Brown Locus Protein Research Articles

Abstract 4742: Anti-tyrosinase related protein 1 (TRP1) antibody combined with cyclophosphamide induces adaptive immunity against melanoma

Abstract Monoclonal antibodies targeting tumor antigens are an effective anti-cancer immunotherapy, with well established benefit against multiple tumor types. They are most potent when given in combination with chemotherapy. The mechanisms underlying the efficacy of combination therapy, however, are poorly understood.1 Tyrosinase-related protein 1 (TRP1) is a protein implicated in pigment formation. TRP1 is found primarily in the melanosome, but surface expression can be detected in vivo. An antibody targeting TRP1 is currently in clinical testing. In mice, anti-TRP1 prophylaxis controls subsequent growth of B16 melanoma, but has minimal activity as a single agent against established disease.2 We have previously shown that anti-TRP1 enhances T cell responses induced by DNA vaccination.3 We now report that anti-TRP1 enhances the cytotoxic effect of cyclophosphamide chemotherapy in the treatment of established cutaneous B16 melanoma. We find considerable efficacy when anti-TRP1 is combined with cyclophosphamide chemotherapy with treatment initiated at timepoints up to day 7 when tumors are palpable with long term disease control observed in some animals. Treatment is ineffective in mice deficient in the Fc receptor common γ chain. Combination therapy is associated with increased infiltration of tumors by CD8+ T lymphocytes visualized by immunofluorescence (IF) and flow cytometry. Mice receiving combination therapy have increased infiltration by CD4+foxp3- T cells, but not by CD4+foxp3+ T cells. Therapeutic efficacy was decreased but not abrogated in animals depleted of CD8+ T cells through injection of anti-CD8 IgG. Surprisingly, although B16 is a non-immunogenic tumor, surviving mice treated with cyclophosphamide and ‘passive’ immunization with anti-TRP1 displayed resistance to repeat inoculation with B16 melanoma. These data strongly implicate a role for adaptive immunity in the therapeutic efficacy of anti-tumor antibodies combined with chemotherapy and has implications for design of future clinical trials using these agents and for the development of novel strategies to induce adaptive anti-tumor immunity. 1. Ferris RL, Jaffee EM, Ferrone S. Tumor antigen-targeted, monoclonal antibody-based immunotherapy: clinical response, cellular immunity, and immunoescape. J Clin Oncol 2010;28:4390-9. 2. Hara I, Takechi Y, Houghton AN. Implicating a role for immune recognition of self in tumor rejection: passive immunization against the brown locus protein. J Exp Med 1995;182:1609-14. 3. Saenger YM, Li Y, Chiou KC, et al. Improved tumor immunity using anti-tyrosinase related protein-1 monoclonal antibody combined with DNA vaccines in murine melanoma. Cancer Res 2008;68:9884-91. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4742. doi:10.1158/1538-7445.AM2011-4742

Rab33A: Characterization, Expression, and Suppression by Epigenetic Modification

Rab33A, a member of the small GTPase superfamily, is an X-linked gene that is expressed in brain, lymphocytes, and normal melanocytes, but is downregulated in melanoma cells. We demonstrate that in normal melanocytes Rab33A colocalizes with melanosomal proteins and that a constitutively active GTPase mutant suppresses their transport to the melanosomes. In the brain, Rab33A is present throughout the cortex, as well as in the hippocampal CA fields. A survey of melanocytic lesions demonstrated that aberrant downregulation of Rab33A is an early event that is already prevalent in melanocytes of giant congenital nevi. Analyses of bisulfite-modified DNA revealed that Rab33A is regulated by DNA methylation of a specific promoter region proximal to the transcription initiation site, and that suppression of Rab33A in melanoma cells recapitulates normal processes that control silencing of X-linked genes, but not tissue specific gene expression. This information is important for understanding carcinogenesis as well as other aberrant processes because Rab33A may have an important role in disorders involving X-chromosome-linked genes associated with vesicular transport.

Coupling and uncoupling of tumor immunity and autoimmunity.

Self-antigens, in the form of differentiation antigens, are commonly recognized by the immune system on melanoma and other cancers. We have shown previously that active immunization of mice against the melanocyte differentiation antigen, a tyrosinase-related protein (TRP) gp75(TRP-1) (the brown locus protein) expressed by melanomas, could induce tumor immunity and autoimmunity manifested as depigmentation. In this system, tumor immunity and autoimmunity were mediated by autoantibodies. Here, we characterize immunity against another tyrosinase family glycoprotein TRP-2 (the slaty locus protein), using the same mouse model and method of immunization. As observed previously for gp75(TRP-1), immunity was induced by DNA immunization against a xenogeneic form of TRP-2, but not against the syngeneic gene, and depended on CD4(+) cells. Immunization against TRP-2 induced autoantibodies and autoreactive cytotoxic T cells. In contrast to immunization against gp75(TRP-1), both tumor immunity and autoimmunity required CD8(+) T cells, but not antibodies. Only autoimmunity required perforin, whereas tumor immunity proceeded in the absence of perforin. Thus, immunity induced against two closely related autoantigens that are highly conserved throughout vertebrate evolution involved qualitatively different mechanisms, i.e., antibody versus CD8(+) T cell. However, both pathways led to tumor immunity and identical phenotypic manifestations of autoimmunity.

Tyrosinase Stabilization by Tyrp1 (the brown Locus Protein)

Mammalian melanogenesis is regulated directly or indirectly by over 85 distinct loci. The Tyr/albino locus, in which mutations cause a lack of pigmentation, encodes tyrosinase (Tyr), the critical and rate-limiting melanogenic enzyme. Other melanogenic enzymes include Tyrp1 (or TRP1) and 3,4-dihydroxyphenylalanine-chrome tautomerase (Dct or TRP2) encoded at the Tyrp1/brown and Dct/slaty loci, respectively. Murine Tyrp1 can oxidize 5, 6-dihydroxyindole-2-carboxylic acid (DHICA) produced by Dct, but mutations in Tyrp1 also affect the catalytic functions of Tyr. All three enzymes are membrane-bound melanosomal proteins with similar structural features and are thought to interact within and stabilize a melanogenic complex. We have now further investigated the effect of a Tyrp1(b) mutation on Tyr stability. Pulse/chase labeling experiments show that Tyr is degraded more quickly in Tyrp1(b) mutant melanocytes than in melanocytes wild type at that locus. This reduced stability of Tyr can be partly rescued by infection with the wild type Tyrp1 gene, and this is accompanied by phenotypic rescue of infected melanocytes. In sum, these results suggest that, in addition to its catalytic function in oxidizing DHICA, Tyrp1 may play an important role in stabilizing Tyr, a second potential role in the regulation of melanin formation.

Tumor immunity and autoimmunity induced by immunization with homologous DNA.

The immune system can recognize self antigens expressed by cancer cells. Differentiation antigens are prototypes of these self antigens, being expressed by cancer cells and their normal cell counterparts. The tyrosinase family proteins are well characterized differentiation antigens recognized by antibodies and T cells of patients with melanoma. However, immune tolerance may prevent immunity directed against these antigens. Immunity to the brown locus protein, gp75/ tyrosinase-related protein-1, was investigated in a syngeneic mouse model. C57BL/6 mice, which are tolerant to gp75, generated autoantibodies against gp75 after immunization with DNA encoding human gp75 but not syngeneic mouse gp75. Priming with human gp75 DNA broke tolerance to mouse gp75. Immunity against mouse gp75 provided significant tumor protection. Manifestations of autoimmunity were observed, characterized by coat depigmentation. Rejection of tumor challenge required CD4(+) and NK1.1(+) cells and Fc receptor gamma-chain, but depigmentation did not require these components. Thus, immunization with homologous DNA broke tolerance against mouse gp75, possibly by providing help from CD4(+) T cells. Mechanisms required for tumor protection were not necessary for autoimmunity, demonstrating that tumor immunity can be uncoupled from autoimmune manifestations.

Indication of a common ancestry for copper tyrosinases and heme catalases revealed by hydrophobic cluster analysis of the brown locus protein sequence.

In mammalian melanocytes, a membrane-anchored high molecular weight protein complex consisting of tyrosinase (monophenol monooxygenase, EC 1.14.18.1) and two tyrosinase-related proteins (TRP-1 and TRP-2, respectively), catalyzes the conversion of tyrosine into the pigment eumelanin. These three proteins are members of a large gene family of binuclear copper proteins (Halaban and Moellmann, 1990). TRP-2 has recently been shown (Tsukamoto et al., 1992; Solano et al., 1996) to possess DOPAchrome tautomerase activity (EC 5.3.2.3). On the other hand, the function of TRP-1, the most abundant glycoprotein expressed in human melanocytes remains controversial. This protein, encoded by the brown locus, exhibits more than 50% sequence identity with tyrosinase (Cohen et al., 1990) and shows some tyrosine hydroxylase activity, but may specifically act as 5,6-dihydroxyindole-2-carboxylic acid (DHICA) oxidase (Jimenez-Cervantes et al., 1994; Kobayashi et al., 1994). However, this protein has also been suggested to act as a melanocyte-specific catalase entitled TRP-1 or catalase B (Halaban and Moellmann, 1990). Several observations support this: the oxidation of 5,6dihydroxyindole and DHICA is associated with the generation of hydrogen peroxide, which is in turn known to destroy melanin and some of its precursors. H2O2 may thus regulate the expression of tyrosinase in melanocytes (Karg et al., 1993), and obviously the course of melanogenesis is strongly affected by factors modulating the level of intracellular peroxides (Nappi and Vass, 1996). Furthermore, H2O2 is a rather potent reversible inhibitor of tyrosinase (Schallreuther et al., 1991), and the oxy-form of this enzyme even shows a limited catalatic potential (Wood and Schallreuther, 1991). The evolutionary relationships among different members of the tyrosinase family have been studied (Morrison et al., 1994), but so far there was no indication of any structural as well as evolutionary relatedness between TRP-1 (catalase B) and the family of heme containing catalases (EC 1.11.1.6). This is caused mainly by the fact that the sequences of brown locus proteins (which obviously bind copper) and those of heme catalases exhibit no easily detectable sequence similarities. For instance, the sequence identity between human TRP-1 (Cohen et al., 1990) and Saccharomyces cerevisiae catalase A (Cohen et al., 1988) is only about 16% in the 400 amino acid overlap (alignment not shown). The presumably related function of these two supposedly structurally unrelated enzymes has evoked the idea on their eventual very distant evolutionary relationship. We have recently shown that one of the most strictly conserved structural parts among heme catalases is formed by the major substrate channel allowing the diffusion

Adenovirus-mediated expression of melanoma antigen gp75 as immunotherapy for metastatic melanoma.

Melanocyte differentiation antigens, such as the brown locus protein gp75, are potential biological targets for immunotherapy. We investigated whether expression of the murine gp75 cDNA mediated by an adenovirus (Ad) vector could induce melanoma rejection using this model self antigen that usually induces tolerance, and whether Ad vector-directed production of interleukin-2 (IL2) might augment this response. To evaluate this approach, Ad vectors were constructed containing the murine gp75 cDNA (Ad.gp75) and the human IL2 cDNA (Ad.IL2). Efficacy was evaluated in C57BI/6 mice challenged i.v. with 10(5) B16 cells, using the number of lung metastases as the efficacy parameter. Naive control mice developed 175 +/- 12 metastases by day 14. Controls receiving intranasal Ad.IL2 1 day after B16 cell injection, intraperitoneal (i.p.) mitomycin-C-treated B16 cells +/- i.p. Ad.IL2 before B16 cell challenge and Ad.beta gal-treated mice had similar numbers of metastases as controls (P > 0.1). In marked contrast, preimmunization with intradermal Ad.gp75 provided dramatic reduction in the number of lung metastases (52 +/- 7, 29% of control). Addition of regional (intranasal delivery to the lung) Ad.IL2 to intradermal Ad.gp75 preimmunization 1 day following tumor challenge provided further protection (18 +/- 6, 10% of control). Depletion of CD4+ and CD8+ T-cell subsets effectively blocked the protective effect seen following immunization. Adoptive transfer of macrophage-depleted splenocytes from Ad.gp75-immunized mice similarly afforded significant protection against B16 tumor cell challenge. Further, serum obtained 21 days following Ad.gp75 immunization showed no detectable anti-gp75 antibody by immunoprecipitation. These results suggest that immunization with Ad.gp75 induces cellular immune responses that are capable of rejecting B16 melanoma in a host that is usually tolerant to gp75 antigen.

The Cytoplasmic Tail of the Mouse Brown Locus Product Determines Intracellular Stability and Export from the Endoplasmic Reticulum

The Cytoplasmic Tail of the Mouse Brown Locus Product Determines Intracellular Stability and Export from the Endoplasmic Reticulum

Fc receptors are required in passive and active immunity to melanoma.

Effective tumor immunity requires recognition of tumor cells coupled with the activation of host effector responses. Fc receptor (FcR) gamma-/- mice, which lack the activating Fc gamma R types I and III, did not demonstrate protective tumor immunity in models of passive and active immunization against a relevant tumor differentiation antigen, the brown locus protein gp75. In wild-type mice, passive immunization with mAb against gp75 or active immunization against gp75 prevented the development of lung metastases. This protective response was completely abolished in FcR gamma-deficient mice. Immune responses were intact in gamma-/- mice because IgG titers against gp75 develop normally in gamma-/- mice immunized with gp75. However, uncoupling of the Fc gamma R effector pathway from antibody recognition of tumor antigens resulted in a loss of protection against tumor challenge. These data demonstrate an unexpected and critical role for FcRs in mediating tumor cytotoxicity in vivo and suggest that enhancement of Fc gamma R-mediated antibody-dependent cellular cytotoxicity by inflammatory cells is a key step in the development of effective tumor immunotherapeutics.

Sorting and Secretion of a Melanosome Membrane Protein, gp75/TRP1

The melanosome is an organelle specialized for melanin synthesis that is derived from the endocytic pathway. Several melanosome membrane proteins have been identified, forming a family of proteins known as tyrosinase-related proteins. Two members of this family, tyrosinase and gp75, are well-characterized melanocyte differentiation antigens. Our previous studies have shown that gp75, the mouse brown locus protein, is sorted to melanosomes along the endocytic pathway, directed by a hexapeptide sorting signal located in the cytoplasmic tail. In this study, we report the unexpected finding that a portion of gp75 is secreted. Substantial levels of secretory gp75 were detected in melanocytic cells. Cell surface expression of gp75 was also detected, representing 2% of cellular gp75. Characterization of secretory gp75 cells showed that it is: (i) a truncated form that lacks the transmembrane region, the cytoplasmic tail where the endosomal sorting signal is located, and a small portion of the lumenal domain; (ii) more extensively glycosylated than endocytic/melanosomal gp75, containing trans-Golgi processed sugar residues; and (iii) generated post-translationally in an acid sensitive compartment after processing in the trans-Golgi, and secreted rapidly after generation. Thus, these endocytic/melanosomal membrane proteins can be processed to abundant secretory forms, probably in an endocytic compartment through a potentially novel secretory pathway.

Implicating a role for immune recognition of self in tumor rejection: passive immunization against the brown locus protein.

The immune system can recognize differentiation antigens that are selectively expressed on malignant cells and their normal cell counterparts. However, it is uncertain whether immunity to differentiation antigens can effectively lead to tumor rejection. The mouse brown locus protein, gp75 or tyrosinase-related protein 1, is a melanocyte differentiation antigen expressed by melanomas and normal melanocytes. The gp75 antigen is recognized by autoantibodies and autoreactive T cells in persons with melanoma. To model autoimmunity against a melanocyte differentiation antigen, mouse antibodies against gp75 were passively transferred into tumor-bearing mice. Passive immunization with a mouse monoclonal antibody against gp75 induced protection and rejection of both subcutaneous tumors and lung metastases in syngeneic C57BL/6 mice, including established tumors. Passive immunity produced coat color alterations but only in regenerating hairs. This system provides a model for autoimmune vitiligo and shows that immune responses to melanocyte differentiation antigens can influence mouse coat color. Immune recognition of a melanocyte differentiation antigen can reject tumors, providing a basis for targeting tissue autoantigens expressed on cancer.

Intracellular sorting and targeting of melanosomal membrane proteins: identification of signals for sorting of the human brown locus protein, gp75.

The structural and functional integrity of cytoplasmic organelles is maintained by intracellular mechanisms that sort and target newly synthesized proteins to their appropriate cellular locations. In melanocytic cells, melanin pigment is synthesized in specialized organelles, melanosomes. A family of melanocyte-specific proteins, known as tyrosinase-related proteins that regulate melanin pigment synthesis, is localized to the melanosomal membrane. The human brown locus protein, tyrosinase-related protein-1 or gp75, is the most abundant glycoprotein in melanocytic cells, and is a prototype for melanosomal membrane proteins. To investigate the signals that allow intracellular retention and sorting of glycoprotein (gp)75, we constructed protein chimeras containing the amino-terminal extracellular domain of the T lymphocyte surface protein CD8, and transmembrane and cytoplasmic domains of gp75. In fibroblast transfectants, chimeric CD8 molecules containing the 36-amino acid cytoplasmic domain of gp75 were retained in cytoplasmic organelles. Signals in the gp75 cytoplasmic tail alone, were sufficient for intracellular retention and targeting of the chimeric proteins to the endosomal/lysosomal compartment. Analysis of subcellular localization of carboxy-terminal deletion mutants of gp75 and the CD8/gp75 chimeras showed that deletion of up amino acids from the gp75 carboxyl terminus did not affect intracellular retention and sorting, whereas both gp75 and CD8/gp75 mutants lacking the carboxyl-terminal 27 amino acids were transported to the cell surface. This region contains the amino acid sequence, asn-gln-pro-leu-leu-thr, and this hexapeptide is conserved among other melanosomal proteins. Further evidence showed that this hexapeptide sequence is necessary for intracellular sorting of gp75 in melanocytic cells, and suggested that a signal for sorting melanosomal proteins along the endosomal/lysosomal pathway lies within this sequence. These data provide evidence for common signals for intracellular sorting of melanosomal and lysosomal proteins, and support the notion that lysosomes and melanosomes share a common endosomal pathway of biogenesis.

Melanocyte Differentiation Marker gp75, the Brown Locus Protein, Can Be Regulated Independently of Tyrosinase and Pigmentation

Human melanoma arises from epidermal melanocytes and displays remarkable phenotypic heterogeneity. This heterogeneity in part reflects the ability of melanoma cells to undergo differentiation along a pathway parallel to differentiation of normal melanocytes. Tyrosinase, encoded by the albino (c), and the tyrosinase-related protein-1 or gp75, encoded by the brown (b) locus, are two of the best-characterized markers for melanocyte differentiation. Both molecules are glycoproteins expressed in melanosomes, the site of pigment synthesis. We studied the regulation of these proteins in human melanoma cells induced by the polar-planar compound hexamethylene bisacetamide (HMBA). In well-differentiated melanoma cell lines, HMBA induced dendritic morphology and specifically regulated the expression of melanosomal glycoproteins (but not a panel of other molecules expressed by melanoma cells). HMBA specifically down-regulated gp75 expression by rapidly decreasing the steady-state level of gp75 mRNA and gp75 synthesis. HMBA was able to down-regulate gp75 expression even in the presence of cholera toxin, which when added alone induced a two- to threefold increase in gp75 expression. In contrast to uniform down-regulation of gp75 expression, HMBA could either up-regulate or down-regulate tyrosinase expression and pigmentation. Based on the differential regulation of gp75 and tyrosinase, melanoma cells could be classified into two groups. In one group, gp75 expression was coordinately regulated with tyrosinase activity and pigmentation. In the other group, gp75 expression and tyrosinase activity and pigmentation were dissociated (with pigmentation coupling to tyrosinase activity, not to gp75 expression). These results show that in mature melanocytic cells, regulation of gp75 expression follows a pattern that can be independent of regulation of tyrosinase and pigmentation.

Functional Analysis of the Tyrosinase Gene and Brown-Locus Protein Gene Promoters.

Functional Analysis of the Tyrosinase Gene and Brown-Locus Protein Gene Promoters.

Functional Analysis of the Tyrosinase Gene and Brown-Locus Protein Gene Promoters

Tyrosinase is a rate-limiting enzyme of melanin biosynthesis and the brown (b)-locus protein is responsible for the formation of black melanin rather than brown. To identify the cis-acting element(s) required for pigment cell-specific gene transcription, we analyzed the promoter function of two pigment cell-specific genes encoding mouse tyrosinase and b-locus protein using a cell-free transcription system prepared from mouse melanoma cells. Functional and structural analysis of both gene promoters reveals that three elements are conserved in both genes at equivalent positions, suggesting that these elements may be responsible for pigment cell-specific transcription. We discuss possible mechanisms for pigment cell-specific expression of the tyrosinase and b-locus protein genes.

Functional Analysis of the Tyrosinase Gene and Brown-Locus Protein Gene Promoters

Functional Analysis of the Tyrosinase Gene and Brown-Locus Protein Gene Promoters