The Hsp104 N-terminal domain enables disaggregase plasticity and potentiation.

The structural basis by which Hsp104 dissolves disordered aggregates and prions is unknown. A single subunit within the Hsp104 hexamer can solubilize disordered aggregates, whereas prion dissolution requires collaboration by multiple Hsp104 subunits. Here, we establish that the poorly understood Hsp104 N-terminal domain (NTD) enables this operational plasticity. Hsp104 lacking the NTD (Hsp104(ΔN)) dissolves disordered aggregates but cannot dissolve prions or be potentiated by activating mutations. We define how Hsp104(ΔN) invariably stimulates Sup35 prionogenesis by fragmenting prions without solubilizing Sup35, whereas Hsp104 couples Sup35 prion fragmentation and dissolution. Volumetric reconstruction of Hsp104 hexamers in ATPγS, ADP-AlFx (hydrolysis transition state mimic), and ADP via small-angle X-ray scattering revealed a peristaltic pumping motion upon ATP hydrolysis, which drives directional substrate translocation through the central Hsp104 channel and is profoundly altered in Hsp104(ΔN). We establish that the Hsp104 NTD enables cooperative substrate translocation, which is critical for prion dissolution and potentiated disaggregase activity.

Tumor-derived exosomes promote carcinogenesis of murine oral squamous cell carcinoma.

Circulating tumor-derived exosomes (TEX) interact with a variety of cells in cancer-bearing hosts, leading to cellular reprogramming which promotes disease progression. To study TEX effects on the development of solid tumors, immunosuppressive exosomes carrying PD-L1 and FasL were isolated from supernatants of murine or human HNSCC cell lines. TEX were delivered (IV) to immunocompetent C57BL/6 mice bearing premalignant oral/esophageal lesions induced by the carcinogen, 4-nitroquinoline 1-oxide (4NQO). Progression of the premalignant oropharyngeal lesions to malignant tumors was monitored. A single TEX injection increased the number of developing tumors (6.2 versus 3.2 in control mice injected with phosphate-buffered saline; P < 0.0002) and overall tumor burden per mouse (P < 0.037). The numbers of CD4+ and CD8+ T lymphocytes infiltrating the developing tumors were coordinately reduced (P < 0.01) in mice injected with SCCVII-derived TEX relative to controls. Notably, TEX isolated from mouse or human tumors had similar effects on tumor development and immune cells. A single IV injection of TEX was sufficient to condition mice harboring premalignant OSCC lesions for accelerated tumor progression in concert with reduced immune cell migration to the tumor.

Optimization of cell culture conditions for exosome isolation using mini-size exclusion chromatography (mini-SEC).

Extracellular vesicles (EVs) are emerging as a major intercellular communication system engaged in a variety of physiological and pathophysiological processes. Tumor-derived exosomes (TEX) are a subset of EVs of special interest as potential cancer biomarkers. Supernatants of tumor cell lines are widely used as the source of pure TEX for molecular/genetic studies. To optimize TEX isolation and characterization for these studies, we evaluated culture conditions for different tumor cell lines and used mini size exclusion chromatography (mini-SEC) for TEX isolation. Each tumor cell line showed unique culture requirements that determined the recovery, purity and total yield of TEX. Culture conditions for optimal TEX purity and recovery by mini-SEC could be modified by altering the media composition and numbers of seeded cells. TEX recovered from mini-SEC fraction #4 under optimized conditions were biologically active, were sized from 30 to 150 nm in diameter, had a typical vesicular morphology and carried endocytic markers. The most critical requirement for reproducible exosome recovery was re-seeding of tumor cells in numbers adjusted to reflect the optimized culture conditions for each tumor cell line. This study provides insights into a cell culture technique, which can be optimized for exosome production by various human or mouse tumor cell lines for isolation by mini-SEC.

Impact of combination immunochemotherapies on progression of 4NQO-induced murine oral squamous cell carcinoma.

Advanced oral squamous cell carcinomas (OSCC) have limited therapeutic options. Although immune therapies are emerging as a potentially effective alternative or adjunct to chemotherapies, the therapeutic efficacy of combination immune chemotherapies has yet to be determined. Using a 4-nitroquinolone-N-oxide (4NQO) orthotopic model of OSCC in immunocompetent mice, we evaluated the therapeutic efficacy of single- and combined-agent treatment with a poly-epitope tumor peptide vaccine, cisplatin and/or an A2AR inhibitor, ZM241385. The monotherapies or their combinations resulted in a partial inhibition of tumor growth and, in some cases, a significant but transient upregulation of systemic anti-tumor CD8+ T cell responses. These responses eroded in the face of expanding immunoregulatory cell populations at later stages of tumor progression. Our findings support the need for the further development of combinatorial therapeutic approaches that could more effectively silence dominant immune inhibitory pathways operating in OSCC and provide novel, more beneficial treatment options for this tumor.

Taking Control of Castleman Disease: Leveraging Precision Medicine Technologies to Accelerate Rare Disease Research.

Castleman disease (CD) is a rare and heterogeneous disorder characterized by lymphadenopathy that may occur in a single lymph node (unicentric) or multiple lymph nodes (multicentric), the latter typically occurring secondary to excessive proinflammatory hypercytokinemia. While a cohort of multicentric Castleman disease (MCD) cases are caused by Human Herpes Virus-8 (HHV-8), the etiology of HHV-8 negative, idiopathic MCD (iMCD), remains unknown. Breakthroughs in "omics" technologies that have facilitated the development of precision medicine hold promise for elucidating disease pathogenesis and identifying novel therapies for iMCD. However, in order to leverage precision medicine approaches in rare diseases like CD, stakeholders need to overcome several challenges. To address these challenges, the Castleman Disease Collaborative Network (CDCN) was founded in 2012. In the past 3 years, the CDCN has worked to transform the understanding of the pathogenesis of CD, funded and initiated genomics and proteomics research, and united international experts in a collaborative effort to accelerate progress for CD patients. The CDCN's collaborative structure leverages the tools of precision medicine and serves as a model for both scientific discovery and advancing patient care.

TGFß+ small extracellular vesicles from head and neck squamous cell carcinoma cells reprogram macrophages towards a pro-angiogenic phenotype.

Transforming growth factor ß (TGFß) is a major component of tumor-derived small extracellular vesicles (TEX) in cancer patients. Mechanisms utilized by TGFß+ TEX to promote tumor growth and pro-tumor activities in the tumor microenvironment (TME) are largely unknown. TEX produced by head and neck squamous cell carcinoma (HNSCC) cell lines carried TGFß and angiogenesis-promoting proteins. TGFß+ TEX stimulated macrophage chemotaxis without a notable M1/M2 phenotype shift and reprogrammed primary human macrophages to a pro-angiogenic phenotype characterized by the upregulation of pro-angiogenic factors and functions. In a murine basement membrane extract plug model, TGFß+ TEX promoted macrophage infiltration and vascularization (p < 0.001), which was blocked by using the TGFß ligand trap mRER (p < 0.001). TGFß+ TEX injected into mice undergoing the 4-nitroquinoline-1-oxide (4-NQO)-driven oral carcinogenesis promoted tumor angiogenesis (p < 0.05), infiltration of M2-like macrophages in the TME (p < 0.05) and ultimately tumor progression (p < 0.05). Inhibition of TGFß signaling in TEX with mRER ameliorated these pro-tumor activities. Silencing of TGFß emerges as a critical step in suppressing pro-angiogenic functions of TEX in HNSCC.

Exosomes from HNSCC Promote Angiogenesis through Reprogramming of Endothelial Cells.

For solid tumors, such as head and neck squamous cell carcinoma (HNSCC), an adequate blood supply is of critical importance for tumor development and metastasis. Tumor-derived exosomes (TEX) accumulate in the tumor microenvironment (TME) and serve as a communication system between tumor and normal stromal cells. This study evaluates in vitro and in vivo effects mediated by TEX that result in promotion of angiogenesis. TEX produced by PCI-13 (HPV-) and UMSCC47 (HPV+) cell lines or from plasma of HNSCC patients were isolated by mini size exclusion chromatography (mini-SEC). TEX morphology, size, numbers, and molecular profile were characterized, and the angiogenesis-inducing potential was measured in arrays and real-time PCR with human endothelial cells (HUVEC). Uptake of labeled TEX by HUVECs was demonstrated by confocal microscopy. Tube formation, proliferation, migration, and adherence by HUVECs in response to TEX were investigated. The 4-nitroquinoline-1-oxide (4-NQO) oral carcinogenesis mouse model was used to confirm that TEX induce the same results in vivo TEX were found to be potent inducers of angiogenesis in vitro and in vivo through functional reprogramming and phenotypic modulation of endothelial cells. TEX carried angiogenic proteins and were internalized by HUVECs within 4 hours. TEX stimulated proliferation (P < 0.001), migration (P < 0.05), and tube formation (P < 0.001) by HUVECs and promoted formation of defined vascular structures in vivo The data suggest that TEX promote angiogenesis and drive HNSCC progression. Future efforts should focus on eliminating or silencing TEX and thereby adding new options for improving existing antiangiogenic therapies.Implications: TEX appear to play an important role in tumor angiogenesis and thus may contribute to tumor growth and metastasis of HNSCC in this context. Mol Cancer Res; 16(11); 1798-808. ©2018 AACR.