Metastatic dissemination is the leading cause of death in cancer patients, which is particularly evident for high-risk sarcomas such as Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma. Previous ...research identified a crucial role for YB-1 in the epithelial-to-mesenchymal transition (EMT) and metastasis of epithelial malignancies. Based on clinical data and two distinct animal models, we now report that YB-1 is also a major metastatic driver in high-risk sarcomas. Our data establish YB-1 as a critical regulator of hypoxia-inducible factor 1α (HIF1α) expression in sarcoma cells. YB-1 enhances HIF1α protein expression by directly binding to and activating translation of HIF1A messages. This leads to HIF1α-mediated sarcoma cell invasion and enhanced metastatic capacity in vivo, highlighting a translationally regulated YB-1-HIF1α axis in sarcoma metastasis.
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•YB-1 expression is elevated in high-risk human sarcomas•YB-1 promotes sarcoma invasion and metastasis•YB-1 regulates HIF1α expression by directly promoting its mRNA translation•YB-1 effects on sarcoma invasion and metastasis are mediated by HIF1α
YB-1 binds DNA and RNA and has been shown to promote epithelial-to-mesenchymal transition and metastasis of carcinomas. El-Naggar et al. show that YB-1 also contributes to metastasis of high-risk sarcomas by binding to HIF1A mRNA and enhancing its translation.
Treatment strategies involving immune-checkpoint blockade (ICB) have significantly improved survival for a subset of patients across a broad spectrum of advanced solid cancers. Despite this, ...considerable room for improving response rates remains. The tumor microenvironment (TME) is a hurdle to immune function, as the altered metabolism-related acidic microenvironment of solid tumors decreases immune activity. Here, we determined that expression of the hypoxia-induced, cell-surface pH regulatory enzyme carbonic anhydrase IX (CAIX) is associated with worse overall survival in a cohort of 449 patients with melanoma. We found that targeting CAIX with the small-molecule SLC-0111 reduced glycolytic metabolism of tumor cells and extracellular acidification, resulting in increased immune cell killing. SLC-0111 treatment in combination with immune-checkpoint inhibitors led to the sensitization of tumors to ICB, which led to an enhanced Th1 response, decreased tumor growth, and reduced metastasis. We identified that increased expression of
is associated with a reduced Th1 response in metastatic melanoma and basal-like breast cancer TCGA cohorts. These data suggest that targeting CAIX in the TME in combination with ICB is a potential therapeutic strategy for enhancing response and survival in patients with hypoxic solid malignancies.
Type II topoisomerase (Top2) poisoning therapy is used to treat a broad range of cancers via induction of double strand breaks (DSBs) in cells undergoing replication and transcription. Preventing the ...repair of DSBs via inhibition of DNA-PK, an inhibitor of non-homologous end-joining (NHEJ), increases cell kill with Top2 poisons and has led to the initiation of several clinical trials. To elucidate the cellular mechanisms leading to synergistic activity of dual DNA-PK/Top2 inhibition we looked at their effects in cycling versus non-cycling cells, in 3D spheroids and in xenograft models. Combined DNA-PK/Top2 inhibition was found to not only increase the cell kill in proliferating cells, the cell population that is typically most vulnerable to Top2 poisoning, but also in non-proliferative but transcriptionally active cells. This effect was observed in both cancer and normal tissue models, killing more cells than high concentrations of etoposide alone. The combination treatment delayed tumor growth in mice compared to Top2 poisoning alone, but also led to increased toxicity. These findings demonstrate sensitization of Top2β-expressing, non-cycling cells to Top2 poisoning by DNA-PK inhibition. Expansion of the target cell population of Top2 poison treatment to include non-proliferating cells via combination with DNA damage repair inhibitors has implications for efficacy and toxicity of these combinations, including for inhibitors of DNA-PK currently in clinical trial.
We aimed to assess the prognostic significance of follicular lymphoma-associated macrophages in the era of rituximab treatment and maintenance.
We applied immunohistochemistry for CD68 and CD163 to ...two large tissue microarrays (TMA). The first TMA included samples from 186 patients from the BC Cancer Agency (BCCA) who had been treated with first-line systemic treatment including rituximab, cyclophosphamide, vincristine, and prednisone. The second contained 395 samples from PRIMA trial patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone, and randomized to rituximab maintenance or observation. Macrophage infiltration was assessed using Aperio image analysis. Each of the two cohorts was randomly split into training/validation sets.
An increased CD163-positive pixel count was predictive of adverse outcome in the BCCA dataset 5-year progression-free survival (PFS) 38% vs. 72%, respectively, P = 0.004 in the training cohort and 5-year PFS 29% vs. 61%, respectively, P = 0.004 in the validation cohort. In the PRIMA trial, an increased CD163 pixel count was associated with favorable outcome (5-year PFS 60% vs. 44%, respectively, P = 0.011 in the training cohort and 5-year PFS 55% vs. 37%, respectively, P = 0.030 in the validation cohort).
CD163-positive macrophages predict outcome in follicular lymphoma, but their prognostic impact is highly dependent on treatment received.
Vascular smooth muscle cells (VSMC) have been suggested to arise from various developmental sources during embryogenesis, depending on the vascular bed. However, evidence also points to a common ...subpopulation of vascular progenitor cells predisposed to VSMC fate in the embryo. In the present study, we use binary transgenic reporter mice to identify a Tie1+CD31dimvascular endothelial (VE)-cadherin–CD45– precursor that gives rise to VSMC in vivo in all vascular beds examined. This precursor does not represent a mature endothelial cell, because a VE-cadherin promoter-driven reporter shows no expression in VSMC during murine development. Blockade of Notch signaling in the Tie1+ precursor cell, but not the VE-cadherin+ endothelial cell, decreases VSMC investment of developing arteries, leading to localized hemorrhage in the embryo at the time of vascular maturation. However, Notch signaling is not required in the Tie1+ precursor after establishment of a stable artery. Thus, Notch activity is required in the differentiation of a Tie1+ local precursor to VSMC in a spatiotemporal fashion across all vascular beds.
Purpose: Limited drug penetration in solid tumors is a potential mechanism of resistance for many anticancer drugs. Taxanes represent
a class of drugs that are currently undergoing a new round of ...development, but with little known of their ability to penetrate
and distribute relative to blood vessels within solid tumors.
Experimental Design: We assessed the tissue penetration of paclitaxel and docetaxel in HCT-116 tumor xenografts and in multilayered cell culture
(MCC), a three-dimensional cell culture model of the tumor extravascular compartment. In xenografts, taxanes were mapped relative
to blood vessels to obtain drug profiles as a function of distance from vasculature. For MCC, cultures were exposed to stirred
drug reservoirs and taxanes measured as a function of depth into tissue.
Results: Both taxanes exhibited limited penetration, with little drug reaching further than 100 μm into the tissue. Of the two, paclitaxel
exhibited up to 2-fold greater penetration than docetaxel. Mapping tumor cell proliferation following treatment allowed the
consequences of limited drug penetration to be assessed. In tumor xenografts where reduced drug exposure to cells far from
vasculature is one of several factors influencing response to treatment, up to a 75% reduction in S-phase cells was achieved
in cells nearest the vessels, but only 50% reduction was observed in the tissue 150 μm away. In MCC-based data, where the
influence of reduced cell proliferation with depth into tissue was circumvented, a 5-fold (paclitaxel) and 10-fold (docetaxel)
increase in reservoir drug concentration was required to produce a response in cells 150 μm into the tissue equivalent to
that seen in cells directly exposed to the drug.
Conclusion: These results indicate that limited distribution is an important mechanism of tumor resistance to taxanes.
Purpose: The high molecular weight and binding affinity of trastuzumab, a monoclonal antibody in use for treatment of breast cancers
overexpressing human epidermal growth factor receptor type 2 ...(HER2), in combination with microenvironmental factors, may limit
its distribution and efficacy. We assessed and mapped the distribution of systemically given, unlabeled trastuzumab at micrometer
resolution in tumor xenografts using immunohistochemistry.
Experimental Design: Mice bearing MDA-435/LCC6 HER2 xenografts were given single doses of 4 or 20 mg/kg unlabeled trastuzumab with tumor harvest at various time points thereafter;
bound trastuzumab was imaged directly in tumor cryosections using fluorescently tagged antihuman secondary antibodies. Combinations
of additional markers, including HER2, 5-bromo-2-deoxyuridine, CD31, DioC 7 (3), desmin, and collagen IV were also mapped on the same tumor sections.
Results: Distribution of trastuzumab in MDA-435/LCC6 HER2 tumors is found to be heterogeneous, with tumor margins saturating more thoroughly in doses and times analyzed. Considerable
intervessel heterogeneity is also seen. For example, in unsaturated tissues, there remain perfused vessels without any trastuzumab
in addition to vessels with a few layers of positively stained perivascular cells, in addition to vessels with bound drug
up to 150 μm away. This heterogeneity is independent of HER2 expression, microvessel density, and perfusion. A slightly greater
proportion of vessels were associated with pericytes in sections with greater trastuzumab saturation, but this would not adequately
account for observed heterogeneous trastuzumab distribution.
Conclusions: Complete penetration of trastuzumab in tumor tissue was not seen in our study, leaving the possibility that inadequate distribution
may represent a mechanism for resistance to trastuzumab.
Conventional chemotherapy targets proliferating cancer cells, but most cells in solid tumors are not in a proliferative state. Thus, strategies to enable conventional chemotherapy to target ...noncycling cells may greatly increase tumor responsiveness. In this study, we used a 3-dimensional tissue culture system to assay diffusible factors that can limit proliferation in the context of the tumor microenvironment, with the goal of identifying targets to heighten proliferative capacity in this setting. We found that supraphysiologic levels of insulin or insulin-like growth factor I (IGF-I) in combination with oxygen supplementation were sufficient to initiate proliferation of quiescence cells in this system. At maximal induction with IGF-I, net tissue proliferation increased 3- to 4-fold in the system such that chemotherapy could trigger a 3- to 6-fold increase in cytotoxicity, compared with control conditions. These effects were confirmed in vivo in colon cancer xenograft models with demonstrations that IGF-I receptor stimulation was sufficient to generate a 45% increase in tumor cell proliferation, along with a 25% to 50% increase in chemotherapy-induced tumor growth delay. Although oxygen was a dominant factor limiting in vitro tumor cell proliferation, we found that oxygen supplementation via pure oxygen breathing at 1 or 2 atmospheres pressure (mimicking hyperbaric therapy) did not decrease hypoxia in the tumor xenograft mouse model and was insufficient to increase tumor proliferation. Thus, our findings pointed to IGF-I receptor stimulation as a rational strategy to successfully increase tumor responsiveness to cytotoxic chemotherapy.
Follicular lymphoma patients display heterogeneous overall survival and variable risk of transformation. Recent studies have highlighted the role of the microenvironment. The contribution of ...microvessel density to follicular lymphoma survival remains controversial. We used a quantitative tumor mapping approach to determine whether the degree of vascularization correlated with outcome in a uniformly treated cohort. Whole-tissue sections of diagnostic biopsies from 84 cases were stained for CD34 and tumor-to-vessel-distance that encompassed 90% of the tumor (TVD(90)) was determined using image analysis. Twenty-one cases with lower TVD(90) showed inferior overall survival (P=0.0001) and high risk of transformation (P=0.01). These cases significantly correlated with increased Lymphoma-Associated Macrophages (χ(2)=0.025). In multivariate analysis macrophages content, IPI and TVD(90) were independent predictors of overall survival (P=0.05, P=0.001 and P=0.01, respectively) and IPI and TVD(90) predicted risk of transformation (P=0.008 and P=0.08, respectively). Increased angiogenesis is an independent marker of inferior survival and may promote transformation.
The angiopoietins Ang1 (ANGPT1) and Ang2 (ANGPT2) are secreted factors that bind to the endothelial cell-specific receptor tyrosine kinase Tie2 (TEK) and regulate angiogenesis. Ang1 activates Tie2 to ...promote blood vessel maturation and stabilization. In contrast, Ang2, which is highly expressed by tumor endothelial cells, is thought to inhibit Tie2 activity and destabilize blood vessels, thereby facilitating VEGF-dependent vessel growth. Here, we show that the inhibition of tumor xenograft growth caused by an Ang2-specific antibody (REGN910) is reversed by systemic administration of the Tie2 agonist Ang1. These results indicate that Ang2 blockade inhibits tumor growth by decreasing Tie2 activity, showing that Ang2 is a Tie2 activator. REGN910 treatment of tumors resulted in increased expression of genes that are repressed by Tie2 activation, providing further evidence that REGN910 inhibits Tie2 signaling. Combination treatment with REGN910 plus the VEGF blocker aflibercept reduced tumor vascularity and tumor perfusion more dramatically than either single agent, resulting in more extensive tumor cell death and more potent inhibition of tumor growth. Challenging the prevailing model of Ang2 as a destabilizing factor, our findings indicate that Ang2 plays a protective role in tumor endothelial cells by activating Tie2, thereby limiting the antivascular effects of VEGF inhibition. Thus, blockade of Ang2 might enhance the clinical benefits currently provided by anti-VEGF agents. .
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