Inhibition of the Axl pathway impairs breast and prostate cancer metastasis to the bones and bone remodeling.

Approximately 90% of cancer-related deaths result from cancer metastasis. In prostate and breast cancers, bone is the most common site of cancer cell dissemination. Key steps in the metastatic cascade are promoted through upregulation of critical cell signaling pathways in neoplastic cells. The present study assessed the role of the receptor tyrosine kinase Axl in prostate and breast cancer cell metastasis to bones using (i) Axl knockdown neoplastic cells and osteoclast progenitor cells in vitro, (ii) intracardiac injection of Axl knockdown tumor cells in vivo, and (iii) selective Axl inhibitor BGB324. Axl inhibition in neoplastic cells significantly decreased their metastatic potential, and suppression of Axl signaling in osteoclast precursor cells also reduced the formation of mature osteoclasts. In vivo, Axl knockdown in prostate and breast cancer cells significantly suppressed the formation and progression of bone metastases. Hence, therapeutic targeting of Axl may impair tumor metastasis to the bones through neoplastic and host cell signaling axes.

Bioprinting on Live Tissue for Investigating Cancer Cell Dynamics.

A challenge in cancer research is the lack of physiologically responsive in vitro models that enable tracking of cancer cells in tissue-like environments. A model that enables real-time investigation of cancer cell migration, fate, and function during angiogenesis does not exist. Current models, such as 2D or 3D in vitro culturing, can contain multiple cell types, but they do not incorporate the complexity of intact microvascular networks. The objective of this study was to establish a tumor microvasculature model by demonstrating the feasibility of bioprinting cancer cells onto excised mouse tissue. Inkjet-printed DiI+ breast cancer cells on mesometrium tissues from C57Bl/6 mice demonstrated cancer cells' motility and proliferation through time-lapse imaging. Colocalization of DAPI+ nuclei confirmed that DiI+ cancer cells remained intact postprinting. Printed DiI+ 4T1 cells also remained viable after printing on Day 0 and after culture on Day 5. Time-lapse imaging over 5 days enabled tracking of cell migration and proliferation. The number of cells and cell area were significantly increased over time. After culture, cancer cell clusters were colocalized with angiogenic microvessels. The number of vascular islands, defined as disconnected endothelial cell segments, was increased for tissues with bioprinted cancer cells, which suggests that the early stages of angiogenesis were influenced by the presence of cancer cells. Bioprinting cathepsin L knockdown 4T1 cancer cells on wild-type tissues or nontarget 4T1 cells on NG2 knockout tissues served to validate the use of the model for probing tumor cell versus microenvironment changes. These results establish the potential for bioprinting cancer cells onto live mouse tissues to investigate cancer microvascular dynamics within a physiologically relevant microenvironment. Impact statement To keep advancing the cancer biology field, tissue engineering has been focusing on developing in vitro tumor biomimetic models that more closely resemble the native microenvironment. We introduce a novel methodology of bioprinting exogenous cancer cells onto mouse tissue that contains multiple cells and systems within native physiology to investigate cancer cell migration and interactions with nearby microvascular networks. This study corroborates the manipulation of different exogenous cells and host microenvironments that impact cancer cell dynamics in a physiologically relevant tissue. Overall, it is a new approach for delineating the effects of the microenvironment on cancer cells and vice versa.

Cathepsin L secretion by host and neoplastic cells potentiates invasion.

The presence of macrophages within breast tumors correlates with metastatic potential. These tumor-associated macrophages often take on a pro-tumorigenic (M2-like) phenotype resulting in the secretion of growth factors and proteases, including the lysosomal protease cathepsin L. Since cathepsin L also is frequently secreted by breast cancer cells and contributes to tumor invasion, metastasis, and angiogenesis, we hypothesized that secretion of cathepsin L by both tumor-associated macrophages and neoplastic cells would facilitate the metastatic phenotype. Our results showed that the novel cathepsin L/K inhibitors KGP94 and KGP207 could inhibit in vitro M2 macrophage invasion and reduce the macrophage-stimulated invasion of 4T1 murine breast cancer cells. KGP94 and KGP207 treatment also reduced the expression of several M2-associated markers, suggesting that cathepsin L activity may be important for IL-4-driven M0 to M2 differentiation. In addition, cathepsin L shRNA knockdown studies revealed that cathepsin L from both the tumor cell and the macrophage population is important for tumor cell invasion. Thus our data suggest that tumor cells and macrophages may both contribute to the cathepsin L-driven metastatic phenotype of breast cancer. Taken together, these studies highlight the importance of cathepsin L in macrophage functions and suggest that cathepsin inhibition strategies may be therapeutically beneficial by impairing the progression of tumors with high infiltration of M2 macrophages.

Stromal cells in breast cancer as a potential therapeutic target.

Breast cancer in the United States is the second most commonly diagnosed cancer in women. About 1 in 8 women will develop invasive breast cancer over the course of her lifetime and breast cancer remains the second leading cause of cancer-related death. In pursuit of novel therapeutic strategies, researchers have examined the tumor microenvironment as a potential anti-cancer target. In addition to neoplastic cells, the tumor microenvironment is composed of several critical normal cell types, including fibroblasts, vascular and lymph endothelial cells, osteoclasts, adipocytes, and immune cells. These cells have important roles in healthy tissue stasis, which frequently are altered in tumors. Indeed, tumor-associated stromal cells often contribute to tumorigenesis, tumor progression, and metastasis. Consequently, these host cells may serve as a possible target in anti-tumor and anti-metastatic therapeutic strategies. Targeting the tumor associated host cells offers the benefit that such cells do not mutate and develop resistance in response to treatment, a major cause of failure in cancer therapeutics targeting neoplastic cells. This review discusses the role of host cells in the tumor microenvironment during tumorigenesis, progression, and metastasis, and provides an overview of recent developments in targeting these cell populations to enhance cancer therapy efficacy.

Epstein-Barr virus stably confers an invasive phenotype to epithelial cells through reprogramming of the WNT pathway.

Epstein-Barr virus (EBV)-associated carcinomas, such as nasopharyngeal carcinoma (NPC), exhibit an undifferentiated and metastatic phenotype. To determine viral contributions involved in the invasive phenotype of EBV-associated carcinomas, EBV-infected human telomerase-immortalized normal oral keratinocytes (NOK) were investigated. EBV-infected NOK were previously shown to undergo epigenetic reprogramming involving CpG island hypermethylation and delayed responsiveness to differentiation. Here, we show that EBV-infected NOK acquired an invasive phenotype that was epigenetically retained after viral loss. The transcription factor lymphoid enhancer factor 1 (LEF1) and the secreted ligand WNT5A, expressed in NPC, were increased in EBV-infected NOK with sustained expression for more than 20 passages after viral loss. Increased LEF1 levels involved four LEF1 variants, and EBV-infected NOK showed a lack of responsiveness to ß-catenin activation. Although forced expression of WNT5A and LEF1 enhanced the invasiveness of parental NOK, LEF1 knockdown reversed the invasive phenotype of EBV-infected NOK in the presence of WNT5A. Viral reprogramming of LEF1 and WNT5A was observed several passages after EBV infection, suggesting that LEF1 and WNT5A may provide a selective advantage to virally-infected cells. Our findings suggest that EBV epigenetically reprogrammed epithelial cells with features of basal, wound healing keratinocytes, with LEF1 contributing to the metastatic phenotype of EBV-associated carcinomas.

Lysosome trafficking is necessary for EGF-driven invasion and is regulated by p38 MAPK and Na+/H+ exchangers.

BACKGROUND: Tumor invasion through a basement membrane is one of the earliest steps in metastasis, and growth factors, such as Epidermal Growth Factor (EGF) and Hepatocyte Growth Factor (HGF), stimulate this process in a majority of solid tumors. Basement membrane breakdown is one of the hallmarks of invasion; therefore, tumor cells secrete a variety of proteases to aid in this process, including lysosomal proteases. Previous studies demonstrated that peripheral lysosome distribution coincides with the release of lysosomal cathepsins. METHODS: Immunofluorescence microscopy, western blot, and 2D and 3D cell culture techniques were performed to evaluate the effects of EGF on lysosome trafficking and cell motility and invasion. RESULTS: EGF-mediated lysosome trafficking, protease secretion, and invasion is regulated by the activity of p38 mitogen activated protein kinase (MAPK) and sodium hydrogen exchangers (NHEs). Interestingly, EGF stimulates anterograde lysosome trafficking through a different mechanism than previously reported for HGF, suggesting that there are redundant signaling pathways that control lysosome positioning and trafficking in tumor cells. CONCLUSIONS: These data suggest that EGF stimulation induces peripheral (anterograde) lysosome trafficking, which is critical for EGF-mediated invasion and protease release, through the activation of p38 MAPK and NHEs. Taken together, this report demonstrates that anterograde lysosome trafficking is necessary for EGF-mediated tumor invasion and begins to characterize the molecular mechanisms required for EGF-stimulated lysosome trafficking.

Identification of small molecules that disrupt vacuolar function in the pathogen Candida albicans.

The fungal vacuole is a large acidified organelle that performs a variety of cellular functions. At least a sub-set of these functions are crucial for pathogenic species of fungi, such as Candida albicans, to survive within and invade mammalian tissue as mutants with severe defects in vacuolar biogenesis are avirulent. We therefore sought to identify chemical probes that disrupt the normal function and/or integrity of the fungal vacuole to provide tools for the functional analysis of this organelle as well as potential experimental therapeutics. A convenient indicator of vacuolar integrity based upon the intracellular accumulation of an endogenously produced pigment was adapted to identify Vacuole Disrupting chemical Agents (VDAs). Several chemical libraries were screened and a set of 29 compounds demonstrated to reproducibly cause loss of pigmentation, including 9 azole antifungals, a statin and 3 NSAIDs. Quantitative analysis of vacuolar morphology revealed that (excluding the azoles) a sub-set of 14 VDAs significantly alter vacuolar number, size and/or shape. Many C. albicans mutants with impaired vacuolar function are deficient in the formation of hyphal elements, a process essential for its pathogenicity. Accordingly, all 14 VDAs negatively impact C. albicans hyphal morphogenesis. Fungal selectivity was observed for approximately half of the VDA compounds identified, since they did not alter the morphology of the equivalent mammalian organelle, the lysosome. Collectively, these compounds comprise of a new collection of chemical probes that directly or indirectly perturb normal vacuolar function in C. albicans.

Zinc finger E-box binding homeobox-1 (Zeb1) drives anterograde lysosome trafficking and tumor cell invasion via upregulation of Na+/H+ Exchanger-1 (NHE1).

Tumor cell invasion through the extracellular matrix is facilitated by the secretion of lysosome-associated proteases. As a common mechanism for secretion, lysosomes must first traffic to the cell periphery (anterograde trafficking), consistent with invasive cells often containing lysosomes closer to the plasma membrane compared to non-invasive cells. Epithelial to mesenchymal transition (EMT) is a transcriptionally driven program that promotes an invasive phenotype, and Zeb1 is one transcription factor that activates the mesenchymal gene expression program. The role of lysosome trafficking in EMT-driven invasion has not been previously investigated. We found that cells with increased levels of Zeb1 displayed lysosomes located closer to the cell periphery and demonstrated increased protease secretion and invasion in 3-dimensional (3D) cultures compared to their epithelial counterparts. Additionally, preventing anterograde lysosome trafficking via pharmacological inhibition of Na+/H+ exchanger 1 (NHE1) or shRNA depletion of ADP-ribosylation like protein 8b (Arl8b) reversed the invasive phenotype of mesenchymal cells, thus supporting a role for lysosome positioning in EMT-mediated tumor cell invasion. Immunoblot revealed that expression of Na+/H+ exchanger 1 correlated with Zeb1 expression. Furthermore, we found that the transcription factor Zeb1 binds to the Na+/H+ exchanger 1 promoter, suggesting that Zeb1 directly controls Na+/H+ transcription. Collectively, these results provide insight into a novel mechanism regulating Na+/H+ exchanger 1 expression and support a role for anterograde lysosome trafficking in Zeb1-driven cancer progression. © 2016 Wiley Periodicals, Inc.

The Arf-like GTPase Arl8b is essential for three-dimensional invasive growth of prostate cancer in vitro and xenograft formation and growth in vivo.

Cancer is a multistep process that requires cells to respond appropriately to the tumor microenvironment, both in early proliferative stages and in later invasive disease. Arl8b is a lysosome localized Arf-like GTPase that controls the spatial distribution of lysosomes via recruitment of kinesin motors. Common features of the tumor microenvironment such as acidic extracellular pH and various growth factors stimulate lysosome trafficking to the cell periphery (anterograde), which is critical for tumor invasion by facilitating the release of lysosomal proteases to promote matrix remodeling. Herein we report for the first time that Arl8b regulates anterograde lysosome trafficking in response to hepatocyte growth factor, epidermal growth factor, and acidic extracellular pH. Depletion of Arl8b results in juxtanuclear lysosome aggregation, and this effect corresponds with both diminished invasive growth and proteolytic extracellular matrix degradation in a three-dimensional model of prostate cancer. Strikingly, we found that depletion of Arl8b abolishes the ability of prostate cancer cells to establish subcutaneous xenografts in mice. We present evidence that Arl8b facilitates lipid hydrolysis to maintain efficient metabolism for a proliferative capacity in low nutrient environments, suggesting a likely explanation for the complete inability of Arl8b-depleted tumor cells to grow in vivo. In conclusion, we have identified two mechanisms by which Arl8b regulates cancer progression: 1) through lysosome positioning and protease release leading to an invasive phenotype and 2) through control of lipid metabolism to support cellular proliferation. These novel roles highlight that Arl8b is a potential target for the development of novel anti-cancer therapeutics.

The oleocanthal-based homovanillyl sinapate as a novel c-Met inhibitor.

The hepatocyte growth factor (HGF)/mesenchymal-epithelial transition factor (c-Met) signaling axis has gained considerable attention as an attractive molecular target for therapeutic blockade of cancer. Inspired by the chemical structure of S (-)-oleocanthal, a natural secoiridoid from extra-virgin olive oil with documented anticancer activity against c-Met-dependent malignancies, the research presented herein reports on the discovery of the novel olive-derived homovanillyl sinapate (HVS) as a promising c-Met inhibitor. HVS was distinguished for its remarkable potency against wild-type c-Met and its oncogenic variant in cell-free assays and confirmed by in silico docking studies. Furthermore, HVS substantially impaired the c-Met-mediated growth across a broad spectrum of breast cancer cells, while similar treatment doses had no effect on the non-tumorigenic mammary epithelial cell growth. In addition, HVS caused a dose-dependent inhibition of HGF-induced, but not epidermal growth factor (EGF)-induced, cell scattering in addition to HGF-mediated migration, invasion, and 3-dimensional (3D) proliferation of tumor cell spheroids. HVS treatment effects were mediated via inhibition of ligand-mediated c-Met activation and its downstream mitogenic signaling and blocking molecular mediators involved in cellular motility across different cellular contexts. An interesting feature of HVS is its good selectivity for c-Met and Abelson murine leukemia viral oncogene homolog 1 (ABL1) when profiled against a panel of kinases. Docking studies revealed interactions likely to impart high dual affinity for both ABL1 and c-Met kinases. HVS markedly reduced tumor growth, showed excellent pharmacodynamics, and suppressed cell proliferation and microvessel density in an orthotopic model of triple negative breast cancer. Collectively, the present findings suggested that the oleocanthal-based HVS is a promising c-Met inhibitor lead entity with excellent therapeutic potential to control malignancies with aberrant c-Met activity.

A Novel High Content Imaging-Based Screen Identifies the Anti-Helminthic Niclosamide as an Inhibitor of Lysosome Anterograde Trafficking and Prostate Cancer Cell Invasion.

Lysosome trafficking plays a significant role in tumor invasion, a key event for the development of metastasis. Previous studies from our laboratory have demonstrated that the anterograde (outward) movement of lysosomes to the cell surface in response to certain tumor microenvironment stimulus, such as hepatocyte growth factor (HGF) or acidic extracellular pH (pHe), increases cathepsin B secretion and tumor cell invasion. Anterograde lysosome trafficking depends on sodium-proton exchanger activity and can be reversed by blocking these ion pumps with Troglitazone or EIPA. Since these drugs cannot be advanced into the clinic due to toxicity, we have designed a high-content assay to discover drugs that block peripheral lysosome trafficking with the goal of identifying novel drugs that inhibit tumor cell invasion. An automated high-content imaging system (Cellomics) was used to measure the position of lysosomes relative to the nucleus. Among a total of 2210 repurposed and natural product drugs screened, 18 "hits" were identified. One of the compounds identified as an anterograde lysosome trafficking inhibitor was niclosamide, a marketed human anti-helminthic drug. Further studies revealed that niclosamide blocked acidic pHe, HGF, and epidermal growth factor (EGF)-induced anterograde lysosome redistribution, protease secretion, motility, and invasion of DU145 castrate resistant prostate cancer cells at clinically relevant concentrations. In an effort to identify the mechanism by which niclosamide prevented anterograde lysosome movement, we found that this drug exhibited no significant effect on the level of ATP, microtubules or actin filaments, and had minimal effect on the PI3K and MAPK pathways. Niclosamide collapsed intralysosomal pH without disruption of the lysosome membrane, while bafilomycin, an agent that impairs lysosome acidification, was also found to induce JLA in our model. Taken together, these data suggest that niclosamide promotes juxtanuclear lysosome aggregation (JLA) via modulation of pathways involved in lysosome acidification. In conclusion, we have designed a validated reproducible high-content assay to screen for drugs that inhibit lysosome trafficking and reduce tumor invasion and we summarize the action of one of these drugs.

The histone deacetylase inhibitor cambinol prevents acidic pHe-induced anterograde lysosome trafficking independently of sirtuin activity.

Common features of the solid tumor microenvironment, such as acidic extracellular pH and growth factors, are known to induce the redistribution of lysosomes from a perinuclear region to a position near the plasma membrane. Lysosome/plasma membrane juxtaposition facilitates invasion by allowing for the release of lysosomal proteases, including cathepsin B, which contribute to matrix degradation. In this study we identified the sirtuin 1/sirtuin 2 (SIRT1/2) inhibitor cambinol acts as a drug that inhibits lysosome redistribution and tumor invasion. Treatment of cells with cambinol resulted in a juxtanuclear lysosome aggregation (JLA) similar to that seen upon treatment with the PPARγ agonist, troglitazone (Tro). Like Tro, cambinol required the activity of ERK1/2 in order to induce this lysosome clustering phenotype. However, cambinol did not require the activity of Rab7, suggesting that this drug causes JLA by a mechanism different from what is known for Tro. Additionally, cambinol-induced JLA was not a result of autophagy induction. Further investigation revealed that cambinol triggered JLA independently of its activity as a SIRT1/2 inhibitor, suggesting that this drug could have effects in addition to SIRT1/2 inhibition that could be developed into a novel anti-cancer therapy.

Supporting a role for the GTPase Rab7 in prostate cancer progression.

Invasion and subsequent metastasis is the major cause of death from most cancers including prostate cancer. Herein we report on the potential tumor suppressive properties of Rab7, a GTPase that regulates trafficking of lysosomes. The movement of lysosomes to the cell surface in response to environmental cues increases the secretion of proteinases and cell invasion. We determined that Troglitazone and other members of the Thiazolidinedione family inhibit cell-surface directed lysosome trafficking and cathepsin B secretion through a Rab7-dependent mechanism. Moreover, Rab7 shRNA expressing cells were found to be more invasive in vitro and in vivo. Increased invasiveness was accompanied by elevated expression of the c-Met receptor and prolonged downstream signaling, thereby supporting a role for Rab7 as a mediator of signaling down-regulation. Taken together, these results suggested that Rab7 acts as a negative regulator of prostate tumor growth and invasion, providing further evidence for its potential as a tumor suppressor.