Systematic therapy for unresectable or metastatic soft-tissue sarcomas: past, present, and future.

Unresectable or metastatic disease occurs in 40% to 60% of soft-tissue sarcoma (STS) patients and portends a poor prognosis. For decades, doxorubicin has formed the backbone of systemic treatment, with response rates of approximately 26%. Patients progressing following first-line therapy were left with few proven options. No other cytotoxic chemotherapy agent or combination has demonstrated superiority to doxorubicin. Advances in targeted therapy of STS have been hindered by STS heterogeneity and poorly understood disease biology. Despite challenges, progress has been made in specific STS subtypes. Here, we highlight the challenges, progress, and lessons learned from STS trials published in the last 20 to 25 years.

Treatment of unresectable and metastatic cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (SCC) is an already common disorder with a rapidly increasing incidence. Treatment of early disease depends primarily on surgery or destructive techniques. In contrast to the frequency of early SCC, unresectable or metastatic SCC is relatively rare, but potentially life-threatening without clearly proven treatment options. Few rigorous studies of the treatment of advanced SCC have been undertaken. In the past, various agents have been explored in a limited fashion, including chemotherapy (cisplatin, fluoropyrimidines, bleomycin, doxorubicin), 13-cis-retinoic acid, and interferon-α2a. Clinical activity has been suggested by these trials, but their small sizes, heterogeneous patient populations, and lack of randomization have hindered the use of their results in defining treatment paradigms. Only one rigorous randomized trial has focused on cutaneous SCC. Enrolling 66 patients, that trial randomized patients at high recurrence risk to either observation or postoperative interferon-α2a and 13-cis-retinoic acid. This treatment did not improve time to recurrence or prevent secondary cutaneous SCC from developing. Though not in the metastatic setting, this study casts doubt on the ability of this regimen to control metastatic disease. Recently, agents targeting the human epidermal growth factor receptor (erlotinib, gefitinib, cetuximab) have displayed preliminary evidence of activity in phase II clinical trials and case series reports. Expression of this receptor is frequent in cutaneous SCC and appears to be prognostically adverse. Only the conduct of rigorous trials, with well-defined endpoints, adequate patient numbers, and preferably randomization, can prove the clinical efficacy of this promising treatment approach and define better therapy for this vexing clinical problem.

Involvement of a specificity proteins-binding element in regulation of basal and estrogen-induced transcription activity of the BRCA1 gene.

INTRODUCTION: Increased estrogen level has been regarded to be a risk factor for breast cancer. However, estrogen has also been shown to induce the expression of the tumor suppressor gene, BRCA1. Upregulation of BRCA1 is thought to be a feedback mechanism for controlling DNA repair in proliferating cells. Estrogens enhance transcription of target genes by stimulating the association of the estrogen receptor (ER) and related coactivators to estrogen response elements or to transcription complexes formed at activator protein (AP)-1 or specificity protein (Sp)-binding sites. Interestingly, the BRCA1 gene lacks a consensus estrogen response element. We previously reported that estrogen stimulated BRCA1 transcription through the recruitment of a p300/ER-alpha complex to an AP-1 site harbored in the proximal BRCA1 promoter. The purpose of the study was to analyze the contribution of cis-acting sites flanking the AP-1 element to basal and estrogen-dependent regulation of BRCA1 transcription. METHODS: Using transfection studies with wild-type and mutated BRCA1 promoter constructs, electromobility binding and shift assays, and DNA-protein interaction and chromatin immunoprecipitation assays, we investigated the role of Sp-binding sites and cAMP response element (CRE)-binding sites harbored in the proximal BRCA1 promoter. RESULTS: We report that in the BRCA1 promoter the AP-1 site is flanked upstream by an element (5'-GGGGCGGAA-3') that recruits Sp1, Sp3, and Sp4 factors, and downstream by a half CRE-binding motif (5'-CGTAA-3') that binds CRE-binding protein. In ER-alpha-positive MCF-7 cells and ER-alpha-negative Hela cells expressing exogenous ER-alpha, mutation of the Sp-binding site interfered with basal and estrogen-induced BRCA1 transcription. Conversely, mutation of the CRE-binding element reduced basal BRCA1 promoter activity but did not prevent estrogen activation. In combination with the AP-1/CRE sites, the Sp-binding domain enhanced the recruitment of nuclear proteins to the BRCA1 promoter. Finally, we report that the MEK1 (mitogen-activated protein kinase kinase-1) inhibitor PD98059 attenuated the recruitment of Sp1 and phosphorylated ER-alpha, respectively, to the Sp and AP-1 binding element. CONCLUSION: These cumulative findings suggest that the proximal BRCA1 promoter segment comprises cis-acting elements that are targeted by Sp-binding and CRE-binding proteins that contribute to regulation of BRCA1 transcription.

The ligand status of the aromatic hydrocarbon receptor modulates transcriptional activation of BRCA-1 promoter by estrogen.

In sporadic breast cancers, BRCA-1 expression is down-regulated in the absence of mutations in the BRCA-1 gene. This suggests that disruption of BRCA-1 expression may contribute to the onset of mammary tumors. Environmental contaminants found in industrial pollution, tobacco smoke, and cooked foods include benzo(a)pyrene [B(a)P] and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which have been shown to act as endocrine disruptors and tumor promoters. In previous studies, we documented that estrogen (E2) induced BRCA-1 transcription through the recruitment of an activator protein-1/estrogen receptor-alpha (ER alpha) complex to the proximal BRCA-1 promoter. Here, we report that activation of BRCA-1 transcription by E2 requires occupancy of the BRCA-1 promoter by the unliganded aromatic hydrocarbon receptor (AhR). The stimulatory effects of E2 on BRCA-1 transcription are counteracted by (a) cotreatment with the AhR antagonist 3'-methoxy-4'-nitroflavone; (b) transient expression in ER alpha-negative HeLa cells of ER alpha lacking the protein-binding domain for the AhR; and (c) mutation of two consensus xenobiotic-responsive elements (XRE, 5'-GCGTG-3') located upstream of the ER alpha-binding region. These results suggest that the physical interaction between the unliganded AhR and the liganded ER alpha plays a positive role in E2-dependent activation of BRCA-1 transcription. Conversely, we show that the AhR ligands B(a)P and TCDD abrogate E2-induced BRCA-1 promoter activity. The repressive effects of TCDD are paralleled by increased recruitment of the liganded AhR and HDAC1, reduced occupancy by p300, SRC-1, and diminished acetylation of H4 at the BRCA-1 promoter region flanking the XREs. We propose that the ligand status of the AhR modulates activation of the BRCA-1 promoter by estrogen.

Quantitative histopathology identifies patients with thin melanomas who are at risk for metastases.

This small exploratory study was designed to test the hypothesis that thin melanoma lesions contain nuclei of two similar phenotypes, in different proportions. In lesions likely to progress to metastatic disease, one of these phenotypes predominates. Histopathological sections from 18 cases of thin melanomas which did not progress to metastasis, and from 10 cases which did progress were imaged and digitized at high resolution, with a total of 2084 and 1148 nuclei, respectively, recorded. Five karyometric features were used to discriminate between nuclei from indolent and from potentially metastatic lesions. For each case, the percentage of nuclei classified by the discriminant function as having come from a potentially metastatic lesion was determined and termed as case classification criterion. Standard histopathological criteria, such as ulceration and high mitotic index, indicated in this material the need for intensive therapy for only one of the 10 participants, as compared with 7/10 identified correctly by the karyometric measure. Using a case classification criterion threshold of 40%, the overall accuracy was 86% in the test set. The proportion of nuclei of an aggressive phenotype may lend itself as an effective prognostic clue for thin melanoma lesions. The algorithm developed in this training set appears to identify those patients at high risk for metastatic disease, and demonstrates a basis for a further study to assess the utility of prognostic clues for thin melanomas.

An estrogen receptor-alpha/p300 complex activates the BRCA-1 promoter at an AP-1 site that binds Jun/Fos transcription factors: repressive effects of p53 on BRCA-1 transcription.

One of the puzzles in cancer predisposition is that women carrying BRCA-1 mutations preferentially develop tumors in epithelial tissues of the breast and ovary. Moreover, sporadic breast tumors contain lower levels of BRCA-1 in the absence of mutations in the BRCA-1 gene. The problem of tissue specificity requires analysis of factors that are unique to tissues of the breast. For example, the expression of estrogen receptor-alpha (ER alpha) is inversely correlated with breast cancer risk, and 90% of BRCA-1 tumors are negative for ER alpha. Here, we show that estrogen stimulates BRCA-1 promoter activity in transfected cells and the recruitment of ER alpha and its cofactor p300 to an AP-1 site that binds Jun/Fos transcription factors. The recruitment of ER alpha/p300 coincides with accumulation in the S-phase of the cell cycle and is antagonized by the antiestrogen tamoxifen. Conversely, we document that overexpression of wild-type p53 prevents the recruitment of ER alpha to the AP-1 site and represses BRCA-1 promoter activity. Taken together, our findings support a model in which an ER alpha/AP-1 complex modulates BRCA-1 transcription under conditions of estrogen stimulation. Conversely, the formation of this transcription complex is abrogated in cells overexpressing p53.

Vorinostat synergizes with ridaforolimus and abrogates the ridaforolimus-induced activation of AKT in synovial sarcoma cells.

BACKGROUND: Curative treatments for patients with metastatic synovial sarcoma (SS) do not exist, and such patients have a poor prognosis. We explored combinations of molecularly-targeted and cytotoxic agents to identify synergistic treatment combinations in SS cells. METHODS: Two SS cell lines (HS-SY-II and SYO-I) were treated with single agents or combinations of molecularly targeted therapies (HDAC inhibitor, vorinostat; mTOR inhibitor, ridaforolimus) and cytotoxic agents. After 72 hours, cell viability was measured using the MTS cell proliferation assay. Combination Indices (CI) were calculated to determine whether each combination was synergistic, additive, or antagonistic. Western Blot analysis assessed alterations in total and phospho-AKT protein levels in response to drug treatment. RESULTS: We determined the single-agent IC50 for ridaforolimus, vorinostat, doxorubicin, and melphalan in HS-SY-II and SYO-I. Synergism was apparent in cells co-treated with ridaforolimus and vorinostat: CI was 0.28 and 0.63 in HS-SY-II and SYO-I, respectively. Ridaforolimus/doxorubicin and ridaforolimus/melphalan exhibited synergism in both cell lines. An additive effect was observed with combination of vorinostat/doxorubicin in both cell lines. Vorinostat/melphalan was synergistic in HS-SY-II and additive in SYO-I. Western blot analysis demonstrated that ridaforolimus increased pAKT-ser473 levels; this effect was abrogated by vorinostat co-treatment. CONCLUSIONS: The combination of ridaforolimus and vorinostat demonstrates in vitro synergism in SS. Addition of vorinostat abrogated ridaforolimus-induced AKT activation. Since AKT activation is a possible mechanism of resistance to mTOR inhibitors, adding vorinostat (or another HDAC inhibitor) may be a route to circumvent AKT-mediated resistance to mTOR inhibitors.

Serum protein acidic and rich in cysteine (SPARC) as a prognostic marker in soft tissue sarcomas.

BACKGROUND: Serum protein acidic and rich in cysteine (SPARC) is a matricellular secreted glycoprotein that performs several cellular functions and has been implicated in tumorigenesis in a variety of tumor types. The chemotherapeutic agent nanoparticle albumin-encapsulated (NAB)-paclitaxel has been postulated to exploit SPARC expression to target neoplastic cells. SPARC's role, and potentially the role of NAB-paclitaxel, in the highly heterogeneous class of soft-tissue sarcomas (STS) has not been investigated. Our objective was to explore the pattern of SPARC expression and its prognostic significance in STS. METHODS: 27 tissue specimens representing various STS histologies were stained for SPARC expression by immunohistochemistry (IHC). Staining intensity was scored blindly. Survival was determined from patients' medical records and analyzed using Kaplan-Meier and log-rank with respect to SPARC expression level. RESULTS: Elevated SPARC expression was observed in 15/27 (56%) specimens. Overall patient survival segregated strongly based on levels of SPARC expression. Patients who expressed low-to-moderate levels of SPARC exhibited median survival of 22.1 months, while the median survival of patients with moderate-to-high expression levels was 4.4 months (log rank; p = 0.0016). CONCLUSIONS: SPARC expression is elevated in a significant proportion of STS specimens analyzed in this study, but it does not appear to correlate with specific STS histologies. Given our limited sample size, we cannot draw definitive conclusions regarding association of SPARC with STS subtype. Overall survival segregates strongly by degree of SPARC expression, with elevated expression being adverse. If validated in a larger study, our results suggest that trials in STS with agents potentially targeting SPARC, such as NAB-paclitaxel, should be stratified by SPARC expression level.

Roles for endothelin receptor B and BCL2A1 in spontaneous CNS metastasis of melanoma.

Metastatic spread of melanoma to the central nervous system (CNS) is a common and devastating manifestation of disease progression, which, despite its clinical importance, remains poorly understood with respect to underlying molecular mechanisms. Using a recently developed preclinical model of spontaneous melanoma CNS metastasis, we have identified alterations in expression of endothelin receptor B (EDNRB) as a potential factor that influences brain metastatic potential. Induced overexpression of this gene mediated enhanced overall metastatic disease, and resulted in an increased incidence of spontaneous CNS metastases. In contrast, the overexpression of other highlighted genes, such as BCL2A1, did not affect the incidence of CNS metastases but nevertheless appears to facilitate intracranial tumor growth. The prometastatic effect in the CNS associated with EDNRB appears to be mediated by the interaction with its ligands resulting in enhanced tumor cell proliferation and thus intracranial melanoma growth. That EDNRB contributes to melanoma metastasis is underscored by the fact that its therapeutic inhibition by the EDNRB-specific inhibitor A192621 translated into improved outcomes when treating mice with either visceral metastases or intracranial tumors. The identification of an influential role of EDNRB in CNS melanoma spontaneous metastasis may provide both a target for therapeutic intervention as well as a potential prognostic marker for patients having an increased predisposition for incidence of CNS melanoma metastases.