From the Cover: PNAS Plus: Functional screen identifies kinases driving prostate cancer visceral and bone metastasis

Mutationally activated kinases play an important role in the progression and metastasis of many cancers. Despite numerous oncogenic alterations implicated in metastatic prostate cancer, mutations of kinases are rare. Several lines of evidence suggest that nonmutated kinases and their pathways are involved in prostate cancer progression, but few kinases have been mechanistically linked to metastasis. Using a mass spectrometry-based phosphoproteomics dataset in concert with gene expression analysis, we selected over 100 kinases potentially implicated in human metastatic prostate cancer for functional evaluation. A primary in vivo screen based on overexpression of candidate kinases in murine prostate cells identified 20 wild-type kinases that promote metastasis. We queried these 20 kinases in a secondary in vivo screen using human prostate cells. Strikingly, all three RAF family members, MERTK, and NTRK2 drove the formation of bone and visceral metastasis confirmed by positron-emission tomography combined with computed tomography imaging and histology. Immunohistochemistry of tissue microarrays indicated that these kinases are highly expressed in human metastatic castration-resistant prostate cancer tissues. Our functional studies reveal the strong capability of select wild-type protein kinases to drive critical steps of the metastatic cascade, and implicate these kinases in possible therapeutic intervention.Metastatic prostate cancer is responsible for the deaths of ∼30,000 men in the United States each year (1, 2). Ninety percent of patients develop bone metastases, and other major sites of metastases include lymph nodes, liver, adrenal glands, and lung (3). First-line treatments for metastatic disease are androgen deprivation therapies that block androgen synthesis or signaling through the androgen receptor (AR) (2). Inevitably, metastatic prostate cancer becomes resistant to androgen blockade. Second-line treatments such as chemotherapy (docetaxel, cabazitaxel) and radiation only extend survival 2–4 mo (4, 5).Identifying new therapeutic targets for metastatic prostate cancer has proven difficult. Exome and whole-genome sequencing of human metastatic prostate cancer tissues have found frequent mutations and/or chromosomal aberrations in numerous genes, including AR, TP53, PTEN, BRCA2, and MYC (6–11). The precise functional contribution of these genes to prostate cancer metastasis remains unknown. Genomic and phosphoproteomic analyses have also revealed that metastatic prostate cancer is molecularly heterogeneous, which has complicated the search for common therapeutic targets (12). Few murine models of prostate cancer develop metastases. Mice having prostate-specific homozygous deletions in SMAD4 and PTEN or expression of mutant KRAS develop metastases in visceral organs but rarely in bone (13–15).Targeting genetically altered constitutively active protein kinases such as BCR-ABL in chronic myelogenous leukemia and BRAF^V600E in melanoma has led to dramatic clinical responses (16). Although numerous oncogenic alterations have been identified in prostate cancer, DNA amplifications, translocations, or other mutations resulting in constitutive activity of kinases are rare (6, 9, 17). Genome sequencing of metastatic prostate cancer tissues from >150 patients found translocations involving the kinases BRAF and CRAF in <1% of patients (8, 18). Although uncommon, these genomic aberrations cause enhanced BRAF and CRAF kinase activity and suggest that kinase-driven pathways can be crucial in prostate cancer. Multiple lines of evidence indicate that nonmutated kinases may contribute to prostate cancer progression, castration resistance, and metastasis. SRC kinase synergizes with AR to drive the progression of early-stage prostatic intraepithelial neoplasia to advanced adenocarcinoma (19). SRC, BMX, and TNK2 kinases promote castration resistance by phosphorylating and stabilizing AR (20–22). Moreover, FGFR1, AKT1, and EGFR kinases activate pathways in prostate cancer cells to drive epithelial-to-mesenchymal transition and angiogenesis, both of which are key steps in metastasis (23–25). Despite the strong evidence implicating kinases in advanced prostate cancer, a systematic analysis of the functional role of kinases in prostate cancer metastasis has been lacking.Metastasis of epithelial-derived cancers encompasses a complex cascade of steps, including (i) migration and invasion through surrounding stroma/basement membrane, (ii) intravasation and survival in circulation/lymphatics, (iii) extravasation through the vasculature, and (iv) survival and growth at a secondary site (26). With the exception of genetically engineered mouse models, no single experimental assay can model all steps of the metastatic cascade. As a result, most screens for genes involved in metastasis have focused on testing one step of the cascade. The migration/invasion step of metastasis is commonly interrogated in vitro by determining the ability of cells to invade through small pores in a membrane (27–29). Genes that function in other steps, or those dependent on the in vivo microenvironment to promote metastasis, are likely to be overlooked in these screens.Multiple groups have performed in vivo screens for regulators of metastasis by manipulating cell lines in vitro with shRNA libraries or using genome editing techniques, and injecting cells either subcutaneously or into the tail vein of mice (30, 31). These methods are advantageous, because they interrogate multiple steps of the metastatic cascade (survival in circulation, extravasation, and colonization and growth at a secondary site) in a physiologically relevant environment. However, the majority of in vivo screens conducted so far have been based on loss-of-function genetics. These screens are limited to inhibiting the function of proteins expressed by a particular cell line. Using a gain-of-function in vivo screen, we sought to identify kinases that activate pathways leading to prostate cancer metastasis.     

CircURI1 interacts with hnRNPM to inhibit metastasis by modulating alternative splicing in gastric cancer

Circular RNAs (circRNAs) have emerged as key regulators of human cancers, yet their modes of action in gastric cancer (GC) remain largely unknown. Here, we identified circURI1 back-spliced from exons 3 and 4 of unconventional prefoldin RPB5 interactor 1 (URI1) from circRNA profiling of five-paired human gastric and the corresponding nontumor adjacent specimens (paraGC). CircURI1 exhibits the significantly higher expression in GC compared with paraGC and inhibitory effects on cell migration and invasion in vitro and GC metastasis in vivo. Mechanistically, circURI1 directly interacts with heterogeneous nuclear ribonucleoprotein M (hnRNPM) to modulate alternative splicing of genes, involved in the process of cell migration, thus suppressing GC metastasis. Collectively, our study expands the current knowledge regarding the molecular mechanism of circRNA-mediated cancer metastasis via modulating alternative splicing.

Gastric cancer (GC) is the fifth most-common malignant tumor and the third leading cause of cancer death worldwide (1, 2). Although diverse treatment options are available, the prognosis for GC patients remains poor, largely due to the presence of metastatic spread in patients (3). Cancer metastasis is a multifactorial, multistep, and complex process influenced by environmental and genetic factors (1). The outlook for metastatic GC patients is very poor, with a median overall survival of ∼8 mo (2). Understanding GC metastasis at the molecular and cellular levels will help to identify potential biomarkers for diagnosis and therapeutic targets for intervention.Circular RNAs (circRNAs) are covalent, closed, single-stranded RNA molecules generated by back-splicing or other RNA circularization mechanisms (4–7). In contrast to linear RNAs, circRNAs lack 5′ and 3′ ends and are resistant to RNA exonuclease, providing them with promising features to serve as potential biomarkers or therapeutic targets (4). Accumulating lines of evidence have illustrated the ectopic expression patterns and fundamental regulatory functions of circRNAs in biological processes, including the cell cycle, cell growth, and metastasis (8–11). Some cytoplasmic circRNAs serve as microRNA (miRNA) sponges to lift the inhibitory effects of miRNAs on their targets (12–15). Another mechanism is that exon–intron circRNAs (EIciRNAs) promote gene expression by binding to the U1 small nuclear ribonucleoprotein complex in the nucleus (16). Furthermore, a small subset of circRNAs undergoes cap-independent translation under certain circumstances, even though the vast majority of circRNAs are thought to be noncoding (17, 18). In GC, circPVT1 promotes cell proliferation by acting as a miR-125b sponge (19). As a nuclear down-regulated noncoding RNA, circHuR suppresses HuR expression and GC progression by inactivating CNBP (20). However, the biological functions and underlying mechanisms of circRNAs in GC progression remain largely elusive.Alternative splicing gives rise to diverse messenger RNA (mRNA) isoforms by the different arrangements of exon organization from precursor mRNAs (pre-mRNAs), leading to encoding structurally and functionally distinct protein variants (21, 22). As a gene expression regulation event in eukaryotes, alternative splicing controlled by splicing factors such as hnRNP proteins plays fundamental roles in the progression of human cancers (23–27). For instance, PTBP1 (hnRNP I) mediates alternative splicing of MEIS2 and PKM to promote lymphatic metastasis and proliferation of bladder cancer (26). Another hnRNP protein hnRNPM is known to regulate breast cancer metastasis via modulating alternative splicing of CD44 (27); actually, the only known molecular role of hnRNPM is to modify alternative splicing (28).To investigate the functional roles of circRNAs in GC, we performed RNA sequencing (RNA-seq) of five-paired GC and the corresponding nontumor adjacent specimens (paraGC) to identify promising circRNA candidates. We found that circURI1 expression levels were remarkably increased in GC compared with paraGC. With a series of molecular, cellular, and biochemical experiments, we demonstrated the roles of circURI1 in the prevention of GC metastasis and further illustrated an elegant molecular pathway, in which circURI1 served as a decoy of hnRNPM to modulate alternative splicing of a subset of genes related to cell migration.     

Loss of glucose 6-phosphate dehydrogenase function increases oxidative stress and glutaminolysis in metastasizing melanoma cells

The pentose phosphate pathway is a major source of NADPH for oxidative stress resistance in cancer cells but there is limited insight into its role in metastasis, when some cancer cells experience high levels of oxidative stress. To address this, we mutated the substrate binding site of glucose 6-phosphate dehydrogenase (G6PD), which catalyzes the first step of the pentose phosphate pathway, in patient-derived melanomas. G6PD mutant melanomas had significantly decreased G6PD enzymatic activity and depletion of intermediates in the oxidative pentose phosphate pathway. Reduced G6PD function had little effect on the formation of primary subcutaneous tumors, but when these tumors spontaneously metastasized, the frequency of circulating melanoma cells in the blood and metastatic disease burden were significantly reduced. G6PD mutant melanomas exhibited increased levels of reactive oxygen species, decreased NADPH levels, and depleted glutathione as compared to control melanomas. G6PD mutant melanomas compensated for this increase in oxidative stress by increasing malic enzyme activity and glutamine consumption. This generated a new metabolic vulnerability as G6PD mutant melanomas were more dependent upon glutaminase than control melanomas, both for oxidative stress management and anaplerosis. The oxidative pentose phosphate pathway, malic enzyme, and glutaminolysis thus confer layered protection against oxidative stress during metastasis.

The pentose phosphate pathway is an important source of NADPH for oxidative stress resistance (1–5). The oxidative branch of the pentose phosphate pathway contains two enzymes that generate NADPH from NADP⁺, glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (PGD) (SI Appendix, Fig. S1). NADPH is an important source of reducing equivalents for oxidative stress resistance because it is used by cells to convert oxidized glutathione (GSSG) to glutathione (GSH), an abundant redox buffer. Complete deficiency for G6PD is embryonic-lethal in mice (2, 6, 7) but hypomorphic G6PD mutations are common in certain human populations, perhaps because they protect against malaria (8, 9). These partial loss-of-function G6PD mutations are well tolerated in adults, though they sensitize red blood cells to hemolysis from oxidative stress under certain circumstances (10).Several studies have reported a lower incidence and mortality for certain cancers in people with hypomorphic mutations in G6PD (11–14), suggesting that cancer cells depend upon G6PD to manage oxidative stress. Cells experience high levels of oxidative stress during certain phases of cancer development and progression, including during metastasis (15–17). Antioxidant mechanisms thus promote the survival of cells during oncogenic transformation (18, 19) as well as during metastasis (15, 16). For example, relative to primary cutaneous tumors, metastasizing melanoma cells exhibit increased dependence upon the folate pathway (15), monocarboxylate transporter-1 (MCT1) (20), and glutathione peroxidase-4 (GPX4) (21), each of which directly or indirectly attenuate oxidative stress. By better understanding the mechanisms that confer oxidative stress resistance in cancer cells, it may be possible to develop pro-oxidant therapies that inhibit cancer progression by exacerbating the oxidative stress experienced by cancer cells.G6PD (22) or PGD deficiency (23–25) reduce the growth of some cancers, including melanoma, but G6PD deficiency has little effect on primary tumor formation by K-Ras–driven epithelial cancers (26). This is at least partly because loss of G6PD in these cancers leads to compensatory increases in the function of other NADPH-generating enzymes, including malic enzyme and isocitrate dehydrogenase (1, 27). Nonetheless, pentose phosphate pathway function may increase during metastasis (20, 28–30) and higher G6PD expression is associated with worse outcomes in several cancers (31–33), raising the question of whether metastasizing cells are particularly dependent upon G6PD. G6PD is not essential for metastasis in a breast cancer cell line but it reduces their capacity to form metastatic tumors (26).Melanoma cells show little evidence of oxidative stress in established primary tumors but exhibit increased levels of reactive oxygen species (ROS) and dependence upon antioxidant mechanisms during metastasis (15, 20, 21). To test if these cells are more dependent upon the pentose phosphate pathway during metastasis, we generated three G6PD mutant melanomas, including two patient-derived xenografts and one human melanoma cell line. Reduced G6PD function had little effect on the formation or growth of primary subcutaneous tumors but significantly increased ROS levels and reduced spontaneous metastasis. G6PD mutant melanomas compensated by increasing malic enzyme activity and glutamine consumption, both to increase oxidative stress resistance and to replenish tricarboxylic acid (TCA) cycle intermediates through anaplerosis. Melanoma cells thus have redundant layers of protection against oxidative stress during metastasis, including the abilities to alter fuel consumption and antioxidant pathway utilization.     

Circulating giant macrophages as a potential biomarker of solid tumors

Tumor-associated macrophages (TAMs) derived from primary tumors are believed to facilitate circulating tumor cell (CTC) seeding of distant metastases, but the mechanisms of these processes are poorly understood. Although many studies have focused on the migration of CTCs, less attention has been given to TAMs that, like CTCs, derive from tumor sites. Using precision microfilters under low-flow conditions, we isolated circulating cancer-associated macrophage-like cells (CAMLs) from the peripheral blood of patients with breast, pancreatic, or prostate cancer. CAMLs, which are not found in healthy individuals, were found to express epithelial, monocytic, and endothelial protein markers and were observed bound to CTCs in circulation. These data support the hypothesis that disseminated TAMs can be used as a biomarker of advanced disease and suggest that they have a participatory role in tumor cell migration.Tumor-associated macrophages (TAMs) are specialized differentiated macrophages found within most tumors, which can be used as prognostic indicators of either tumor invasiveness or tumor suppression (1–3). TAMs, recruited to the stroma from circulating monocytes, are required for tumor cell intravasation, migration, extravasation, and angiogenesis (2–7). Tumors attract monocytes via chemoattractants (e.g., MCP-1, CCL-2) (2–4). In turn TAMs secrete cytokines and growth factors (e.g., MMP-1, CXCL12) which stimulate tumor cells with the potential to become circulating tumor cells (CTCs) (2–4). TAMs and CTCs then migrate via the lymphatic system or intravasate across intratumor capillary barriers into peripheral circulation (4–9).Pathological evidence detailing the dissemination of CTCs via a metastatic cascade remains inconclusive. Typically, cancer cell dissemination requires three steps: CTC separation from the tumor, movement away from the parent mass, and migration into the circulatory system (10). Although various theories have explained selected aspects of this dissemination and involved various cell types in this process, including endothelial progenitor cells (EPCs), cancer mesenchymal stem cells, and hybrid cancer cells (10–12), among others, none of these single components explains the entire metastatic process. Recent, in vivo studies have shown that circulating monocytic cells are intricately involved in tumor cell invasiveness, motility, and metastatic potential (1–6). Interactions between myeloid-lineage cells and tumor cells have been documented in patients and modeled in mice, suggesting that the pathway for cancer cell intravasation occurs in conjunction with macrophages via transendothelial migration (4–7).Here we report evidence of the existence of highly differentiated giant circulating (macrophage-like) cells isolated from the peripheral blood of patients with breast, prostate, or pancreatic cancer, which we hypothesize to be disseminated TAMs (DTAMs). Although giant cells resembling these have been observed sporadically in the past, only now have their systematic isolation, identification, and characterization for proper in-depth study become technologically possible (13–15). We isolated this cell type by developing a low-pressure filtration system equipped with precision microfilters, allowing histological identification of cellular morphology (16). We term this giant cell a “circulating cancer-associated macrophage-like cell” (CAML), because it exhibits CD14⁺ expression and vacuoles of phagocytosed material and has been observed exclusively in cancer patients (Fig. 1 and Table S1). We propose that this cell population, which is not detected in healthy individuals, could serve as a robust cellular biomarker of a previously undefined innate immune response to cancer presence and of cancer aggressiveness and could be useful in monitoring chemotherapy-induced responses. Observations of these giant cells interacting with CTCs while in circulation support evidence that a patient’s immune cells have an observable effect on the migration or elimination of CTCs. Furthermore, angiopoietin-1 receptor (TIE-2) positive markers expressed by macrophages (4, 5) suggest that CAMLs have a possible a role as cellular initiators of neovascularization within tumor metastases. We have uncovered supporting in vivo evidence that CAMLs may play an associated role in the migration of CTCs in circulation.Open in a separate windowFig. 1.(Upper) Representative collage of the five morphologies, signal variation, and cytoplasmic diameters. (A, F, and G) Pancreatic cells. (B, C, and D) breast cells. (E, H, and I) Prostate cells. (J) Typical WBCs. Morphology variants are as follows: amorphous (A), oblong (B and G), spindle-shaped (C, F, and I), round (D), and tadpole-shaped (E and H). Color differences result from varying degrees of staining for DAPI (blue), cytokeratins (green), EpCAM (red), and CD45 (violet) (Fig. S1). (Scale bar, 50 μm.) (K, Lower) Whisker plot of cytoplasmic diameters showing diameters of WBCs, CTCs, and CAMLs (n = 75) from pooled prostate, breast, and pancreatic samples.     

miR-182 targeting reprograms tumor-associated macrophages and limits breast cancer progression

The protumor roles of alternatively activated (M2) tumor-associated macrophages (TAMs) have been well established, and macrophage reprogramming is an important therapeutic goal. However, the mechanisms of TAM polarization remain incompletely understood, and effective strategies for macrophage targeting are lacking. Here, we show that miR-182 in macrophages mediates tumor-induced M2 polarization and can be targeted for therapeutic macrophage reprogramming. Constitutive miR-182 knockout in host mice and conditional knockout in macrophages impair M2-like TAMs and breast tumor development. Targeted depletion of macrophages in mice blocks the effect of miR-182 deficiency in tumor progression while reconstitution of miR-182-expressing macrophages promotes tumor growth. Mechanistically, cancer cells induce miR-182 expression in macrophages by TGFβ signaling, and miR-182 directly suppresses TLR4, leading to NFκb inactivation and M2 polarization of TAMs. Importantly, therapeutic delivery of antagomiR-182 with cationized mannan-modified extracellular vesicles effectively targets macrophages, leading to miR-182 inhibition, macrophage reprogramming, and tumor suppression in multiple breast cancer models of mice. Overall, our findings reveal a crucial TGFβ/miR-182/TLR4 axis for TAM polarization and provide rationale for RNA-based therapeutics of TAM targeting in cancer.

It is well known that the nonmalignant stromal components in tumors play pivotal roles in tumor progression and therapeutic responses (1, 2). Macrophages are a major component of tumor microenvironment and display considerable phenotypic plasticity in their effects toward tumor progression (3–5). Classically activated (M1) macrophages often exert direct tumor cytotoxic effects or induce antitumor immune responses by helping present tumor-related antigens (6, 7). In contrast, tumoral cues can polarize macrophages toward alternative activation with immunosuppressive M2 properties (6–8). Numerous studies have firmly established the protumor effects of M2-like tumor-associated macrophages (TAMs) and the association of TAMs with poor prognosis of human cancer (9–11). However, how tumors induce the coordinated molecular and phenotypic changes in TAMs for M2 polarization remains incompletely understood, impeding the designing of TAM-targeting strategies for cancer intervention. In addition, drug delivery also represents a hurdle for therapeutic macrophage reprogramming.Noncoding RNAs, including microRNAs, have been shown to play vital roles in various pathological processes of cancer (12). The microRNA miR-182 has been implicated in various developmental processes and disease conditions (13–15). Particularly, it receives extensive attention in cancer studies. Prevalent chromosomal amplification of miR-182 locus and up-regulation of its expression in tumors have been observed in numerous cancer types including breast cancer, gastric cancer, lung adenocarcinoma, colorectal adenocarcinoma, ovarian carcinoma, and melanoma (16–21). miR-182 expression is also linked to higher risk of metastasis and shorter survival of patients (20, 22–24). Functional studies showed that miR-182 expression in cancer cells plays vital roles in various aspects of cancer malignancy, including tumor proliferation (25–29), migration (30, 31), invasion (16, 32, 33), epithelial-mesenchymal transition (34–36), metastasis (21, 37, 38), stemness (30, 39, 40), and therapy resistance (41, 42). A number of target genes, including FOXO1/3 (18, 21, 43–45), CYLD (46), CADM1 (47), BRCA1 (27, 48), MTSS1 (34), PDK4 (49), and SMAD7 (35), were reported to be suppressed by miR-182 in cancer cells. Our previous work also proved that tumoral miR-182 regulates lipogenesis in lung adenocarcinoma and promotes metastasis of breast cancer (34, 35, 49). Although miR-182 was established as an important regulator of cancer cell malignancy, previous studies were limited, with analyses of miR-182 in cultured cancer cells and transplanted tumors. Thus, the consequences of miR-182 regulation in physiologically relevant tumor models, such as genetically modified mice, have not been shown. More importantly, whether miR-182 also plays a role in tumor microenvironmental cell components is unknown.In this study, we show that miR-182 expression in macrophages can be induced by breast cancer cells and regulates TAM polarization in various tumor models of mice. In addition, miR-182 inhibition with TAM-targeting exosomes demonstrates promising efficacy for cancer treatment.     

Polyclonal breast cancer metastases arise from collective dissemination of keratin 14-expressing tumor cell clusters

Recent genomic studies challenge the conventional model that each metastasis must arise from a single tumor cell and instead reveal that metastases can be composed of multiple genetically distinct clones. These intriguing observations raise the question: How do polyclonal metastases emerge from the primary tumor? In this study, we used multicolor lineage tracing to demonstrate that polyclonal seeding by cell clusters is a frequent mechanism in a common mouse model of breast cancer, accounting for >90% of metastases. We directly observed multicolored tumor cell clusters across major stages of metastasis, including collective invasion, local dissemination, intravascular emboli, circulating tumor cell clusters, and micrometastases. Experimentally aggregating tumor cells into clusters induced a >15-fold increase in colony formation ex vivo and a >100-fold increase in metastasis formation in vivo. Intriguingly, locally disseminated clusters, circulating tumor cell clusters, and lung micrometastases frequently expressed the epithelial cytoskeletal protein, keratin 14 (K14). RNA-seq analysis revealed that K14⁺ cells were enriched for desmosome and hemidesmosome adhesion complex genes, and were depleted for MHC class II genes. Depletion of K14 expression abrogated distant metastases and disrupted expression of multiple metastasis effectors, including Tenascin C (Tnc), Jagged1 (Jag1), and Epiregulin (Ereg). Taken together, our findings reveal K14 as a key regulator of metastasis and establish the concept that K14⁺ epithelial tumor cell clusters disseminate collectively to colonize distant organs.During metastasis, cancer cells escape the primary tumor, travel through the circulation, and colonize distant organs. Conventional models of cancer progression propose that each metastasis arises from the clonal outgrowth of a single tumor cell and this conceptual framework is a foundation for models, such as epithelial-mesenchymal transition (EMT) and migratory cancer stem cells (1).Challenging the generality of the single-cell/single-metastasis model are long-standing clinical observations that tumor cell clusters (also termed “tumor clumps”) are also observed across the stages of metastasis. Tumor cell clusters are detected in the bloodstream of cancer patients (2), clusters can efficiently seed metastases (3), and though rare, circulating tumor cell (CTC) clusters have prognostic significance (4, 5). Furthermore, metastases are composed of multiple genetically distinct tumor cell clones, in mouse models of breast, pancreas, and small cell carcinoma (5–7), and in human metastatic prostate cancer patients (8). Taken together, these observations provide accumulating evidence that tumor cell clusters contribute to metastasis. However, they leave unresolved two important questions: how do tumor cell clusters emerge from the primary tumor, and which molecular features identify cell clusters that metastasize?An important clinical observation is that cancer cells invade the surrounding stroma as cohesive clusters in the majority of epithelial tumors, a process termed “collective invasion” (9, 10). In breast cancer, collective invasion is facilitated by invasive leader cells, a subpopulation of tumor cells that highly express keratin 14 (K14) and other basal epithelial markers (11). K14⁺ cells are migratory, protrusive, and lead trailing K14⁻ cells, while maintaining cell–cell cohesion and E-cadherin–based cell contacts.In this study, we sought to understand how these K14⁺ cells exit collective invasion strands in the primary tumor and travel to distant organs (12). One hypothesis is that collective invasion is an intermediate step toward eventual single-cell dissemination and monoclonal metastasis. However, tumor cell clusters are detected in circulation (5) and primary human breast tumors can disseminate collectively into the surrounding extracellular matrix in ex vivo assays (13–15). These data prompted an alternative hypothesis, that collectively invading K14⁺ cancer cells could initiate and complete the metastatic process as a cohesive multicellular unit. Here we define the clonal nature of metastases in a spontaneous mouse model of metastasis to the lungs (16, 17), in which the predominant invasive form in the primary tumor is collective invasion strands led by K14⁺ cells (11). We establish that the majority of metastases arise from polyclonal seeds, and show that disseminated tumor cell clusters are predominantly composed of K14⁺ cells. We propose a mechanism for polyclonal metastasis via the collective invasion, dissemination, and colonization of clusters of K14⁺ cancer cells.     

Mechanism-based heparanase inhibitors reduce cancer metastasis in vivo

Heparan sulfate proteoglycans (HSPGs) mediate essential interactions throughout the extracellular matrix (ECM), providing signals that regulate cellular growth and development. Altered HSPG composition during tumorigenesis strongly aids cancer progression. Heparanase (HPSE) is the principal enzyme responsible for extracellular heparan sulfate catabolism and is markedly up-regulated in aggressive cancers. HPSE overactivity degrades HSPGs within the ECM, facilitating metastatic dissemination and releasing mitogens that drive cellular proliferation. Reducing extracellular HPSE activity reduces cancer growth, but few effective inhibitors are known, and none are clinically approved. Inspired by the natural glycosidase inhibitor cyclophellitol, we developed nanomolar mechanism-based, irreversible HPSE inhibitors that are effective within physiological environments. Application of cyclophellitol-derived HPSE inhibitors reduces cancer aggression in cellulo and significantly ameliorates murine metastasis. Mechanism-based irreversible HPSE inhibition is an unexplored anticancer strategy. We demonstrate the feasibility of such compounds to control pathological HPSE-driven malignancies.

Cancer progression is accompanied by extensive changes to the tumor microenvironment, which support the proliferation and dissemination of malignant cells. Extracellular matrix (ECM) remodeling by cancer and associated stromal cells utilizes a host of enzymes that act upon both proteins and carbohydrates within the ECM, causing profound compositional changes that can drive growth of the primary tumor, as well as prime distant sites for metastasis (1).Heparan sulfate proteoglycans (HSPGs) are a fundamental class of ECM constituent, comprised of pericellular and extracellular core proteins conjugated to one or more chains of the glycosaminoglycan polysaccharide heparan sulfate (HS) (2). HSPGs mediate myriad biological processes, including signaling (3), developmental patterning (4), adhesion (5), barrier formation (6, 7), endocytosis (8), and viral entry (9–11). These processes largely depend upon the HS polysaccharides adorning the core protein (12), whose heterogeneous structures allow for interaction with multiple diverse partners.Given the importance of HSPGs within the ECM, tightly coordinated biosynthesis and breakdown mechanisms act to regulate their composition (13, 14). HSPG breakdown is mediated (in part) by endo- and exo-glycosidases, which hydrolytically cleave within or at the termini of HS polysaccharide chains, respectively (14). Heparanase (HPSE) is a mammalian endo-β-D-glucuronidase (hereafter, endo-β-glucuronidase) that plays a key role in HS polysaccharide degradation (15). Initially produced as a proenzyme (proHPSE), HPSE is matured within lysosomes by proteolysis of a linker peptide that obstructs its active site. Mature HPSE remains active within lysosomes, but can also be secreted into the extracellular space, where it aids ECM remodeling during tissue development and homeostasis. In contrast to its physiological role in ECM maintenance, pathological HPSE overexpression strongly drives the growth of aggressive metastatic cancers (16): excessive HSPG degradation within the ECM directly facilitates cancer cell migration to and from the vasculature (17, 18), while growth factors and cytokines liberated upon HSPG degradation stimulate proliferation and angiogenesis (19, 20) (Fig. 1A). HPSE overexpression can also increase the formation of tumor-derived exosomes, which circulate to distal tissues to establish premetastatic niche environments primed for colonization (21–23).Open in a separate windowFig. 1.Design and development of HS-configured cyclophellitol pseudodisaccharides. (A) Biological effects of HPSE overexpression in the extracellular space. Excessive degradation of HSPG networks in basement membranes facilitates cell migration to and from the vasculature. Release of HSPG sequestered growth factors stimulates cell proliferation. (B) Preferred HPSE target sequence, comprising a GlcA residue flanked by two sulfated glucosamine residues. (C) Inhibitors and probes used in this study; atom reference positions are shown on inhibitor 2. Full structures of 7–9, including Cy5 linker, are shown in the SI Appendix. (Bottom) Principal of pseudodisaccharide HPSE selectivity via steric occlusion of exo-β-glucuronidase binding.HPSE is the sole human enzyme responsible for extracellular HS polysaccharide degradation. Accordingly, there is intense interest in its inhibition as an anticancer strategy. A plethora of small-molecule (24–27), saccharide (28–31), neoproteoglycan (32, 33), and antibody (34) based HPSE inhibitors have been reported (35), although, to date, only four compounds have progressed to clinical trials (SI Appendix, Fig. S1) (28–31). Notably, these current “best-in-class” inhibitors are all polyanionic oligo-/polysaccharide derivatives that mimic the physicochemical properties of the natural HPSE inhibitor heparin, a glycosaminoglycan polysaccharide closely related to HS. The heparin-like properties of these best-in-class inhibitors, along with their structural heterogeneity, can produce unwanted pleiotropic effects, such as anticoagulation and growth factor binding, which complicate their clinical use (36).Comparatively few small-molecule HPSE inhibitors with in vivo efficacy have been described (35). The active site of HPSE has proven challenging for small-molecule pharmacological intervention, given its extensive interaction surface evolved to bind large HS polysaccharides. Such challenging sites are often well targeted by mechanism-based covalent inhibitors, which may still react with an enzyme despite weak or transient initial binding. We previously reported that derivatives of the naturally occurring β-glucosidase inhibitor cyclophellitol yield highly active mechanism-based retaining glycosidase inhibitors and probes (37–40). Enzymatic attack by glycosidases upon the electrophilic epoxide “warhead” of cyclophellitol-derived molecules leads to alkylation of a catalytically essential nucleophile residue, resulting in covalent and irreversible inhibition (SI Appendix, Fig. S2). Tuning the functionality and stereochemistry of cyclophellitol-derived scaffolds alters target selectivity, in principle allowing inhibition of any retaining glycosidase enzyme (41).Herein, we present HS-configured pseudodisaccharides, featuring a cyclitol epoxide designed to bind selectively to the HPSE active site and react with its essential catalytic nucleophile residue. We show that these HS-configured pseudodisaccharides are nanomolar, irreversible HPSE inhibitors in vitro and markedly reduce cancer aggression in in cellulo and in vivo cancer models. Covalent irreversible inhibition remains an underexploited avenue for therapeutic exploitation. Our results demonstrate that compounds acting by this mechanism can deliver effective treatments for addressing HPSE-driven cancer aggression.     

Combination of tucatinib and neural stem cells secreting anti-HER2 antibody prolongs survival of mice with metastatic brain cancer

Brain metastases are a leading cause of death in patients with breast cancer. The lack of clinical trials and the presence of the blood–brain barrier limit therapeutic options. Furthermore, overexpression of the human epidermal growth factor receptor 2 (HER2) increases the incidence of breast cancer brain metastases (BCBM). HER2-targeting agents, such as the monoclonal antibodies trastuzumab and pertuzumab, improved outcomes in patients with breast cancer and extracranial metastases. However, continued BCBM progression in breast cancer patients highlighted the need for novel and effective targeted therapies against intracranial metastases. In this study, we engineered the highly migratory and brain tumor tropic human neural stem cells (NSCs) LM008 to continuously secrete high amounts of functional, stable, full-length antibodies against HER2 (anti-HER2Ab) without compromising the stemness of LM008 cells. The secreted anti-HER2Ab impaired tumor cell proliferation in vitro in HER2+ BCBM cells by inhibiting the PI3K-Akt signaling pathway and resulted in a significant benefit when injected in intracranial xenograft models. In addition, dual HER2 blockade using anti-HER2Ab LM008 NSCs and the tyrosine kinase inhibitor tucatinib significantly improved the survival of mice in a clinically relevant model of multiple HER2+ BCBM. These findings provide compelling evidence for the use of HER2Ab-secreting LM008 NSCs in combination with tucatinib as a promising therapeutic regimen for patients with HER2+ BCBM.

Breast cancer metastasis is one of the leading causes of cancer-related deaths among women worldwide (1). This is especially true in the context of the brain, where the presence of the blood–brain barrier (BBB) significantly decreases the efficacy of the existing systemic therapies (2). The burden of brain metastatic breast cancer is further compounded by the fact that the current standard treatment is palliative and primarily local, whereby surgical resection, stereotactic radiosurgery, and/or whole-brain radiation therapy achieve limited survival benefits (3). In addition, some intracranial lesions, such as diffused multiple micrometastases or metastases close to the eloquent areas in the brain, are not suitable for surgical resection (4).The overexpression of the human epidermal growth factor receptor 2 (HER2), a tyrosine kinase receptor, is observed in about 30% of patients with breast cancer and is known to be associated with advanced disease and decreased overall survival (5). In addition, up to 50% of patients with HER2+ overexpressing breast cancer will develop central nervous system (CNS) metastases, resulting in a median survival of 11 to 18 mo after diagnosis (6–9). Trastuzumab (Herceptin), a humanized monoclonal antibody (mAb) targeting HER2, was the first clinically approved targeted therapy for the treatment of HER2+ overexpressing breast cancer and is now used routinely as the first-line therapy (10, 11). The antitumor mechanisms of trastuzumab therapy are complex, which include antibody-dependent cell-mediated cytotoxicity, inhibition of cleavage of the extracellular domain of HER2, inhibition of ligand-independent HER2 receptor dimerization, impaired activation of HER2 downstream pathways, induction of cell cycle arrest and apoptosis, inhibition of angiogenesis, and interference with DNA repair (12–14). Pertuzumab is another mAb that binds to the HER2 dimerization domain, inhibiting its heterodimerization with other HER family receptors (15, 16). In combination with trastuzumab, pertuzumab further improves invasive disease-free survival among patients with HER2+ breast cancer (17). More recently, the combination of both agents was reported to improve the response rate in patients with HER2+ metastatic breast cancer and progressive CNS metastases (18). However, the large molecular sizes of trastuzumab or pertuzumab and their weak permeability through the BBB require high dosages that can lead to toxicity. A clinical trial that evaluated the effect of tucatinib in combination with trastuzumab and capecitabine in patients with HER2+ metastatic breast cancer (HER2CLIMB) showed promising results in patients with brain metastases (19). Tucatinib (Tukysa) is a Food and Drug Administration (FDA)-approved oral tyrosine kinase inhibitor (TKI) that is highly selective for the kinase domain of HER2 with minimal inhibition of the epidermal growth factor receptor, limited low-grade toxicity, strong ability to cross the BBB, and notable antitumor activity in heavily pretreated HER2+ metastatic breast cancer patients (19–22). The risk of disease progression or death was decreased by 52% in patients with HER2+ breast cancer brain metastasis (BCBM) receiving the tucatinib combination treatment compared to those patients in the placebo combination group (23). Nevertheless, the limited efficacy of tucatinib as a monotherapy for HER2+ BCBM (24) underscores the need for innovative therapeutic regimens and delivery platforms that can improve clinical outcomes in patients with brain metastases.Human neural stem cell (NSC)-based therapies have emerged in the last few years as promising strategies for the treatment of CNS malignancies. Proof-of-concept preclinical and clinical studies have demonstrated the efficacy and feasibility of these NSCs for targeted delivery of therapeutic agents and oncolytic viruses (25–28). Our group has previously demonstrated the ability of the v-MYC immortalized HB1.F3 NSC line to deliver functional anti-HER2 antibodies (anti-HER2Ab) when injected directly in the CNS, improving significantly the survival of mice bearing breast cancer cells in the brain (4). The present study utilized the L-MYC–immortalized human NSC line LM-NSC008 (LM008), previously described as nontumorigenic in vivo and with tumor cell tropism and high migratory properties (29). Transduction of NSCs with L-MYC reduces the risk of oncogenic transformation, enhances their in vivo engraftment and migration capabilities, and results in a complete absence of tumorigenicity for up to 9 mo when injected in mouse brains (30, 31). After modifying the LM008 cells to secrete stable and high amounts of anti-HER2Ab (LM008–HER2Ab cells), we analyzed their efficacy when delivered locally in the brain and systemically in a model of multiple HER2+ BCBM. Our results demonstrate a significant survival benefit in mice injected with LM008–HER2Ab cells that was further improved when these mAb-secreting NSCs were used in combination with tucatinib. Thus, this study provides compelling evidence for the use of LM008–HER2Ab NSCs in combination with tucatinib for the treatment of HER2+ overexpressing BCBM.     

VEGF-B promotes cancer metastasis through a VEGF-A–independent mechanism and serves as a marker of poor prognosis for cancer patients

The biological functions of VEGF-B in cancer progression remain poorly understood. Here, we report that VEGF-B promotes cancer metastasis through the remodeling of tumor microvasculature. Knockdown of VEGF-B in tumors resulted in increased perivascular cell coverage and impaired pulmonary metastasis of human melanomas. In contrast, the gain of VEGF-B function in tumors led to pseudonormalized tumor vasculatures that were highly leaky and poorly perfused. Tumors expressing high levels of VEGF-B were more metastatic, although primary tumor growth was largely impaired. Similarly, VEGF-B in a VEGF-A–null tumor resulted in attenuated primary tumor growth but substantial pulmonary metastases. VEGF-B also led to highly metastatic phenotypes in Vegfr1 tk^−/− mice and mice treated with anti–VEGF-A. These data indicate that VEGF-B promotes cancer metastasis through a VEGF-A–independent mechanism. High expression levels of VEGF-B in two large-cohort studies of human patients with lung squamous cell carcinoma and melanoma correlated with poor survival. Taken together, our findings demonstrate that VEGF-B is a vascular remodeling factor promoting cancer metastasis and that targeting VEGF-B may be an important therapeutic approach for cancer metastasis.Although genetic alterations of malignant cells govern the intrinsic features of invasiveness, host-derived cellular and molecular components may play predominant roles in cancer invasion and metastasis (1). For example, the tumor vasculature is essential for tumor growth and metastasis (2), and blocking tumor angiogenesis has been used successfully for treatment of animal and human cancers (3–5). Similarly, targeting other nonvascular host components, including inflammatory cells and stromal cells, also provides effective therapeutic options for treatment of cancer (6). Hematogenous metastasis is a complex process that involves intimate interactions between malignant cells and various host cells. At the primary tumor site, tumor cells must intravasate through the vessel wall into the circulation, and intravasation requires cooperative and coordinated interactions between tumor cells, perivascular cells such as pericytes, endothelial cells, and possibly inflammatory cells (7–9). In addition to their physical interactions, tumor cells and host cells produce various signaling molecules that modulate cell morphology, migration, proliferation, production of proteases, and adhesion molecules. Consequently, the vascular endothelium within and surrounding primary tumors undergoes structural changes that permit tumor cell invasion. After arriving at distal organs, tumor cells need to extravasate from the circulation. Again, circulating tumor cells interact closely with endothelial cells and perivascular cells to manipulate vascular structures for extravasation (10). The subsequent formation of metastatic niches and regrowth of metastatic nodules to clinically detectable masses are dependent on angiogenesis and vascular remodeling.Tumors often express angiogenic factors at high levels to induce neovascularization (11). Multiple growth factors/cytokines and their signaling receptors often coexist in the same tumor microenvironment and collectively modulate tumor growth, invasiveness and metastasis (12). Among all known angiogenic factors, vascular endothelial growth factor A (VEGF-A), which modulates angiogenesis, vascular permeability, vessel survival, and vascular remodeling, is probably the best characterized (13, 14). Although VEGF-A binds to VEGF receptor 1 (VEGFR1) and VEGFR2, two tyrosine kinase receptors, it is believed that VEGFR2 mediates most of these VEGF-A–triggered vascular functions (15). Unlike VEGF-A, VEGF-B binds only to VEGFR1, which also is considered a decoy receptor that transduces negative signals for angiogenesis (16, 17). Despite its early discovery, the biological functions, and especially the vascular functions, of VEGF-B remain an enigma (18, 19). Initially, VEGF-B was shown to stimulate endothelial cell activity and angiogenesis (18). However, later studies do not support these claims, and opposing results that VEGF-B inhibits tumor angiogenesis have been reported (20, 21). The roles of VEGF-B in tumor invasion and metastasis have not been studied.In this work, we report, for the first time to our knowledge, the crucial role of VEGF-B in modulating the vascular remodeling that facilitates tumor metastasis in human and mouse tumor models. Surprisingly, VEGF-B expression is reversely correlated with primary tumor growth, demonstrating that it negatively regulates tumor angiogenesis. Despite retarded growth rates of primary tumors, VEGF-B markedly promotes metastasis. Thus, primary tumor growth and metastasis are separate events, and the latter process is dependent on vascular alterations that become permissive for tumor invasion. Our present work provides compelling experimental evidence that VEGF-B is a metastatic factor and that targeting VEGF-B may be an important approach for the treatment of cancer invasion and metastasis.     

Human 3D vascularized organotypic microfluidic assays to study breast cancer cell extravasation

A key aspect of cancer metastases is the tendency for specific cancer cells to home to defined subsets of secondary organs. Despite these known tendencies, the underlying mechanisms remain poorly understood. Here we develop a microfluidic 3D in vitro model to analyze organ-specific human breast cancer cell extravasation into bone- and muscle-mimicking microenvironments through a microvascular network concentrically wrapped with mural cells. Extravasation rates and microvasculature permeabilities were significantly different in the bone-mimicking microenvironment compared with unconditioned or myoblast containing matrices. Blocking breast cancer cell A₃ adenosine receptors resulted in higher extravasation rates of cancer cells into the myoblast-containing matrices compared with untreated cells, suggesting a role for adenosine in reducing extravasation. These results demonstrate the efficacy of our model as a drug screening platform and a promising tool to investigate specific molecular pathways involved in cancer biology, with potential applications to personalized medicine.Dissemination of cancer cells from a primary tumor to secondary loci is responsible for more than 90% of cancer-related mortality (1). Despite significant advances in diagnostics and therapy, most of the available treatments are not effective, because the disseminated cells are resistant to conventional agents (2). Invasion and metastasization are complex and multistep processes guided by a wide spectrum of genetic and biochemical determinants (3). A key aspect of metastases is reflected in the interactions between specific cancer cell types and different secondary organs. Although circulatory patterns and flow rates may play some role in cancer cell dissemination, it appears that the organ specificity of metastases is primary due to the cross-talk between specific cancer cells and biologically unique tissues: the seed-and-soil paradigm (4). For example, breast and prostate cancers are known to metastasize frequently to bone (5), with 70% of advanced breast cancer patients affected by skeletal metastases, leading to high rates of morbidity and mortality (6). Moreover, it has been recently demonstrated that breast cancer cells can reseed from bone to other sites including the breast, further emphasizing the key role of the bone microenvironment in the metastatic process (7).Metastasis organ specificity and extravasation appear to be tightly coupled because specific chemo-attractant molecules are secreted by organ-specific stromal cells (8). Furthermore, positive interactions with circulating noncancer cells, e.g., platelets, leukocytes, and monocytes/macrophages, promote cancer cell transendothelial migration into surrounding tissues (9).In vivo and ex vivo studies have been performed to investigate cancer cell extravasation in mouse models through liver sinusoids and pulmonary circulation (10) or in zebrafish embryos (11). Recently, Schumacher et al. have shown the influence of platelet-secreted nucleotides playing a crucial role in the transendothelial migration of cancer cells in the lung of mouse models (12). In vivo models have been developed to study breast cancer metastases to bone by means of i.v. and skeletal injection of breast cancer cells in mice (13). Although in vivo models play an essential role in replicating physiological conditions, they lack the possibility to analyze highly specific interactions between human cancer cells, human blood vessels, and tissues, and they are not well suited to perform reproducible parametric studies. To remedy this, several in vitro models have been developed to analyze cell migration mechanisms and particularly the invasive potential of cancer cells (14). However, these models are often highly simplified, such as the Boyden chamber or wound assays (15), which fail to allow the analysis of complex cell–cell and cell–matrix interactions, are limited in their capability to tightly control the local microenvironment, and offer only limited imaging.Microfluidics overcomes some of the technical limitations of traditional assays (16), allowing the study of cancer metastases under biochemically and biophysically controlled 3D microenvironments coupled with high-resolution real-time imaging (17). Various microfluidic models have been developed for studying tumor angiogenesis (18), transition to invasion (19), intravasation (20), the role of interstitial flow (21) and matrix stiffness (22) on cancer cell migration, adhesion (23), and extravasation (24, 25). Recently, our group presented a microfluidic model to investigate the specificity of breast cancer metastasis to bone, providing quantitative data on cancer cell extravasation rate and reproducing the effects of the CXCL5–CXCR2 interaction between bone cells and metastatic breast cancer cells observed in vivo (26). However, in that system, the vascular wall was represented by an endothelial monolayer on the side of a central gel region. With the recent attempts in inducing vasculogenesis (27, 28), vascular networks have been generated inside the gel region either by coculture with human lung fibroblasts in separate gel regions or by interstitial flow. Despite the tremendous advances in modeling angiogenesis and vasculogenesis, these models have not previously been used to study metastasis organ specificity and investigate the role of human organ-specific microenvironments.Here we present an organ-specific human 3D microfluidic model that enables the study of human metastatic breast cancer cell extravasation within a perfusable human microvascularized bone-mimicking (BMi) microenvironment. The resulting model represents a functional human quad-culture in which breast cancer cells flow into, adhere to, and metastasize through human microvascular networks. These networks are supported by primary human bone marrow-derived mesenchymal stem cells (hBM-MSCs) that have undergone phenotypical transition toward the smooth muscle cell lineage, embedded in a BMi microenvironment with homogeneously distributed osteo-differentiated (OD) primary hBM-MSCs.     

Intravital imaging reveals distinct responses of depleting dynamic tumor-associated macrophage and dendritic cell subpopulations

Tumor-infiltrating inflammatory cells comprise a major part of the stromal microenvironment and support cancer progression by multiple mechanisms. High numbers of tumor myeloid cells correlate with poor prognosis in breast cancer and are coupled with the angiogenic switch and malignant progression. However, the specific roles and regulation of heterogeneous tumor myeloid populations are incompletely understood. CSF-1 is a major myeloid cell mitogen, and signaling through its receptor CSF-1R is also linked to poor outcomes. To characterize myeloid cell function in tumors, we combined confocal intravital microscopy with depletion of CSF-1R–dependent cells using a neutralizing CSF-1R antibody in the mouse mammary tumor virus long-terminal region-driven polyoma middle T antigen breast cancer model. The depleted cells shared markers of tumor-associated macrophages and dendritic cells (M-DCs), matching the phenotype of tumor dendritic cells that take up antigens and interact with T cells. We defined functional subgroups within the M-DC population by imaging endocytic and matrix metalloproteinase activity. Anti–CSF-1R treatment altered stromal dynamics and impaired both survival of M-DCs and accumulation of new M-DCs, but did not deplete Gr-1⁺ neutrophils or block doxorubicin-induced myeloid cell recruitment, and had a minimal effect on lung myeloid cells. Nevertheless, prolonged treatment led to delayed tumor growth, reduced vascularity, and decreased lung metastasis. Because the myeloid infiltrate in metastatic lungs differed significantly from that in mammary tumors, the reduction in metastasis may result from the impact on primary tumors. The combination of functional analysis by intravital imaging with cellular characterization has refined our understanding of the effects of experimental targeted therapies on the tumor microenvironment.Cells of the myeloid lineage, including macrophages, monocytes, neutrophils, mast cells, and immature myeloid cells, are major components of the complex stromal microenvironment of solid tumors (1, 2). Abundant evidence from human and experimental tumor types shows that myeloid cells support tumor growth and progression by a wide range of mechanisms, including stimulation of angiogenesis, secretion of factors inducing tumor growth, survival and cell migration, remodeling of the extracellular matrix to facilitate growth and invasion, recruitment of additional support cells, and suppression of the antitumor immune response (3–5). In human breast cancer, as in most other solid tumors, large numbers of myeloid cells, characterized as tumor-associated macrophages (TAMs), correlate with poor prognosis, as does high expression of the myeloid cell mitogen colony-stimulating factor-1 (CSF-1, M-CSF) or its receptor CSF-1R (CD115, c-fms), and gene-expression profiles reflecting myeloid involvement or CSF-1 signaling (3, 5–8).The marked effects of myeloid cells on tumor growth, invasion, and metastasis identify them and the signaling pathways mediating cancer cell–myeloid cell interactions as potentially attractive targets for treatment of aggressive cancer. TAM depletion by clodronate-encapsulated liposomes reduces tumor growth in various grafted tumors (3, 5, 9). Genetic ablation, antisense and antibody approaches for inhibition of CSF-1/CSF-1R signaling lead to macrophage depletion and reduce malignancy in several models, with abrogated tumor angiogenesis in most cases (3, 5, 9, 10, and references therein). A null mutation in CSF-1, which leads to the lifelong absence of the majority of macrophages, results in delayed tumor progression in the transgenic mouse mammary tumor virus long terminal region-driven polyoma middle T antigen (MMTV-PyMT) model of aggressive breast cancer, evidenced by more differentiated histology and decreased lung metastasis, apparently because of decreased tumor angiogenesis (11–13).Our understanding of the properties and functions of myeloid cell types in primary and metastatic tumors is still incomplete, because of the broad specificity and varying use of cellular markers, as well as myeloid cell plasticity and contrasting results obtained from a wide variety of tumor models (4, 14–16). We and others have used intravital microscopy to define real-time dynamics of several distinct subpopulations of stromal cells in different stages of mammary tumor progression and tumor response to chemotherapy, or relapse thereafter (17–20). In this study, we sought to define the characteristics and importance of CSF-1–dependent myeloid cells in the MMTV-PyMT breast cancer model by using the neutralizing monoclonal CSF-1R antibody M279 (21) to deplete this population at different stages of tumor progression. We then analyzed the tumor response, phenotype of the depleted cells, and cell dynamics in the tumor microenvironment.     

MicroRNA-224 promotes tumor progression in nonsmall cell lung cancer

Lung cancer is the leading cause of cancer-related deaths worldwide. Despite advancements and improvements in surgical and medical treatments, the survival rate of lung cancer patients remains frustratingly poor. Local control for early-stage nonsmall cell lung cancer (NSCLC) has dramatically improved over the last decades for both operable and inoperable patients. However, the molecular mechanisms of NSCLC invasion leading to regional and distant disease spread remain poorly understood. Here, we identify microRNA-224 (miR-224) to be significantly up-regulated in NSCLC tissues, particularly in resected NSCLC metastasis. Increased miR-224 expression promotes cell migration, invasion, and proliferation by directly targeting the tumor suppressors TNFα-induced protein 1 (TNFAIP1) and SMAD4. In concordance with in vitro studies, mouse xenograft studies validated that miR-224 functions as a potent oncogenic miRNA in NSCLC in vivo. Moreover, we found promoter hypomethylation and activated ERK signaling to be involved in the regulation of miR-224 expression in NSCLC. Up-regulated miR-224, thus, facilitates tumor progression by shifting the equilibrium of the partially antagonist functions of SMAD4 and TNFAIP1 toward enhanced invasion and growth in NSCLC. Our findings indicate that targeting miR-224 could be effective in the treatment of certain lung cancer patients.Lung cancer is the second most common cancer and the leading cause of cancer-related death worldwide. In 2013, there were an estimated 228,190 new cases of lung cancer and 159,480 deaths in the United States. Despite advancements and improvements in surgical and medical treatments, the 5-y survival rate of lung cancer patients remains frustratingly poor (1). Although local control for early-stage nonsmall cell lung cancer (NSCLC) has dramatically improved over the last decades for both operable and inoperable patients (2, 3), ∼20% of early-stage patients, however, are developing distant metastasis (4, 5), and 10–15% of patients undergoing stereotactic ablative body radiation fail regionally (6). The molecular mechanisms of NSCLC invasion leading to regional and distant disease spread remain poorly understood. Understanding the molecular mechanisms that regulate invasion and disease spread would help to identify promising therapeutic targets and could be exploited to refine patient selection for already existing therapies.MicroRNAs (miRNAs) are small endogenous noncoding RNAs that negatively regulate mRNA stability and/or repress mRNA translation (7). miRNAs have been proven to play essential roles in the initiation and progression of certain cancer types, such as chronic lymphocytic leukemia (8), breast cancer (9), and lung cancer (10, 11). Several miRNA expression profiling studies have shown that miRNAs could be used as diagnostic and prognostic biomarkers. For example, high expression levels of miR-155 and low levels of let-7a expression correlate with poor prognosis of lung cancer (10). In colorectal cancer (CRC), up-regulated miR-135b correlates with tumor stage and poor clinical outcome (12). Recently, we conducted genome-wide miRNA sequencing in primary lung cancer tissue from patients with lung adenocarcinoma (ADC), and we identified that miR-31 promotes lymph node metastasis and negatively correlates with survival in patients with lung ADC (13), emphasizing the impact of miRNAs in NSCLC biology.TNFα-induced protein 1 (TNFAIP1) was originally identified as a TNFα- and LPS-inducible gene (14). It has been reported that TNFAIP1 interacts with the proliferating cell nuclear antigen and the small subunit of DNA polymerase-δ (P50) (15), suggesting that TNFAIP1 might be involved in DNA synthesis and apoptosis. Indeed, TNFAIP1 elicited proapoptotic activity, and coexpression of TNFAIP1 and RhoB markedly increased apoptosis in HeLa cells (16). SMAD4 plays a central role in the TGF-β family signaling pathways and is the only member of the SMAD family that is involved in TGF-β, activing, and bone morphogenetic protein signaling pathways (17, 18). SMAD4 functions as a tumor suppressor; loss of SMAD4 was frequently seen in pancreatic cancers and CRCs. Approximately 55% of pancreatic cancers have deletions or mutations in the SMAD4 locus (19), and about 30% of biallelic loss of SMAD4 was found in metastatic CRCs (20). To date, several studies have reported that TNFAIP1 and SMAD4 are targets of miRNAs in certain cancer types. For instance, oncogenic miRNAs, such as the miR-130a/301a/454 family, target SMAD4 in CRC, and miR-182 targets SMAD4 in bladder cancer (21, 22). In gastric cancer, miR-372/373 targets TNFAIP1, promoting carcinogenesis (23, 24).Here, we show that increased miR-224 in NSCLC promotes cell migration, invasion, and proliferation by direct targeting of TNFAIP1 and SMAD4. We further show that aberrant miR-224 expression is partially controlled by hypomethylation of its promoter region and activated ERK signaling in NSCLC. Through both in vitro and in vivo analyses, we revealed the mechanisms of miR-224 up-regulation and its oncogenic role in NSCLC pathogenesis.     

Lung inflammation promotes metastasis through neutrophil protease-mediated degradation of Tsp-1

Inflammation is inextricably associated with primary tumor progression. However, the contribution of inflammation to tumor outgrowth in metastatic organs has remained underexplored. Here, we show that extrinsic inflammation in the lungs leads to the recruitment of bone marrow-derived neutrophils, which degranulate azurophilic granules to release the Ser proteases, elastase and cathepsin G, resulting in the proteolytic destruction of the antitumorigenic factor thrombospondin-1 (Tsp-1). Genetic ablation of these neutrophil proteases protected Tsp-1 from degradation and suppressed lung metastasis. These results provide mechanistic insights into the contribution of inflammatory neutrophils to metastasis and highlight the unique neutrophil protease–Tsp-1 axis as a potential antimetastatic therapeutic target.The contribution of inflammation to primary tumor progression is well documented (1); however, little is known about its role in metastatic outgrowth in distant organs. The lung, which is a frequent site of metastasis from extrapulmonary neoplasms, is susceptible to inflammatory insults. Bacterial infection-induced, metastasis-conducive environments in the lung (2, 3) and cigarette smoke-induced inflammation were associated with pulmonary metastasis from breast cancer (2, 4).Bacterial lipopolysaccharide (LPS) is a well-characterized inducer of inflammation because its binding to toll-like receptor 4 (TLR4) results in nuclear factor kappa B (NF-κB) activation and expression of proinflammatory cytokines, including interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), and IL-6 (5). LPS-induced acute lung injury is marked by increased neutrophil influx and up-regulation of proinflammatory cytokines. Similar phenotypes are observed in other lung inflammatory conditions, including asthma (6), chronic obstructive pulmonary disease (7), and pneumonia (8, 9). LPS-mediated lung inflammation is associated with breast and colon cancer metastasis to the lungs (10–12).The mechanisms by which inflammation contributes to metastatic outgrowth in distant organs have remained underexplored. From a clinical perspective, although blocking primary tumor invasion and blocking dissemination are considered effective approaches in suppressing metastasis, an important question is how best to treat patients whose tumor has already metastasized. Thus, approaches are required to block tumor outgrowth in secondary organs for effective treatment of metastatic cancers. In this study, using two independent models of lung inflammation, we show enhanced recruitment of neutrophils, which degranulate to release the Ser proteases, neutrophil elastase (NE) and cathepsin G (CG), to degrade thrombospondin-1 (Tsp-1) in the lung microenvironment, enhancing metastatic outgrowth. Protease deficiency protected Tsp-1 from proteolysis and suppressed metastasis, providing a previously unidentified mechanism of Tsp-1 regulation in the metastatic organ.