Keila E. Torres

Vimentin Is a Novel Anti-Cancer Therapeutic Target; Insights from In Vitro and In Vivo Mice Xenograft Studies

Background Vimentin is a ubiquitous mesenchymal intermediate filament supporting mechano-structural integrity of quiescent cells while participating in adhesion, migration, survival, and cell signaling processes via dynamic assembly/disassembly in activated cells. Soft tissue sarcomas and some epithelial cancers exhibiting “epithelial to mesenchymal transition” phenotypes express vimentin. Withaferin-A, a naturally derived bioactive compound, may molecularly target vimentin, so we sought to evaluate its effects on tumor growth in vitro and in vivo thereby elucidating the role of vimentin in drug-induced responses. Methods and Findings Withaferin-A elicited marked apoptosis and vimentin cleavage in vimentin-expressing tumor cells but significantly less in normal mesenchymal cells. This proapoptotic response was abrogated after vimentin knockdown or by blockade of caspase-induced vimentin degradation via caspase inhibitors or overexpression of mutated caspase-resistant vimentin. Pronounced anti-angiogenic effects of Withaferin-A were demonstrated, with only minimal effects seen in non-proliferating endothelial cells. Moreover, Withaferin-A significantly blocked soft tissue sarcoma growth, local recurrence, and metastasis in a panel of soft tissue sarcoma xenograft experiments. Apoptosis, decreased angiogenesis, and vimentin degradation were all seen in Withaferin-A treated specimens. Conclusions In light of these findings, evaluation of Withaferin-A, its analogs, or other anti-vimentin therapeutic approaches in soft tissue sarcoma and “epithelial to mesenchymal transition” clinical contexts is warranted.

Recurrent PTPRB and PLCG1 mutations in angiosarcoma

Angiosarcoma is an aggressive malignancy that arises spontaneously or secondarily to ionizing radiation or chronic lymphoedema. Previous work has identified aberrant angiogenesis, including occasional somatic mutations in angiogenesis signaling genes, as a key driver of angiosarcoma. Here we employed whole-genome, whole-exome and targeted sequencing to study the somatic changes underpinning primary and secondary angiosarcoma. We identified recurrent mutations in two genes, PTPRB and PLCG1, which are intimately linked to angiogenesis. The endothelial phosphatase PTPRB, a negative regulator of vascular growth factor tyrosine kinases, harbored predominantly truncating mutations in 10 of 39 tumors (26%). PLCG1, a signal transducer of tyrosine kinases, encoded a recurrent, likely activating p.Arg707Gln missense variant in 3 of 34 cases (9%). Overall, 15 of 39 tumors (38%) harbored at least one driver mutation in angiogenesis signaling genes. Our findings inform and reinforce current therapeutic efforts to target angiogenesis signaling in angiosarcoma.

Universal Marker and Detection Tool for Human Sarcoma Circulating Tumor Cells

To date, no specific marker exists for the detection of circulating tumor cells (CTC) from different types of sarcomas, though tools are available for detection of CTCs in peripheral blood of patients with cancer for epithelial cancers. Here, we report cell-surface vimentin (CSV) as an exclusive marker on sarcoma CTC regardless of the tissue origin of the sarcoma as detected by a novel monoclonal antibody. Utilizing CSV as a probe, we isolated and enumerated sarcoma CTCs with high sensitivity and specificity from the blood of patients bearing different types of sarcoma, validating their phenotype by single cell genomic amplification, mutation detection, and FISH. Our results establish the first universal and specific CTC marker described for enumerating CTCs from different types of sarcoma, thereby providing a key prognosis tool to monitor cancer metastasis and relapse.

Activated MET is a molecular prognosticator and potential therapeutic target for malignant peripheral nerve sheath tumors

Purpose: MET signaling has been suggested a potential role in malignant peripheral nerve sheath tumors (MPNST). Here, MET function and blockade were preclinically assessed. Experimental Design: Expression levels of MET, its ligand hepatocyte growth factor (HGF), and phosphorylated MET (pMET) were examined in a clinically annotated MPNST tissue microarray (TMA) incorporating univariable and multivariable statistical analyses. Human MPNST cells were studied in vitro and in vivo; Western blot (WB) and ELISA were used to evaluate MET and HGF expression, activation, and downstream signaling. Cell culture assays tested the impact of HGF-induced MET activation and anti-MET–specific siRNA inhibition on cell proliferation, migration, and invasion; in vivo gel-foam assays were used to evaluate angiogenesis. Cells stably transduced with anti-MET short hairpin RNA (shRNA) constructs were tested for growth and metastasis in severe combined immunodeficient (SCID) mice. The effect of the tyrosine kinase inhibitor XL184 (Exelixis) targeting MET/VEGFR2 (vascular endothelial growth factor receptor 2) on local and metastatic MPNST growth was examined in vivo. Results: All three markers were expressed in MPNST human samples; pMET expression was an independent prognosticator of poor patient outcome. Human MPNST cell lines expressed MET, HGF, and pMET. MET activation increased MPNST cell motility, invasion, angiogenesis, and induced matrix metalloproteinase-2 (MMP2) and VEGF expression; MET knockdown had inverse effects in vitro and markedly decreased local and metastatic growth in vivo. XL184 abrogated human MPNST xenograft growth and metastasis in SCID mice. Conclusions: Informative prognosticators and novel therapies are crucially needed to improve MPNST management and outcomes. We show an important role for MET in MPNST, supporting continued investigation of novel anti-MET therapies in this clinical context. Clin Cancer Res; 17(12); 3943–55. ©2011 AACR.

Autophagic Survival in Resistance to Histone Deacetylase Inhibitors: Novel Strategies to Treat Malignant Peripheral Nerve Sheath Tumors

Histone deacetylase inhibitors (HDACi) show promise as cancer therapeutics; however, the full scope of their utility remains unknown. Here we report findings that strongly rationalize clinical evaluation of HDACis in malignant peripheral nerve sheath tumors (MPNST), a class of highly aggressive, therapeutically resistant, and commonly fatal malignancies that occur sporadically or in patients with the inherited neurofibromatosis type-1 (NF1) syndrome. We evaluated the effects of the chemical HDACis PCI-24781, suberoylanilide hydroxamic acid, and MS-275 on a panel of human NF1-associated and sporadic MPNSTs in vitro and in vivo. A subset of MPNSTs was found to be highly sensitive to HDACis, especially to PCI-24781. All cell lines in this group were NF1-associated. Significant proapoptotic effects were noted in vitro and in vivo and were independent of p53 mutational status. In contrast, as a group the sporadic-MPNST cells were markedly resistant to HDACi treatment. HDACis were found to induce productive autophagy in MPNST cells. Genetic and/or pharmacologic autophagy blockade resulted in significant HDACi-induced apoptosis in cells defined as resistant or sensitive, leading to abrogated growth of primary tumors and lung metastases in tumor xenograft assays. Among autophagy-associated genes expressed in response to HDACi, the immunity-related GTPase family, M was validated as a critical target in mediating HDACi-induced autophagy and enhanced apoptosis. Taken together, our findings strongly support the evaluation of HDACi currently in clinical trials as an important new therapeutic strategy to treat MPNST, including in combination with autophagy blocking combination regimens in particular for patients with sporadic MPNST.

Extensive Survey of STAT6 Expression in a Large Series of Mesenchymal Tumors

OBJECTIVES Expression of strong nuclear STAT6 is thought to be a specific marker for solitary fibrous tumors (SFTs). Little is known about subtle expression patterns in other mesenchymal lesions. METHODS We performed immunohistochemical studies against the C-terminus of STAT6 in tissue microarrays and whole sections, comprising 2366 mesenchymal lesions. RESULTS Strong nuclear STAT6 was expressed in 285 of 2,021 tumors, including 206 of 240 SFTs, 49 of 408 well-differentiated/dedifferentiated liposarcomas, eight of 65 unclassified sarcomas, and 14 of 184 desmoid tumors, among others. Expression in SFTs was predominately limited to the nucleus. Other positive tumors typically expressed both nuclear and cytoplasmic STAT6. Complete absence of STAT6 was most common in pleomorphic liposarcoma and alveolar soft part sarcoma (60% and 72% cases negative, respectively). CONCLUSIONS Strong nuclear STAT6 is largely specific for SFTs. Physiologic low-level cytoplasmic/nuclear expression is common in mesenchymal neoplasia and is of uncertain significance.

TRAIL and Doxorubicin Combination Induces Proapoptotic and Antiangiogenic Effects in Soft Tissue Sarcoma In vivo

Purpose: Novel therapeutic approaches for complex karyotype soft tissue sarcoma (STS) are crucially needed. Consequently, we assessed the efficacy of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), in combination with chemotherapy, on local and metastatic growth of human STS xenografts in vivo. Experimental Design: TRAIL was evaluated alone and combined with low-dose doxorubicin in two human STS severe combined immunodeficient mouse xenograft models using fibrosarcoma (HT1080; wild-type p53) and leiomyosarcoma (SKLMS1; mutated p53), testing for effects on local growth, metastasis, and overall survival. Magnetic resonance imaging was used to evaluate local growth and bioluminescence was used to longitudinally assess lung metastases. Tissues were evaluated through immunohistocemistry and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling staining for treatment effects on tumor cell proliferation, apoptosis, angiogenesis, angiogenic factors, and TRAIL receptor expression. Quantitative real-time polymerase chain reaction (QRTPCR) angiogenesis array was used to assess therapy-induced gene expression changes. Results: TRAIL/doxorubicin combination induced marked STS local and metastatic growth inhibition in a p53-independent manner. Significantly increased (P < 0.001) host survival was also demonstrable. Combined therapy induced significant apoptosis, decreased tumor cell proliferation, and increased TRAIL receptor (DR4 and DR5) expression in all treated tumors. Moreover, decreased microvessel density was observed, possibly secondary to increased expression of the antiangiogenic factor CXCL10 and decreased proangiogenic interleukin-8 cytokine in response to TRAIL/doxorubicin combination, as was also observed in vitro. Conclusions: Given the urgent need for better systemic approaches to STS, clinical trials evaluating TRAIL in combination with low-dose chemotherapy are potentially warranted. Clin Cancer Res; 16(9); 2591–604. ©2010 AACR.

Involvement of the PI3K/Akt Pathway in Myxoid/Round Cell Liposarcoma

The molecular determinants involved in the progression of myxoid liposarcoma to increased cellularity/round cell change are poorly understood. We studied the PI3K/Akt pathway in myxoid and round cell liposarcomas using a tissue microarray composed of 165 tumors from 111 patients, and mutational analysis of PIK3CA in 44 cases. Activating PIK3CA mutations were found in 6/44 cases, 14%; mutations were more frequent in round cell vs myxoid tumors (5/15, 33% vs 1/29, 3%; P=0.013). Complete loss of PTEN, an alternative mechanism for PI3K/Akt activation, was found in 13/111 (12%) cases and was mutually exclusive with PIK3CA mutation. Strong IGF1R expression was demonstrated in 14/39 (36%) of round cell and 11/58 (19%) of myxoid tumors (P=0.062). Activation of the PI3K pathway was confirmed using immunohistochemical analysis for downstream targets phospho-S6 ribosomal protein and phospho-4EBP1. Phospho-4EBP1 was increased in round cell tumors compared with myxoid tumors (24/30, 80% vs 25/44, 57%; P=0.038) or tumors with treatment effect (10/24, 42%; P=0.02). Phospho-S6 was highly expressed in both myxoid and round cell tumors (29/47, 62% and 14/30, 47%, respectively; P=0.2). In tumors with PIK3CA mutation, any IGF1R expression, or loss of PTEN expression, phospho-4EBP1 was more frequently elevated compared with tumors without a known activating event in the PI3K pathway (55/72; 76% vs 3/8, 38%; P=0.033). These findings suggest that activation of the PI3K/Akt pathway via activating mutation of PIK3CA, loss of PTEN, or IGF1R expression have a role in round cell transformation. The PI3K/Akt pathway may therefore provide a therapeutic target in round cell liposarcoma.

Localized and metastatic myxoid/round cell liposarcoma: Clinical and molecular observations

Myxoid liposarcoma (MLPS), a disease especially of young adults with potential for local recurrence and metastasis, currently lacks solid prognostic factors and therapeutic targets. The authors of this report evaluated the natural history and outcome of patients with MLPS and commonly deregulated protein biomarkers.

Integrative genomic characterization and a genomic staging system for gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) historically were grouped with leiomyosarcomas (LMSs) based on their morphologic similarities; however, recently, GIST was established unequivocally as a distinct type of sarcoma based on its molecular features and response to imatinib treatment.