Radiation-activated secretory proteins of Scgb1a1+ club cells increase the efficacy of immune checkpoint blockade in lung cancer.

Radiation therapy (RT) in combination with immune checkpoint inhibitor (ICI) represents a promising regimen for non-small cell lung cancer (NSCLC), however, the underlying mechanisms are poorly characterized. We identified a specific dose of RT that conferred tumor regression and improved survival in NSCLC models when combined with ICI. The immune-modulating functions of RT was ascribed to activated lung-resident Scgb1a1+ club cells. Importantly, mice with club cell-specific knockout of synaptosome-associated protein 23 failed to benefit from the combination treatment, indicating a pivotal role of club cell secretome. We identified 8 club cells secretory proteins, which inhibited immunosuppressive myeloid cells, reduced pro-tumor inflammation, and enhanced anti-tumor immunity. Notably, CC10, a member of club cell secretome was increased in plasma of NSCLC patients responding to the combination therapy. By revealing an immune-regulatory role of club cells, our studies have the potential to guide future clinical trials of ICI in NSCLC.

Copper depletion modulates mitochondrial oxidative phosphorylation to impair triple negative breast cancer metastasis.

Copper serves as a co-factor for a host of metalloenzymes that contribute to malignant progression. The orally bioavailable copper chelating agent tetrathiomolybdate (TM) has been associated with a significant survival benefit in high-risk triple negative breast cancer (TNBC) patients. Despite these promising data, the mechanisms by which copper depletion impacts metastasis are poorly understood and this remains a major barrier to advancing TM to a randomized phase II trial. Here, using two independent TNBC models, we report a discrete subpopulation of highly metastatic SOX2/OCT4+ cells within primary tumors that exhibit elevated intracellular copper levels and a marked sensitivity to TM. Global proteomic and metabolomic profiling identifies TM-mediated inactivation of Complex IV as the primary metabolic defect in the SOX2/OCT4+ cell population. We also identify AMPK/mTORC1 energy sensor as an important downstream pathway and show that AMPK inhibition rescues TM-mediated loss of invasion. Furthermore, loss of the mitochondria-specific copper chaperone, COX17, restricts copper deficiency to mitochondria and phenocopies TM-mediated alterations. These findings identify a copper-metabolism-metastasis axis with potential to enrich patient populations in next-generation therapeutic trials.

Tetrathiomolybdate (TM)-associated copper depletion influences collagen remodeling and immune response in the pre-metastatic niche of breast cancer.

Tetrathiomolybdate (TM) is a novel, copper-depleting compound associated with promising survival in a phase II study of patients with high-risk and triple-negative breast cancer. We sought to elucidate the mechanism of TM by exploring its effects on collagen processing and immune function in the tumor microenvironment (TME). Using an exploratory cohort, we identified markers of collagen processing (LOXL2, PRO-C3, C6M, and C1M) that differed between those with breast cancer versus controls. We measured these collagen biomarkers in TM-treated patients on the phase II study and detected evidence of decreased collagen cross-linking and increased degradation over formation in those without disease compared to those who experienced disease progression. Preclinical studies revealed decreased collagen deposition, lower levels of myeloid-derived suppressor cells, and higher CD4+ T-cell infiltration in TM-treated mice compared with controls. This study reveals novel mechanisms of TM targeting the TME and immune response with potential applications across cancer types.

Thrombospondin in Tumor Microenvironment.

Thrombospondins (TSPs) are multifaceted proteins that contribute to physiologic as well as pathologic conditions. Due to their multiple receptor-binding domains, TSPs display both oncogenic and tumor-suppressive qualities and are thus essential components of the extracellular matrix. Known for their antiangiogenic capacity, TSPs are an important component of the tumor microenvironment. The N- and C-terminal domains of TSP are, respectively, involved in cell adhesion and spreading, an important feature of wound healing as well as cancer cell migration. Previously known for the activation of TGF-ß to promote tumor growth and inflammation, TSP-1 has recently been found to be transcriptionally induced by TGF-ß, implying the presence of a possible feedback loop. TSP-1 is an endogenous inhibitor of T cells and also mediates its immunosuppressive effects via induction of Tregs. Given the diverse roles of TSPs in the tumor microenvironment, many therapeutic strategies have utilized TSP-mimetic peptides or antibody blockade as anti-metastatic approaches. This chapter discusses the diverse structural domains, functional implications, and anti-metastatic therapies in the context of the role of TSP in the tumor microenvironment.

Differential Contributions of Pre- and Post-EMT Tumor Cells in Breast Cancer Metastasis.

Metastases are responsible for the majority of breast cancer-associated deaths. The contribution of epithelial-to-mesenchymal transition (EMT) in the establishment of metastases is still controversial. To obtain in vivo evidence of EMT in metastasis, we established an EMT lineage tracing (Tri-PyMT) model, in which tumor cells undergoing EMT would irreversibly switch their fluorescent marker from RFP+ to GFP+ due to mesenchymal-specific Cre expression. Surprisingly, we found that lung metastases were predominantly derived from the epithelial compartment of breast tumors. However, concerns were raised on the fidelity and sensitivity of RFP-to-GFP switch of this model in reporting EMT of metastatic tumor cells. Here, we evaluated Tri-PyMT cells at the single-cell level using single-cell RNA-sequencing and found that the Tri-PyMT cells exhibited a spectrum of EMT phenotypes, with EMT-related genes concomitantly expressed with the activation of GFP. The fluorescent color switch in these cells precisely marked an unequivocal change in EMT status, defining the pre-EMT and post-EMT compartments within the tumor. Consistently, the pre-EMT cells played dominant roles in metastasis, while the post-EMT cells were supportive in promoting tumor invasion and angiogenesis. Importantly, the post-EMT (GFP+) cells in the Tri-PyMT model were not permanently committed to the mesenchymal phenotype; they were still capable of reverting to the epithelial phenotype and giving rise to secondary tumors, suggesting their persistent EMT plasticity. Our study addressed major concerns with the Tri-PyMT EMT lineage tracing model, which provides us with a powerful tool to investigate the dynamic EMT process in tumor biology. SIGNIFICANCE: These findings confirm the fidelity and sensitivity of the EMT lineage tracing (Tri-PyMT) model and highlight the differential contributions of pre- and post-EMT tumor cells in breast cancer metastasis.See related commentary by Bunz, p. 153.

Copper Depletion as a Therapeutic Strategy in Cancer.

Copper is an essential trace element that plays a critical role in a variety of basic biological functions, and serves as a key component in a number of copper-dependent enzymes that regulate such processes as cell proliferation, angiogenesis, and motility. A growing body of preclinical work has demonstrated that copper is essential to metastatic cancer progression, and may have a role in tumor growth, epithelial-mesenchymal transition, and the formation of the tumor microenvironment and pre-metastatic niche. As a result, copper depletion has emerged as a novel therapeutic strategy in the treatment of metastatic cancer. We present a review of the physiologic role of copper with a discussion of relevant enzymes of the copper proteome in both normal tissue and in cancer. We conducted a comprehensive review of the available preclinical data of several copper chelation agents, including penicillamine, trientine, disulfiram, clioquinol, and tetrathiomolybdate (TM), across a variety of tumor types. We also present the existing early phase clinical trial data for the use of the copper chelator TM in the treatment of breast cancer and other malignancies.

Nanoparticle Delivery of miR-708 Mimetic Impairs Breast Cancer Metastasis.

Triple-negative breast cancer (TNBC) patients exhibit the worst clinical outcome due to its aggressive clinical course, higher rate of recurrence, and a conspicuous lack of FDA-approved targeted therapies. Here, we show that multilayered nanoparticles (NPs) carrying the metastasis suppressor microRNA miR-708 (miR708-NP) localize to orthotopic primary TNBC, and efficiently deliver the miR-708 cargo to reduce lung metastasis. Using a SOX2/OCT4 promoter reporter, we identified a population of miR-708low cancer cells with tumor-initiating properties, enhanced metastatic potential, and marked sensitivity to miR-708 treatment. In vivo, miR708-NP directly targeted the SOX2/OCT4-mCherry+ miR-708low tumor cells to impair metastasis. Together, our preclinical findings provide a mechanism-based antimetastatic therapeutic approach for TNBC, with a marked potential to generate miR-708 replacement therapy for high-risk TNBC patients in the clinic. To our knowledge, this gold nanoparticle-based delivery of microRNA mimetic is the first oligonucleotide-based targeted therapy for TNBC.

Activation of carbonic anhydrase IX by alternatively spliced tissue factor under late-stage tumor conditions.

Molecules of the coagulation pathway predispose patients to cancer-associated thrombosis and also trigger intracellular signaling pathways that promote cancer progression. The primary transcript of tissue factor, the main physiologic trigger of blood clotting, can undergo alternative splicing yielding a secreted variant, termed asTF (alternatively spliced tissue factor). asTF is not required for normal hemostasis, but its expression levels positively correlate with advanced tumor stages in several cancers, including pancreatic adenocarcinoma. The asTF-overexpressing pancreatic ductal adenocarcinoma cell line Pt45.P1/asTF+ and its parent cell line Pt45.P1 were tested for growth and mobility under normoxic conditions that model early-stage tumors, and in the hypoxic environment of late-stage cancers. asTF overexpression in Pt45.P1 cells conveys increased proliferative ability. According to cell cycle analysis, the major fraction of Pt45.P1/asTF+ cells reside in the dividing G2/M phase of the cell cycle, whereas the parental Pt45.P1 cells are mostly confined to the quiescent G0/G1 phase. asTF overexpression is also associated with significantly higher mobility in cells plated under either normoxia or hypoxia. A hypoxic environment leads to upregulation of carbonic anhydrase IX (CAIX), which is more pronounced in Pt45.P1/asTF+ cells. Inhibition of CAIX by the compound U-104 significantly decreases cell growth and mobility of Pt45.P1/asTF+ cells in hypoxia, but not in normoxia. U-104 also reduces the growth of Pt45.P1/asTF+ orthotopic tumors in nude mice. CAIX is a novel downstream mediator of asTF in pancreatic cancer, particularly under hypoxic conditions that model late-stage tumor microenvironment.

Antibody-based targeting of alternatively spliced tissue factor: a new approach to impede the primary growth and spread of pancreatic ductal adenocarcinoma.

Alternatively spliced Tissue Factor (asTF) is a secreted form of Tissue Factor (TF), the trigger of blood coagulation whose expression levels are heightened in several forms of solid cancer, including pancreatic ductal adenocarcinoma (PDAC). asTF binds to ß1 integrins on PDAC cells, whereby it promotes tumor growth, metastatic spread, and monocyte recruitment to the stroma. In this study, we determined if targeting asTF in PDAC would significantly impact tumor progression. We here report that a novel inhibitory anti-asTF monoclonal antibody curtails experimental PDAC progression. Moreover, we show that tumor-derived asTF is able to promote PDAC primary growth and spread during early as well as later stages of the disease. This raises the likelihood that asTF may comprise a viable target in early- and late-stage PDAC. In addition, we show that TF expressed by host cells plays a significant role in PDAC spread. Together, our data demonstrate that targeting asTF in PDAC is a novel strategy to stem PDAC progression and spread.

Interactions between osteopontin and vascular endothelial growth factor: Implications for skeletal disorders.

Osteopontin (OPN) and vascular endothelial growth factor (VEGF) are characterized by a convergence in function for maintaining the homeostasis of the skeletal and renal systems (the bone-renal-vascular axis regulates bone metabolism). The two cytokines contribute to bone remodeling, dental healing, kidney function, and the adjustment to microgravity. Often, they are co-expressed or one molecule induces the other, however, in some settings OPN-associated pathways and VEGF-associated pathways are distinct. In bone remodeling, OPN and VEGF are regulated under the influence of growth factors and hormones, hypoxia and inflammation, the micro-environment, and various physical forces. Their abundance can be affected by drug treatment. OPN and VEGF are variably associated with kidney disease. Their balanced levels are critical for restoring endothelial cell function and ameliorating the adverse effects of microgravity. Here, we review the relevant 83 papers of 257 articles published, and listed in PubMed under the key words OPN and VEGF.

Interactions between osteopontin and vascular endothelial growth factor: Implications for cancer.

For this comprehensive review, 257 publications with the keywords "osteopontin" or "OPN" and "vascular endothelial growth factor" or "VEGF" in PubMed were screened (time frame from year 1996 to year 2014). 37 articles were excluded because they were not focused on the interactions between these molecules, and papers relevant for transformation-related phenomena were selected. Osteopontin (OPN) and vascular endothelial growth factor (VEGF) are characterized by a convergence in function for regulating cell motility and angiogenesis, the response to hypoxia, and apoptosis. Often, they are co-expressed or one molecule induces the other, however, in some settings OPN-associated pathways and VEGF-associated pathways are distinct. Their relationships affect the pathogenesis in cancer, where they contribute to progression and angiogenesis and serve as markers for poor prognosis. The inhibition of OPN may reduce VEGF levels and suppress tumor progression. In vascular pathologies, these two cytokines mediate remodeling, but may also perpetuate inflammation and narrowing of the arteries. OPN and VEGF are elevated and contribute to vascularization in inflammatory diseases.

An osteopontin promoter polymorphism is associated with aggressiveness in breast cancer.

Metastasis-related genes are deregulated in cancer by aberrant expression or splicing. Here, we analyzed polymorphic sites in the osteopontin promoter as potential contributors to aberrant expression in breast cancers. This study comprised 241 breast cancer specimens, for which DNA from normal surrounding tissue was available for 111, and 65 healthy breast samples. The polymorphic site in position -443 of the promoter was associated with tumor grade. As expected, there was no association between promoter single nucleotide polymorphisms (SNPs) and tumor stage or in situ carcinoma versus cancer, as stage and early transformation are determined by the sampling time more than by tumor genetics. In a subset of samples, osteopontin RNA expression levels had previously been obtained. The allelic distribution in positions -443 and -1748 was distinct between high and low expressors, confirming the importance of promoter SNPs. These two sites also form a haplotype. Osteopontin expression has been associated with breast cancer progression, regardless of the histological subtype of the cancer. Remarkably, the polymorphic site at -443, but not -1748 or -1776, showed differences between ER-positive and ER-negative breast cancers and between PR-positive and PR-negative breast cancers, but there was no association with HER2 status. In five cases, the genotype of the tumor was different from the genotype of the host, implying the possibility of somatic mutations in the osteopontin promoter that may affect expression. Our results corroborate that the osteopontin promoter SNPs -443 (rs11730582) and -1748 (rs2728127) are important for gene expression and breast cancer aggressiveness.