CD38-Mediated Immunosuppression as a Mechanism of Tumor Cell Escape from PD-1/PD-L1 Blockade.

Although treatment with immune checkpoint inhibitors provides promising benefit for patients with cancer, optimal use is encumbered by high resistance rates and requires a thorough understanding of resistance mechanisms. We observed that tumors treated with PD-1/PD-L1 blocking antibodies develop resistance through the upregulation of CD38, which is induced by all-trans retinoic acid and IFNß in the tumor microenvironment. In vitro and in vivo studies demonstrate that CD38 inhibits CD8+ T-cell function via adenosine receptor signaling and that CD38 or adenosine receptor blockade are effective strategies to overcome the resistance. Large data sets of human tumors reveal expression of CD38 in a subset of tumors with high levels of basal or treatment-induced T-cell infiltration, where immune checkpoint therapies are thought to be most effective. These findings provide a novel mechanism of acquired resistance to immune checkpoint therapy and an opportunity to expand their efficacy in cancer treatment.Significance: CD38 is a major mechanism of acquired resistance to PD-1/PD-L1 blockade, causing CD8+ T-cell suppression. Coinhibition of CD38 and PD-L1 improves antitumor immune response. Biomarker assessment in patient cohorts suggests that a combination strategy is applicable to a large percentage of patients in whom PD-1/PD-L1 blockade is currently indicated. Cancer Discov; 8(9); 1156-75. ©2018 AACR.See related commentary by Mittal et al., p. 1066This article is highlighted in the In This Issue feature, p. 1047.

Thy-1+ Cancer-associated Fibroblasts Adversely Impact Lung Cancer Prognosis.

Cancer-associated fibroblasts (CAFs) regulate diverse intratumoral biological programs and can promote or inhibit tumorigenesis, but those CAF populations that negatively impact the clinical outcome of lung cancer patients have not been fully elucidated. Because Thy-1 (CD90) marks CAFs that promote tumor cell invasion in a murine model of KrasG12D-driven lung adenocarcinoma (KrasLA1), here we postulated that human lung adenocarcinomas containing Thy-1+ CAFs have a worse prognosis. We first examined the location of Thy-1+ CAFs within human lung adenocarcinomas. Cells that co-express Thy-1 and α-smooth muscle actin (αSMA), a CAF marker, were located on the tumor periphery surrounding collectively invading tumor cells and in perivascular regions. To interrogate a human lung cancer database for the presence of Thy-1+ CAFs, we isolated Thy-1+ CAFs and normal lung fibroblasts (LFs) from the lungs of KrasLA1 mice and wild-type littermates, respectively, and performed global proteomic analysis on the murine CAFs and LFs, which identified 425 proteins that were differentially expressed. Used as a probe to identify Thy-1+ CAF-enriched tumors in a compendium of 1,586 lung adenocarcinomas, the presence of the 425-gene signature predicted a significantly shorter survival. Thus, Thy-1 marks a CAF population that adversely impacts clinical outcome in human lung cancer.

Epithelial-to-mesenchymal transition drives a pro-metastatic Golgi compaction process through scaffolding protein PAQR11.

Tumor cells gain metastatic capacity through a Golgi phosphoprotein 3-dependent (GOLPH3-dependent) Golgi membrane dispersal process that drives the budding and transport of secretory vesicles. Whether Golgi dispersal underlies the pro-metastatic vesicular trafficking that is associated with epithelial-to-mesenchymal transition (EMT) remains unclear. Here, we have shown that, rather than causing Golgi dispersal, EMT led to the formation of compact Golgi organelles with improved ribbon linking and cisternal stacking. Ectopic expression of the EMT-activating transcription factor ZEB1 stimulated Golgi compaction and relieved microRNA-mediated repression of the Golgi scaffolding protein PAQR11. Depletion of PAQR11 dispersed Golgi organelles and impaired anterograde vesicle transport to the plasma membrane as well as retrograde vesicle tethering to the Golgi. The N-terminal scaffolding domain of PAQR11 was associated with key regulators of Golgi compaction and vesicle transport in pull-down assays and was required to reconstitute Golgi compaction in PAQR11-deficient tumor cells. Finally, high PAQR11 levels were correlated with EMT and shorter survival in human cancers, and PAQR11 was found to be essential for tumor cell migration and metastasis in EMT-driven lung adenocarcinoma models. We conclude that EMT initiates a PAQR11-mediated Golgi compaction process that drives metastasis.

Growth and metastasis of lung adenocarcinoma is potentiated by BMP4-mediated immunosuppression.

Cancer cells modulate the recruitment and function of inflammatory cells to create an immunosuppressive microenvironment that favors tumor growth and metastasis. However, the tumor-derived regulatory programs that promote intratumoral immunosuppression remain poorly defined. Here, we show in a KrasLA1/+p53R172HΔg/+-based mouse model that bone morphogenetic protein-4 (BMP4) augments the expression of the T cell co-inhibitory receptor ligand PD-L1 in the mesenchymal subset of lung cancer cells, leading to profound CD8+ T cell-mediated immunosuppression, producing tumor growth and metastasis. We previously reported in this model that BMP4 functions as a pro-tumorigenic factor regulated by miR-200 via GATA4/6. Thus, BMP4-mediated immunosuppression is part of a larger miR-200-directed gene expression program in tumors that promotes tumor progression, which could have important implications for cancer treatment.

Ultrahigh-throughput Generation and Characterization of Cellular Aggregates in Laser-ablated Microwells of Poly(dimethylsiloxane).

Aggregates of cells, also known as multicellular aggregates (MCAs), have been used as microscale tissues in the fields of cancer biology, regenerative medicine, and developmental biology for many decades. However, small MCAs (fewer than 100 cells per aggregate) have remained challenging to manufacture in large quantities at high uniformity. Forced aggregation into microwells offers a promising solution for forming consistent aggregates, but commercial sources of microwells are expensive, complicated to manufacture, or lack the surface packing densities that would significantly improve MCA production. To address these concerns, we custom-modified a commercial laser cutter to provide complete control over laser ablation and directly generate microwells in a poly(dimethylsiloxane) (PDMS) substrate. We achieved ultra rapid microwell production speeds (>50,000 microwells/hr) at high areal packing densities (1,800 microwells/cm2) and over large surface areas for cell culture (60 cm2). Variation of the PDMS substrate distance from the laser focal plane during ablation allowed for the generation of microwells with a variety of sizes, contours, and aspect ratios. Casting of high-fidelity microneedle masters in polyurethane allowed for non-ablative microwell reproduction through replica molding. MCAs of human bone marrow derived mesenchymal stem cells (hMSCs), murine 344SQ metastatic adenocarcinoma cells, and human C4-2 prostate cancer cells were generated in our system with high uniformity within 24 hours, and computer vision software aided in the ultra-high-throughput analysis of harvested aggregates. Moreover, MCAs maintained invasive capabilities in 3D migration assays. In particular, 344SQ MCAs demonstrated epithelial lumen formation on Matrigel, and underwent EMT and invasion in the presence of TGF-ß. We expect this technique to find broad utility in the generation and cultivation of cancer cell aggregates, primary cell aggregates, and embryoid bodies.

The microRNA-200/Zeb1 axis regulates ECM-dependent ß1-integrin/FAK signaling, cancer cell invasion and metastasis through CRKL.

Tumor cell metastasis is a complex process that has been mechanistically linked to the epithelial-mesenchymal transition (EMT). The double-negative feedback loop between the microRNA-200 family and the Zeb1 transcriptional repressor is a master EMT regulator, but there is incomplete understanding of how miR-200 suppresses invasion. Our recent efforts have focused on the tumor cell-matrix interactions essential to tumor cell activation. Herein we utilized both our Kras/p53 mutant mouse model and human lung cancer cell lines to demonstrate that upon miR-200 loss integrin ß1-collagen I interactions drive 3D in vitro migration/invasion and in vivo metastases. Zeb1-dependent EMT enhances tumor cell responsiveness to the ECM composition and activates FAK/Src pathway signaling by de-repression of the direct miR-200 target, CRKL. We demonstrate that CRKL serves as an adaptor molecule to facilitate focal adhesion formation, mediates outside-in signaling through Itgß1 to drive cell invasion, and inside-out signaling that maintains tumor cell-matrix contacts required for cell invasion. Importantly, CRKL levels in pan-cancer TCGA analyses were predictive of survival and CRKL knockdown suppressed experimental metastases in vivo without affecting primary tumor growth. Our findings highlight the critical ECM-tumor cell interactions regulated by miR-200/Zeb1-dependent EMT that activate intracellular signaling pathways responsible for tumor cell invasion and metastasis.

Cancer-Associated Fibroblasts Induce a Collagen Cross-link Switch in Tumor Stroma.

UNLABELLED: Intratumoral collagen cross-links heighten stromal stiffness and stimulate tumor cell invasion, but it is unclear how collagen cross-linking is regulated in epithelial tumors. To address this question, we used Kras(LA1) mice, which develop lung adenocarcinomas from somatic activation of a Kras(G12D) allele. The lung tumors in Kras(LA1) mice were highly fibrotic and contained cancer-associated fibroblasts (CAF) that produced collagen and generated stiffness in collagen gels. In xenograft tumors generated by injection of wild-type mice with lung adenocarcinoma cells alone or in combination with CAFs, the total concentration of collagen cross-links was the same in tumors generated with or without CAFs, but coinjected tumors had higher hydroxylysine aldehyde-derived collagen cross-links (HLCC) and lower lysine-aldehyde-derived collagen cross-links (LCCs). Therefore, we postulated that an LCC-to-HLCC switch induced by CAFs promotes the migratory and invasive properties of lung adenocarcinoma cells. To test this hypothesis, we created coculture models in which CAFs are positioned interstitially or peripherally in tumor cell aggregates, mimicking distinct spatial orientations of CAFs in human lung cancer. In both contexts, CAFs enhanced the invasive properties of tumor cells in three-dimensional (3D) collagen gels. Tumor cell aggregates that attached to CAF networks on a Matrigel surface dissociated and migrated on the networks. Lysyl hydroxylase 2 (PLOD2/LH2), which drives HLCC formation, was expressed in CAFs, and LH2 depletion abrogated the ability of CAFs to promote tumor cell invasion and migration. IMPLICATIONS: CAFs induce a collagen cross-link switch in tumor stroma to influence the invasive properties of tumor cells.

Metastasis is regulated via microRNA-200/ZEB1 axis control of tumour cell PD-L1 expression and intratumoral immunosuppression.

Immunosuppression of tumour-infiltrating lymphocytes (TIL) is a common feature of advanced cancer, but its biological basis has remained obscure. We demonstrate here a molecular link between epithelial-to-mesenchymal transition (EMT) and CD8(+) TIL immunosuppression, two key drivers of cancer progression. We show that microRNA-200 (miR-200), a cell-autonomous suppressor of EMT and metastasis, targets PD-L1. Moreover, ZEB1, an EMT activator and transcriptional repressor of miR-200, relieves miR-200 repression of PD-L1 on tumour cells, leading to CD8(+) T-cell immunosuppression and metastasis. These findings are supported by robust correlations between the EMT score, miR-200 levels and PD-L1 expression in multiple human lung cancer datasets. In addition to revealing a link between EMT and T-cell dysfunction, these findings also show that ZEB1 promotes metastasis through a heretofore unappreciated cell non-autonomous mechanism, and suggest that subgroups of patients in whom malignant progression is driven by EMT activators may respond to treatment with PD-L1 antagonists.

Fibulin-2 is a driver of malignant progression in lung adenocarcinoma.

The extracellular matrix of epithelial tumors undergoes structural remodeling during periods of uncontrolled growth, creating regional heterogeneity and torsional stress. How matrix integrity is maintained in the face of dynamic biophysical forces is largely undefined. Here we investigated the role of fibulin-2, a matrix glycoprotein that functions biomechanically as an inter-molecular clasp and thereby facilitates supra-molecular assembly. Fibulin-2 was abundant in the extracellular matrix of human lung adenocarcinomas and was highly expressed in tumor cell lines derived from mice that develop metastatic lung adenocarcinoma from co-expression of mutant K-ras and p53. Loss-of-function experiments in tumor cells revealed that fibulin-2 was required for tumor cells to grow and metastasize in syngeneic mice, a surprising finding given that other intra-tumoral cell types are known to secrete fibulin-2. However, tumor cells grew and metastasized equally well in Fbln2-null and -wild-type littermates, implying that malignant progression was dependent specifically upon tumor cell-derived fibulin-2, which could not be offset by other cellular sources of fibulin-2. Fibulin-2 deficiency impaired the ability of tumor cells to migrate and invade in Boyden chambers, to create a stiff extracellular matrix in mice, to cross-link secreted collagen, and to adhere to collagen. We conclude that fibulin-2 is a driver of malignant progression in lung adenocarcinoma and plays an unexpected role in collagen cross-linking and tumor cell adherence to collagen.

A synthetic matrix with independently tunable biochemistry and mechanical properties to study epithelial morphogenesis and EMT in a lung adenocarcinoma model.

Better understanding of the biophysical and biochemical cues of the tumor extracellular matrix environment that influence metastasis may have important implications for new cancer therapeutics. Initial exploration into this question has used naturally derived protein matrices that suffer from variability, poor control over matrix biochemistry, and inability to modify the matrix biochemistry and mechanics. Here, we report the use of a synthetic polymer-based scaffold composed primarily of poly(ethylene glycol), or PEG, modified with bioactive peptides to study murine models of lung adenocarcinoma. In this study, we focus on matrix-derived influences on epithelial morphogenesis of a metastatic cell line (344SQ) that harbors mutations in Kras and p53 (trp53) and is prone to a microRNA-200 (miR-200)-dependent epithelial-mesenchymal transition (EMT) and metastasis. The modified PEG hydrogels feature biospecific cell adhesion and cell-mediated proteolytic degradation with independently adjustable matrix stiffness. 344SQ encapsulated in bioactive peptide-modified, matrix metalloproteinase-degradable PEG hydrogels formed lumenized epithelial spheres comparable to that seen with three-dimensional culture in Matrigel. Altering both matrix stiffness and the concentration of cell-adhesive ligand significantly influenced epithelial morphogenesis as manifest by differences in the extent of lumenization, in patterns of intrasphere apoptosis and proliferation, and in expression of epithelial polarity markers. Regardless of matrix composition, exposure to TGF-ß induced a loss of epithelial morphologic features, shift in expression of EMT marker genes, and decrease in mir-200 levels consistent with EMT. Our findings help illuminate matrix-derived cues that influence epithelial morphogenesis and highlight the potential utility that this synthetic matrix-mimetic tool has for cancer biology.

Map2k4 functions as a tumor suppressor in lung adenocarcinoma and inhibits tumor cell invasion by decreasing peroxisome proliferator-activated receptor γ2 expression.

MAP2K4 encodes a dual-specificity kinase (mitogen-activated protein kinase kinase 4, or MKK4) that is mutated in a variety of human malignancies, but the biochemical properties of the mutant kinases and their roles in tumorigenesis have not been fully elucidated. Here we showed that 8 out of 11 cancer-associated MAP2K4 mutations reduce MKK4 protein stability or impair its kinase activity. On the basis of findings from bioinformatic studies on human cancer cell lines with homozygous MAP2K4 loss, we posited that MKK4 functions as a tumor suppressor in lung adenocarcinomas that develop in mice owing to expression of mutant Kras and Tp53. Conditional Map2k4 inactivation in the bronchial epithelium of mice had no discernible effect alone but increased the multiplicity and accelerated the growth of incipient lung neoplasias induced by oncogenic Kras. MKK4 suppressed the invasion and metastasis of Kras-Tp53-mutant lung adenocarcinoma cells. MKK4 deficiency increased peroxisomal proliferator-activated receptor γ2 (PPARγ2) expression through noncanonical MKK4 substrates, and PPARγ2 enhanced tumor cell invasion. We conclude that Map2k4 functions as a tumor suppressor in lung adenocarcinoma and inhibits tumor cell invasion by decreasing PPARγ2 levels.

miR-200 Inhibits lung adenocarcinoma cell invasion and metastasis by targeting Flt1/VEGFR1.

The microRNA-200 (miR-200) family is part of a gene expression signature that predicts poor prognosis in lung cancer patients. In a mouse model of K-ras/p53-mutant lung adenocarcinoma, miR-200 levels are suppressed in metastasis-prone tumor cells, and forced miR-200 expression inhibits tumor growth and metastasis, but the miR-200 target genes that drive lung tumorigenesis have not been fully elucidated. Here, we scanned the genome for putative miR-200 binding sites and found them in the 3'-untranslated region (3'-UTR) of 35 genes that are amplified in human cancer. Mining of a database of resected human lung adenocarcinomas revealed that the levels of one of these genes, Flt1/VEGFR1, correlate inversely with duration of survival. Forced miR-200 expression suppressed Flt1 levels in metastasis-prone lung adenocarcinoma cells derived from K-ras/p53-mutant mice, and negatively regulated the Flt1 3'-UTR in reporter assays. Cancer-associated fibroblasts (CAFs) isolated from murine lung adenocarcinomas secreted abundant VEGF and enhanced tumor cell invasion in coculture studies. CAF-induced tumor cell invasion was abrogated by VEGF neutralization or Flt1 knockdown in tumor cells. Flt1 knockdown decreased the growth and metastasis of tumor cells in syngeneic mice. We conclude that miR-200 suppresses lung tumorigenesis by targeting Flt1.

Identification of secreted proteins that mediate cell-cell interactions in an in vitro model of the lung cancer microenvironment.

Non-small cell lung cancer (NSCLC) cells with somatic mutations in K-ras recruit to the tumor a variety of cell types (hereafter collectively termed "stromal cells") that can promote or inhibit tumorigenesis by mechanisms that have not been fully elucidated. Here, we postulated that stromal cells in the tumor microenvironment alter the tumor cell secretome, including those proteins required for tumor growth and dissemination, and we developed an in vitro model to test this hypothesis. Coculturing a murine K-ras mutant lung adenocarcinoma cell line (LKR-13) with a murine lung stromal cell (macrophage, endothelial cell, or fibroblast) enhanced stromal cell migration, induced endothelial tube formation, increased LKR-13 cell proliferation, and regulated the secretion of proteins involved in angiogenesis, inflammation, cell proliferation, and epithelial-to-mesenchymal transition. Among these proteins, CXCL1 has been reported to promote NSCLC development, whereas interleukin-18 (IL-18) has an undefined role. Genetic and pharmacologic strategies to inhibit CXCL1 and IL-18 revealed that stromal cell migration, LKR-13 cell proliferation, and LKR-13 cell tumorigenicity required one or both of these proteins. We conclude that stromal cells enhanced LKR-13 cell tumorigenicity partly through their effects on the secretome of LKR-13 cells. Strategies to inhibit tumor/stromal cell interactions may be useful as therapeutic approaches in NSCLC patients.