The epoxyketone-based proteasome inhibitors carfilzomib and orally bioavailable oprozomib have anti-resorptive and bone-anabolic activity in addition to anti-myeloma effects.

Proteasome inhibitors (PIs), namely bortezomib, have become a cornerstone therapy for multiple myeloma (MM), potently reducing tumor burden and inhibiting pathologic bone destruction. In clinical trials, carfilzomib, a next generation epoxyketone-based irreversible PI, has exhibited potent anti-myeloma efficacy and decreased side effects compared with bortezomib. Carfilzomib and its orally bioavailable analog oprozomib, effectively decreased MM cell viability following continual or transient treatment mimicking in vivo pharmacokinetics. Interactions between myeloma cells and the bone marrow (BM) microenvironment augment the number and activity of bone-resorbing osteoclasts (OCs) while inhibiting bone-forming osteoblasts (OBs), resulting in increased tumor growth and osteolytic lesions. At clinically relevant concentrations, carfilzomib and oprozomib directly inhibited OC formation and bone resorption in vitro, while enhancing osteogenic differentiation and matrix mineralization. Accordingly, carfilzomib and oprozomib increased trabecular bone volume, decreased bone resorption and enhanced bone formation in non-tumor bearing mice. Finally, in mouse models of disseminated MM, the epoxyketone-based PIs decreased murine 5TGM1 and human RPMI-8226 tumor burden and prevented bone loss. These data demonstrate that, in addition to anti-myeloma properties, carfilzomib and oprozomib effectively shift the bone microenvironment from a catabolic to an anabolic state and, similar to bortezomib, may decrease skeletal complications of MM.

The CXCR4/SDF-1 chemokine axis: a potential therapeutic target for bone metastases?

Chemokines and chemokine receptors play diverse roles in homeostasis. The chemokine stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4 have critical functions in the immune, circulatory, and central nervous systems and have also been implicated in tumor biology and metastasis. Here we review the current data regarding the role of the CXCR4/SDF-1 chemokine axis in the development of bone metastases, derived from tumor models of breast or prostate cancers. There is substantial evidence that CXCR4 and SDF-1 directly influence the survival and proliferation of tumor cells. In regards to bone metastases, the CXCR4/SDF-1 axis also appears to facilitate tumor cell recruitment to the bone marrow microenvironment via a homing mechanism. This makes disruption of the chemokine axis an attractive therapeutic target for the prevention of tumor cell spread to bone. However, within the bone microenvironment, SDF-1 and CXCR4 appear to have conflicting roles. While genetic disruption of CXCR4 enhances osteoclast activity and therefore stimulates tumor cell growth in the bone - likely via release of bone-derived growth factors - SDF-1 has been shown to have either a stimulatory effect or no effect on osteoclasts. In short, the effects of the CXCR4/SDF-1 axis on tumor cell growth within the bone are not yet fully defined. Further, there are theoretical risks that blockade of this chemokine axis could impair immune function or mobilize tumor cells leading to other sites of metastasis. As such, caution should be taken when designing therapeutic strategies targeting this chemokine axis.

Multiple facets of junD gene expression are atypical among AP-1 family members.

JunD is a versatile AP-1 transcription factor that can activate or repress a diverse collection of target genes. Precise control of junD expression and JunD protein-protein interactions modulate tumor angiogenesis, cellular differentiation, proliferation and apoptosis. Molecular and clinical knowledge of two decades has revealed that precise JunD activity is elaborated by interrelated layers of constitutive transcriptional control, complex post-transcriptional regulation and a collection of post-translational modifications and protein-protein interactions. The stakes are high, as inappropriate JunD activity contributes to neoplastic, metabolic and viral diseases. This article deconvolutes multiple layers of control that safeguard junD gene expression and functional activity. The activity of JunD in transcriptional activation and repression is integrated into a regulatory network by which JunD exerts a pivotal role in cellular growth control. Our discussion of the JunD regulatory network integrates important open issues and posits new therapeutic targets for the neoplastic, metabolic and viral diseases associated with JunD/AP-1 expression.

Microphthalmia transcription factor: a sensitive and specific marker for malignant melanoma in cytologic specimens.

BACKGROUND: The diagnosis of melanoma can be difficult because of shared cytomorphology with other malignant neoplasms. The most commonly used melanocytic markers, anti-S-100 protein and HMB-45 antigen, have limited specificity and sensitivity, respectively. Microphthalmia transcription factor (Mitf) is a nuclear transcription factor critical for the development and survival of melanocytes and has been shown as a sensitive and specific marker for melanoma in histologic specimens. METHODS: To evaluate the efficacy of Mitf as a marker for melanoma in cytologic preparations, 81 cell blocks from 44 patients with melanoma and 37 patients with nonmelanoma malignancies (29 patients with carcinoma, 4 patients with mesotheliomas, 2 patients with lymphoma, and 2 patients with islet cell tumors) were stained with monoclonal antibodies against Mitf (clone D5), S-100 protein, and HMB-45 antigen. The staining was evaluated blindly by three independent observers. The presence of nuclear staining for Mitf and cytoplasmic staining for S-100 protein or HMB-45 antigen in > 10% of tumor cells was considered positive staining for each antigen. RESULTS: Forty-four melanomas (100%), including all 3 spindle-cell melanomas, were positive for Mitf. All nonmelanoma neoplasms were negative with only one exception: One mammary carcinoma showed rare (< 10%), weak nuclear staining with Mitf. The sensitivity and specificity of Mitf as a marker for melanoma were both 100%, whereas the sensitivity of HMB-45 antigen was 90.4%, and the specificity of S-100 protein was 70.3%. CONCLUSIONS: Mitf is a sensitive and specific marker for malignant melanoma, including the spindle-cell variant, in cytologic specimens and may be superior to the current standard melanocytic markers, S-100 protein and HMB-45 antigen.

Linkage of M-CSF signaling to Mitf, TFE3, and the osteoclast defect in Mitf(mi/mi) mice.

Osteoclasts are multinucleated hematopoietic cells essential for bone resorption. Macrophage colony-stimulating factor (M-CSF) is critical for osteoclast development and function, although its nuclear targets in osteoclasts are largely unknown. Mitf and TFE3 are two closely related helix-loop-helix (HLH) transcription factors previously implicated in osteoclast development and function. We demonstrate that cultured Mitf(mi/mi) osteoclasts are immature, mononuclear, express low levels of TRAP, and fail to mature upon M-CSF stimulation. In addition, M-CSF induces phosphorylation of Mitf and TFE3 via a conserved MAPK consensus site, thereby triggering their recruitment of the coactivator p300. Furthermore, an unphosphorylatable mutant at the MAPK consensus serine is specifically deficient in formation of multinucleated osteoclasts, mimicking the defect in Mitf(mi/mi) mice. These results identify a signaling pathway that appears to coordinate cytokine signaling with the expression of genes vital to osteoclast development.

Microphthalmia transcription factor: not a sensitive or specific marker for the diagnosis of desmoplastic melanoma and spindle cell (non-desmoplastic) melanoma.

Microphthalmia transcription factor (Mitf), a melanocytic nuclear protein critical for the embryonic development and postnatal viability of melanocytes, is a master lineage regulator and modulates extracellular signals. Recently, Mitf expression was shown to be both a sensitive and specific marker of epithelioid melanoma. Because loss of specific melanocytic markers in melanomas with spindle cell morphology is more common compared with those tumors with epithelioid morphology, we investigated the sensitivity of D5, an anti-Mitf antibody, for diagnosis in this diagnostically problematic subset of melanomas. Twenty of 21 (95%) spindle cell and desmoplastic melanomas examined were reactive for S-100 protein. Only 4 of 21 (19%) spindle cell and desmoplastic melanomas were reactive for HMB-45. Six of 21 tumors (29%) were reactive for D5, including one case that was non-reactive for S-100 and HMB-45. Melan-A reactivity was seen in 2 of 13 cases (15%) studied. Eight of 24 (33%) non-melanocytic spindle cell tumors were reactive for D5, including 4 of 6 dermatofibromas, 1 of 6 schwannomas, 1 of 2 leiomyomas, and 2 of 6 leiomyosarcomas. Although D5 was shown in a previous study to be a highly sensitive and specific marker for epithelioid melanomas, the results of this study show it is not a sensitive or specific marker of spindle cell and desmoplastic melanomas. Nevertheless, we believe that diffuse positive staining for D5 when taken in clinical, histologic and immunohistochemical context may be diagnostically useful in selected cases of melanoma.

Linking osteopetrosis and pycnodysostosis: regulation of cathepsin K expression by the microphthalmia transcription factor family.

Various genetic conditions produce dysfunctional osteoclasts resulting in osteopetrosis or osteosclerosis. These include human pycnodysostosis, an autosomal recessive syndrome caused by cathepsin K mutation, cathepsin K-deficient mice, and mitf mutant rodent strains. Cathepsin K is a highly expressed cysteine protease in osteoclasts that plays an essential role in the degradation of protein components of bone matrix. Cathepsin K also is expressed in a significant fraction of human breast cancers where it could contribute to tumor invasiveness. Mitf is a member of a helix-loop-helix transcription factor subfamily, which contains the potential dimerization partners TFE3, TFEB, and TFEC. In mice, dominant negative, but not recessive, mutations of mitf, produce osteopetrosis, suggesting a functional requirement for other family members. Mitf also has been found-and TFE3 has been suggested-to modulate age-dependent changes in osteoclast function. This study identifies cathepsin K as a transcriptional target of Mitf and TFE3 via three consensus elements in the cathepsin K promoter. Additionally, cathepsin K mRNA and protein were found to be deficient in mitf mutant osteoclasts, and overexpression of wild-type Mitf dramatically up-regulated expression of endogenous cathepsin K in cultured human osteoclasts. Cathepsin K promoter activity was disrupted by dominant negative, but not recessive, mouse alleles of mitf in a pattern that closely matches their osteopetrotic phenotypes. This relationship between cathepsin K and the Mitf family helps explain the phenotypic overlap of their corresponding deficiencies in pycnodysostosis and osteopetrosis and identifies likely regulators of cathepsin K expression in bone homeostasis and human malignancy.

Clear cell sarcoma shows immunoreactivity for microphthalmia transcription factor: further evidence for melanocytic differentiation.

Microphthalmia transcription factor, a melanocytic nuclear protein critical for the embryonic development and postnatal viability of melanocytes, is a master regulator in modulating extracellular signals. Recently, microphthalmia transcription factor expression was shown to be both a sensitive and specific marker of epithelioid melanoma. We investigated the sensitivity of D5, an anti-microphthalmia transcription factor antibody, for diagnosis of clear cell sarcoma (also known as malignant melanoma of soft parts). Immunoreactivity in a nuclear pattern for D5 was present in 8 of 12 (75%) tumors. D5 staining was strong in three tumors, moderate in two, and weak in three. S-100 protein expression was seen in all 12 cases that had clear cell sarcoma examined. HMB-45 staining was seen in 11 of 12 (92%) tumors. Focal Melan-A positivity was seen in 3 of 7 (43%) tumors. Although D5 was shown in a previous study to be a highly sensitive and specific marker for epithelioid melanomas, the results of this study expand the spectrum of tumors showing immunoreactivity for D5. D5 immunoreactivity in clear cell sarcoma provides further evidence for melanocytic differentiation in this unusual tumor.

Microphthalmia transcription factor expression in cutaneous benign, malignant melanocytic, and nonmelanocytic tumors.

The protein encoded by the microphthalmia (mi) gene is a transcription factor essential for the development and survival of melanocytes. Using a monoclonal antibody generated against human Mi transcription factor protein (Mitf) the authors previously demonstrated that Mitf expression is conserved in primary and metastatic malignant melanomas, and appears to be a highly sensitive and specific melanocytic marker. Mitf expression in various cutaneous nevi and cutaneous nonmelanocytic tumors has not been documented systematically. The authors evaluated Mitf immunostaining in 62 benign nevi, 58 primary cutaneous melanomas, and 53 nonmelanocytic tumors. Mitf immunostaining was conserved in all benign nevi, with Spitz nevi and neurotized nevi demonstrating decreased staining intensity. With the exception of desmoplastic melanomas, all primary cutaneous melanomas were immunopositive regardless of the cell type. Only one of 14 desmoplastic melanomas was Mitf positive. None of the nonmelanocytic tumors was immunopositive, including those lesions that may resemble melanoma histologically (spindle cell carcinomas, atypical fibroxanthomas, and leiomyosarcomas). The results demonstrate that Mitf antibody expression is conserved in the majority of benign and malignant melanocytic lesions, and that it may be helpful in the diagnosis of primary melanocytic skin lesions. Its use in desmoplastic melanomas is limited and is reflective of other melanocyte-associated antigens. Mitf discriminates between spindle cell nonmelanocytic tumors and melanomas with a spindle cell morphology, and is useful in a panel with other appropriate antibodies.

Mechanisms of osteoporosis after hematopoietic cell transplantation.

Osteopenia and osteoporosis are common complications of bone marrow and peripheral blood stem cell transplantation. Bone loss occurs in 50% to 60% of patients treated with the most common preparatory regimens. The major causes of transplant-related bone loss are primary hypogonadism (low estrogen and testosterone), secondary hyperparathyroidism due to low serum calcium, and posttransplant steroid therapy. Other transplant-related treatments that induce bone loss are discussed. Trabecular bone is particularly vulnerable to transplant-related therapies. The spine and hip contain 50% to 75% trabecular bone and are most at risk for fracture after hematopoietic cell transplantation (HCT). The structure of bone and the bone cells that are involved in maintaining skeletal integrity are discussed, followed by a discussion of the transplant-related therapies that have been shown to cause damage to bone and lead to bone loss. Recommendations for patients undergoing HCT include (1) evaluation of bone mineral density either shortly before or shortly after transplantation and appropriate intervention and monitoring based on the results; and (2) evaluation of estrogen and testosterone levels after HCT and replacement when appropriate; and (3) administration of bisphosphonate therapy to all patients on steroids for >2 months. Early intervention and prevention of bone loss can have a tremendous clinical impact for patients undergoing HCT because once significant bone loss has occurred, it is difficult to reverse.

Microphthalmia transcription factor. A sensitive and specific melanocyte marker for MelanomaDiagnosis.

Malignant melanomas do not uniformly retain expression of melanocytic gene products-an observation associated with diagnostic dilemmas. Microphthalmia transcription factor (Mitf) is a melanocytic nuclear protein critical for the embryonic development and postnatal viability of melanocytes. It serves as a master regulator in modulating extracellular signals, such as those triggered by alpha-MSH and c-Kit ligand. Because of its central role in melanocyte survival and to assess its potential use as a histopathological marker for melanoma, Mitf expression was examined in histologically confirmed human melanoma specimens. Western blot analysis of melanoma cell lines revealed consistent expression of two Mitf protein isoforms differing by MAP kinase-mediated phosphorylation. In a series of 76 consecutive human melanoma surgical specimens, 100% stained positively for Mitf with a nuclear pattern of reactivity. In a side-by-side comparison, Mitf staining was positive in melanomas that failed to stain for either HMB-45 or S-100, the most common currently used melanoma markers. Of 60 non-melanoma tumors, none displayed nuclear Mitf staining and two displayed cytoplasmic staining. Although Mitf does not distinguish benign from malignant melanocytic lesions, for invasive neoplasms it appears to be a highly sensitive and specific histopathological melanocyte marker for melanoma.

alpha-Melanocyte-stimulating hormone signaling regulates expression of microphthalmia, a gene deficient in Waardenburg syndrome.

The pituitary peptide alpha-melanocyte-stimulating hormone (alpha-MSH) stimulates melanocytes to up-regulate cAMP, but the downstream targets of cAMP are not well understood mechanistically. One consequence of alpha-MSH stimulation is increased melanization attributable to induction of pigmentation enzymes, including tyrosinase, which catalyzes a rate-limiting step in melanin synthesis. The tyrosinase promoter is a principle target of the melanocyte transcription factor Microphthalmia (Mi), a factor for which deficiency in humans causes Waardenburg syndrome II. We show here that both alpha-MSH and forskolin, a drug that increases cAMP, stimulate a rapid increase in Mi mRNA and protein levels in both melanoma cell lines and primary melanocytes. This up-regulation requires a cAMP-responsive element within the Mi promoter, and the pathway leading to Mi stimulation is subject to tight homeostatic regulation. Although cAMP signaling is ubiquitous, the Mi promoter was seen to be cAMP-responsive in melanocytes but not in nonmelanocytes. Moreover, dominant negative interference with Mi impeded successful alpha-MSH stimulation of tyrosinase. The regulation of Mi expression via alpha-MSH thus provides a direct mechanistic link to pigmentation. In addition, because the human melanocyte and deafness condition Waardenburg syndrome is sometimes caused by haploinsufficiency of Mi, its modulation by alpha-MSH suggests therapeutic strategies targeted at up-regulating the remaining wild type Mi allele.

Age-resolving osteopetrosis: a rat model implicating microphthalmia and the related transcription factor TFE3.

Microphthalmia (Mi) is a basic helix-loop-helix-leucine zipper (b-HLH-ZIP) transcription factor implicated in pigmentation, mast cells, and bone development. Two dominant-negative mi alleles (mi/mi and Mior/Mior) in mice cause osteopetrosis. In contrast, osteopetrosis has not been observed in a number of recessive mi alleles, suggesting the existence of Mi protein partners important in osteoclast function. An osteopetrotic rat of unknown genetic defect (mib) has been described whose skeletal sclerosis improves dramatically with age and that is associated with pigmentation defects reminiscent of mouse mi alleles. Here we report that this rat strain harbors a large genomic deletion encompassing the 3' half of mi including most of the b-HLH-ZIP region. Osteoclasts from these animals lack Mi protein in contrast to wild-type rat, mouse, and human osteoclasts. Mi is not detectable in primary osteoblasts. In addition TFE3, a b-HLH-ZIP transcription factor related to Mi, was found to be expressed in osteoclasts, but not osteoblasts, and to coimmunoprecipitate with Mi. These results demonstrate the existence of members of a family of biochemically related transcription factors that may cooperate to play a central role in osteoclast function and possibly in age-related osteoclast homeostasis.

Long-term human hematopoiesis in the SCID-hu mouse.

Coimplantation of small fragments of human fetal thymus and fetal liver into immunodeficient SCID mice resulted in the formation of a unique structure (Thy/Liv). Thereafter, the SCID-hu mice showed reproducible and long-term reconstitution of human hematopoietic activity. For periods lasting 5-11 mo after transplantation, active T lymphopoiesis was observed inside the grafts and cells that were negative for T cell markers were found to have colony-forming units for granulocyte/macrophage (CFU-GM) and erythroid burst-forming unit (BFU-E) activity in the methylcellulose colony assay. In addition, structures similar to normal human bone marrow were observed inside the Thy/Liv grafts, consisting of blast cells, mature and immature forms of myelomonocytic cells, and megakaryocytes. These data indicate long-term maintenance, in vivo, of human progenitor cells for the T lymphoid, myelomonocytic, erythroid, and megakaryocytic lineages. The role of the implanted fetal liver fragments was analyzed using HLA-mismatched Thy/Liv implants. The HLA type of the liver donor was found on T cells and macrophages in the graft. In addition, cells grown in the methylcellulose colony assay and cells in a bone marrow-like structure, the "thymic isle," expressed the HLA type of the liver donor. Thus, the Thy/Liv implants provided a microenvironment in which to follow human hematopoietic progenitor cells for multiple lineages. The formation of the Thy/Liv structures also results in a continuous source of human T cells in the peripheral circulation of the SCID-hu mouse. Though present for 5-11 mo, these cells did not engage in a xenograft (graft-versus-host) reaction. This animal model, the first in which multilineage human hematopoietic activity is maintained for long periods of time, should be useful for the analysis of human hematopoiesis in vivo.