb and p16 status. We now show that cells that are CD44(+) and CD24(-) , a phenotype associated with stem-like breast cancer cells, are more abundant in ER(-) /p16(-) breast cancer cell lines than in ER(-) /p16(+) lines. It was also found that p16 expression was downregulated in mammospheres from an ER-negative breast cancer cell line. Depletion of p16 by RNA interference in ER-negative breast cancer cells increased the percentage of CD44(+) /CD24(-) cells and increased the expression of mRNA of the ES-like genes Nanog, Oct4, and Sox2 through an Rb-independent pathway. Furthermore, such depletion of p16 reduced chemosensitivity. The loss of p16 expression may thus reduce the response of ER-negative breast cancer cells to chemotherapy by conferring cancer stem cell-like properties. Consistent with this conclusion, immunohistochemical analysis of the clinical samples suggests that low p16 expression in TNBC is associated with resistance to preoperative chemotherapy.

Bone marrow transplantation restores epidermal basement membrane protein expression and rescues epidermolysis bullosa model mice.

Attempts to treat congenital protein deficiencies using bone marrow-derived cells have been reported. These efforts have been based on the concepts of stem cell plasticity. However, it is considered more difficult to restore structural proteins than to restore secretory enzymes. This study aims to clarify whether bone marrow transplantation (BMT) treatment can rescue epidermolysis bullosa (EB) caused by defects in keratinocyte structural proteins. BMT treatment of adult collagen XVII (Col17) knockout mice induced donor-derived keratinocytes and Col17 expression associated with the recovery of hemidesmosomal structure and better skin manifestations, as well improving the survival rate. Both hematopoietic and mesenchymal stem cells have the potential to produce Col17 in the BMT treatment model. Furthermore, human cord blood CD34(+) cells also differentiated into keratinocytes and expressed human skin component proteins in transplanted immunocompromised (NOD/SCID/gamma(c)(null)) mice. The current conventional BMT techniques have significant potential as a systemic therapeutic approach for the treatment of human EB.

Topical application of anti-angiogenic peptides based on pigment epithelium-derived factor can improve psoriasis.

BACKGROUND: Psoriasis is a common chronic inflammatory skin disorder with a high prevalence (3-5%) in the Caucasian population. Although the number of capillary vessels increases in psoriatic lesions, there have been few reports that have specifically examined the role of angiogenesis in psoriasis. Angiogenic factors, such as vascular endothelial growth factor (VEGF), may dominate the activity of anti-angiogenic factors and accelerate angiogenesis in psoriatic skin. OBJECTIVE: We investigated to identify small peptide mimetics of PEDF that might show anti-angiogenic potential for the topical treatment for psoriasis. METHODS: We examined the expression of PEDF in skin by immunohistochemical staining, immunoblotting, and RT-PCR. To identify potential PEDF peptides, we screened peptides derived from the proteolytic fragmentation of PEDF for their anti-proliferative action. Anti-psoriatic functions of these peptides were analyzed using a mouse graft model of psoriasis. RESULTS: The specific low-molecular weight peptides (MW<850 Da) penetrated the skin and showed significant anti-angiogenic activity in vitro. Topical application of these peptides in a severe combined immunodeficient mouse model of psoriatic disease led to reduced angiogenesis and epidermal thickness. CONCLUSIONS: These data suggest that low-molecular PEDF peptides with anti-angiogenic activity may be a novel therapeutic strategy for psoriasis.

Decreased expression of neurofibromin contributes to epithelial-mesenchymal transition in neurofibromatosis type 1.

Plexiform and/or dermal neurofibromas are nerve sheath tumors of the peripheral nervous system that are usually present in individuals with neurofibromatosis type 1 (NF1). Neurofibromas arise from Schwann cells with biallelic inactivation of NF1, the gene that encodes neurofibromin. This protein is responsible for regulation of the Ras-mediated pathway, which has been shown to play a crucial role in epithelial-to-mesenchymal transition (EMT). EMT is a biological process that occurs during embryogenesis and wound healing and is involved in pathological processes such as organ fibrosis and cancer metastasis. However, the relationship between neurofibromin and EMT has not been elucidated. We investigated whether the EMT-related signaling pathway was upregulated in NF1-associated neurofibromas and Schwann cells by assessing the expression levels of the EMT-related transcription factors Snail, Slug, Twist, ZEB1 and ZEB2. Immunohistochemical studies and quantitative reverse transcription polymerase chain reaction revealed an increase in the expression levels of EMT-related transcription factors in neurofibroma specimens and NF1-derived Schwann cells (sNF96.2). In addition, the silencing of NF1 by siRNA induced the expression of EMT-related transcription factors in normal human Schwann cells and in epithelial-like breast cancer cells. Our findings suggest that the loss of neurofibromin activated the EMT-related signaling pathway and that the excessive mesenchymal reaction may play a key role in the development of NF1-associated neurofibromas.

DNA vaccination against macrophage migration inhibitory factor improves atopic dermatitis in murine models.

BACKGROUND: Atopic dermatitis (AD) is a common chronic inflammatory skin disease. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that has been implicated in the pathogenesis of AD. Recently, we developed a novel DNA vaccine that generates neutralizing endogenous anti-MIF antibodies. OBJECTIVE: This study explores the preventive and therapeutic effects of this MIF-DNA vaccine in mouse models of AD. METHODS: Two different AD model mice (DS-Nh and NC/Nga) received MIF-DNA vaccination to analyze preventive and therapeutic effects, as assessed by clinical skin scores, histologic findings, and serum IgE levels. RESULTS: In murine models of AD, MIF-DNA vaccination prevented the occurrence of the AD skin phenotype. Furthermore, administration of MIF-DNA vaccine to mice that had already developed AD produced a rapid improvement in AD skin manifestation. There were reduced histologic signs of inflammation and lower serum IgE levels in treated mice compared with those seen in control animals. Finally, passive transfer of IgG from MIF-DNA vaccinated mice to AD mice also produced a significant therapeutic effect. These results demonstrate that MIF-DNA vaccination not only prevents the development of AD but also improves the symptoms of pre-existing AD. CONCLUSION: Taken together, the induction of an anti-MIF autoantibody response using MIF-DNA vaccination appears to be a useful approach in the treatment of AD.

Bone marrow-derived cells are not the origin of the cancer stem cells in ultraviolet-induced skin cancer.

Several lines of evidence have demonstrated that various cancers are derived from cancer stem cells (CSCs), which are thought to originate from either tissue stem or progenitor cells. However, recent studies have suggested that the origin of CSCs could be bone marrow-derived cells (BMDCs); for example, gastric cancer, which follows persistent gastric inflammation, appears to originate from BMDCs. Although our previous research showed the capability of BMDCs to differentiate into epidermal keratinocytes, it has yet to be determined whether skin CSCs originate from BMDCs. To assess the possibility that BMDCs could be the origin of CSCs in skin squamous cell carcinoma (SCC), we used a mouse model of UVB-induced skin SCC. We detected a low percentage of BMDCs in the lesions of epidermal dysplasia (0.59%), SCC in situ (0.15%), and SCC (0.03%). Furthermore, we could not find any evidence of clonal BMDC expansion. In SCC lesions, we also found that most of the BMDCs were tumor-infiltrating hematopoietic cells. In addition, BMDCs in the SCC lesions lacked characteristics of epidermal stem cells, including expression of stem cell markers (CD34, high alpha6 integrin) and the potential retention of BrdU label. These results indicate that BMDCs are not a major source of malignant keratinocytes in UVB-induced SCC. Therefore, we conclude that BMDCs are not the origin of CSCs in UVB-induced SCC.

Mesenchymal stem cells are recruited into wounded skin and contribute to wound repair by transdifferentiation into multiple skin cell type.

Mesenchymal stem cells (MSCs) can differentiate not only into mesenchymal lineage cells but also into various other cell lineages. As MSCs can easily be isolated from bone marrow, they can be used in various tissue engineering strategies. In this study, we assessed whether MSCs can differentiate into multiple skin cell types including keratinocytes and contribute to wound repair. First, we found keratin 14-positive cells, presumed to be keratinocytes that transdifferentiated from MSCs in vitro. Next, we assessed whether MSCs can transdifferentiate into multiple skin cell types in vivo. At sites of mouse wounds that had been i.v. injected with MSCs derived from GFP transgenic mice, we detected GFP-positive cells associated with specific markers for keratinocytes, endothelial cells, and pericytes. Because MSCs are predominantly located in bone marrow, we investigated the main MSC recruitment mechanism. MSCs expressed several chemokine receptors; especially CCR7, which is a receptor of SLC/CCL21, that enhanced MSC migration. Finally, MSC-injected mice underwent rapid wound repaired. Furthermore, intradermal injection of SLC/CCL21 increased the migration of MSCs, which resulted in an even greater acceleration of wound repair. Taken together, we have demonstrated that MSCs contribute to wound repair via processes involving MSCs differentiation various cell components of the skin.

CTACK/CCL27 accelerates skin regeneration via accumulation of bone marrow-derived keratinocytes.

Recent studies have suggested that bone marrow (BM) cells transdifferentiate to regenerate a variety of cellular lineages. Due to the relatively small population of BM-derived cells in each organ, it is still controversial whether these BM-derived cells are really present in sufficient numbers for effective function. Conversely, it is speculated that chemokine/chemokine receptor interactions mediate this migration of the tissue-specific precursor cells from BM into the target tissue. Here, we show that cutaneous T-cell attracting chemokine (CTACK)/CCL27 is the major regulator involved in the migration of keratinocyte precursor cells from BM into skin. By screening various chemokine expression patterns, we demonstrated that CTACK is constitutively expressed in normal skin and upregulated in wounds and that approximately 20% of CD34(+) BM cells expressed CCR10, the ligand for CTACK. Intradermal injection of CTACK/CCL27 into the periphery of skin wounds significantly enhanced BM-derived keratinocyte (BMDK) migration, and CTACK/CCL27 neutralizing antibody inhibited this BMDK migration. Furthermore, increased BMDK migration caused by CTACK/CCL27 significantly accelerated the wound-healing process without any influence over either angiogenesis or keratinocyte proliferation. These results provide direct evidence that recruitment of BM keratinocyte precursor cells to the skin is regulated by specific chemokine/chemokine receptor interactions, making possible the development of new regenerative therapeutic strategies.

Presence of circulating CCR10+ T cells and elevated serum CTACK/CCL27 in the early stage of mycosis fungoides.

PURPOSE: Mycosis fungoides (MF), a common type of cutaneous T cell lymphoma with an indolent clinical course, has the characteristic that malignant T cell clones are recruited into the skin from the early disease stages. The mechanisms of recruitment have been suggested from our knowledge of various chemokine-chemokine receptor interactions. Recently, CCR10 and CTACK/CCL27 were proposed to play a role in the recruitment of other types of cutaneous T cell lymphoma. We examined the expression of CCR10 in peripheral blood and serum CTACK/CCL27 levels in patients with MF. EXPERIMENTAL DESIGN: Eighteen patients with MF, six patients with atopic dermatitis, and nine healthy volunteers were enrolled in our investigation. We investigated the differences in CCR10+ CD4+ expression in peripheral blood mononuclear cells by flow cytometry. Serum CTACK/CCL27 levels were determined using a CTACK/CCL27 ELISA assay kit. RESULTS: The number of circulating CCR10+ CD4+ cells was significantly higher in MF peripheral blood than in controls, even during the early stages. In lesional MF skin, infiltrating tumor cells also showed extensive expression of CCR10. The serum level of CTACK/CCL27 was higher in patients with MF than normal controls, but no statistical difference was found compared with atopic dermatitis patients. CONCLUSIONS: CCR10-CTACK/CCL27 interactions between circulating T cells and keratinocytes would seem to play an important role in the pathophysiology of MF from the early disease stages.