Search

Significance Isolation of sufficient numbers of circulating tumor cells (CTCs) in cancer patients could provide an alternative to invasive tumor biopsies, providing multianalyte cell-based biomarkers that are not available from current plasma circulating tumor DNA sequencing. Given the average prevalence at one CTC per billion blood cells, very large blood volumes must be screened to provide enough CTCs for reliable clinical applications. By creating an ultrahigh-throughput magnetic sorter, we demonstrate the efficient removal of leukocytes from near whole blood volume equivalents. Combined with leukapheresis to initially concentrate blood mononuclear cells, this LPCTC-iChip platform will enable noninvasive sampling of cancer cells in sufficient numbers for clinical applications, ranging from real-time pharmacokinetic monitoring of drug response to tissue-of-origin determination in early-stage cancer screening., Circulating tumor cell (CTC)-based liquid biopsies provide unique opportunities for cancer diagnostics, treatment selection, and response monitoring, but even with advanced microfluidic technologies for rare cell detection the very low number of CTCs in standard 10-mL peripheral blood samples limits their clinical utility. Clinical leukapheresis can concentrate mononuclear cells from almost the entire blood volume, but such large numbers and concentrations of cells are incompatible with current rare cell enrichment technologies. Here, we describe an ultrahigh-throughput microfluidic chip, LPCTC-iChip, that rapidly sorts through an entire leukapheresis product of over 6 billion nucleated cells, increasing CTC isolation capacity by two orders of magnitude (86% recovery with 105 enrichment). Using soft iron-filled channels to act as magnetic microlenses, we intensify the field gradient within sorting channels. Increasing magnetic fields applied to inertially focused streams of cells effectively deplete massive numbers of magnetically labeled leukocytes within microfluidic channels. The negative depletion of antibody-tagged leukocytes enables isolation of potentially viable CTCs without bias for expression of specific tumor epitopes, making this platform applicable to all solid tumors. Thus, the initial enrichment by routine leukapheresis of mononuclear cells from very large blood volumes, followed by rapid flow, high-gradient magnetic sorting of untagged CTCs, provides a technology for noninvasive isolation of cancer cells in sufficient numbers for multiple clinical and experimental applications.

[This corrects the article DOI: 10.1371/journal.pone.0054014.].

Mitochondrial metabolism is an attractive target for cancer therapy1,2. Reprogramming metabolic pathways could improve the ability of metabolic inhibitors to suppress cancers with limited treatment options, such as triple-negative breast cancer (TNBC)1,3. Here we show that BTB and CNC homology1 (BACH1)4, a haem-binding transcription factor that is increased in expression in tumours from patients with TNBC, targets mitochondrial metabolism. BACH1 decreases glucose utilization in the tricarboxylic acid cycle and negatively regulates transcription of electron transport chain (ETC) genes. BACH1 depletion by shRNA or degradation by hemin sensitizes cells to ETC inhibitors such as metformin5,6, suppressing growth of both cell line and patient-derived tumour xenografts. Expression of a haem-resistant BACH1 mutant in cells that express a short hairpin RNA for BACH1 rescues the BACH1 phenotype and restores metformin resistance in hemin-treated cells and tumours7. Finally, BACH1 gene expression inversely correlates with ETC gene expression in tumours from patients with breast cancer and in other tumour types, which highlights the clinical relevance of our findings. This study demonstrates that mitochondrial metabolism can be exploited by targeting BACH1 to sensitize breast cancer and potentially other tumour tissues to mitochondrial inhibitors. The transcription factor BACH1, which targets mitochondrial metabolism, is expressed at high levels in several types of cancer; reducing its expression in tumours makes them more susceptible to treatment with mitochondrial inhibitors.

Simple Summary About 10–20 percent of patients with breast cancer are diagnosed with triple-negative breast cancer (TNBC). These tumors are named for their lack of expression of estrogen receptor (ER), progesterone receptor (PR), and amplification of human epidermal growth factor receptor 2 (HER2). These genes are targeted by therapies in other breast cancer patients. However, most TNBC patients recur within 5 years. Understanding how and why these tumors metastasize will help clinicians better treat these underserved cancer patients. TNBC tumors are highly infiltrated by tumor-associated macrophages (TAMs) that promote tumorigenesis and metastasis. Our study elucidates how the tumor co-opts macrophages recruited to the tumor through extracellular vesicles (EVs), further increasing tumor metastasis. Expression of tumor CCL5 regulates EV secretion and cargo that further alters macrophage phenotype to drive tumor metastasis. Together, our data suggest a more extensive role of EVs in the biology of tumor metastasis as well as their potential use as biomarkers. Abstract Purpose: To understand how tumor cells alter macrophage biology once they are recruited to triple-negative breast cancer (TNBC) tumors by CCL5. Method: Mouse bone marrow derived macrophage (BMDMs) were isolated and treated with recombinant CCL5 protein alone, with tumor cell conditioned media, or with tumor extracellular vesicles (EVs). Media from these tumor EV-educated macrophages (TEMs) was then used to determine how these macrophages affect TNBC invasion. To understand the mechanism, we assayed the cytokine secretion from these macrophages to determine how they impact tumor cell invasion. Tumor CCL5 expression was varied in tumors to determine its role in regulating macrophage biology through EVs. Results: Tumor EVs are a necessary component for programming naïve macrophages toward a pro-metastatic phenotype. CCL5 expression in the tumor cells regulates both EV biogenesis/secretion/cargo and macrophage EV-education toward a pro-metastatic phenotype. Analysis of the tumor EV-educated macrophages (TEMs) showed secretion of a variety of factors including CXCL1, CTLA-4, IFNG, OPN, HGF, TGFB, and CCL19 capable of remodeling the surrounding tumor stroma and immune infiltrate. Injection of tumor cells with macrophages educated by metastatic tumor cell EVs into mice increased tumor metastasis to the lung. Conclusion: These results demonstrate that tumor-derived EVs are key mediators of macrophage education and likely play a more complex role in modulating tumor therapeutic response by regulating the tumor immune infiltrate.

SummaryTriple-negative breast cancers (TNBC) are highly infiltrated by tumor-associated macrophages (TAMs) that promote tumor growth, survival, metastasis and therapeutic resistance. Although cytokines such as CCL5 have been implicated in TAM recruitment to TNBC tumors, the mechanism by which tumor cells educate TAMs is not understood. Here we show that tumor EVs are both necessary and sufficient for programming TAMs toward a pro-metastatic phenotype. The mechanism involves CCL5 regulation of tumor extracellular vesicles (EVs), which activate TLR2 and TLR3, leading to secretion of a common set of cytokines that further stimulate tumor cell invasion and metastasis as well as alter the tumor microenvironment. Cytokine expression is significantly correlated to CCL5 expression and up-regulated in TNBC patient tumors. These results demonstrate for the first time that tumor EVs are key mediators of TAM education, phenocopy the pro-metastatic and drug resistant state of the tumors to TAMs, and illustrate the potential clinical relevance of these findings to TNBC patients.HighlightsTumor extracellular vesicles (EVs) are required for pro-metastatic programming of tumor-associated macrophages (TAMs)Tumor CCL5 and macrophage TLR signaling mediate tumor EV programming of TAMs in TNBCsTumor EVs mediate drug resistance in TAMs and alter recruitment of regulatory T-cells.Cytokines expressed by EV-educated TAMs are enriched and correlate with CCL5 in human TNBC patients.eTOCChemokines such as CCL5 recruit tumor-associated macrophages (TAMs) that are required for metastasis, but TAM programming is not understood. Rabe et al. show that tumor extracellular vesicles (EVs) are required for programming TAMs via Toll-like Receptors (TLRs) to phenocopy the tumor, rewire the microenvironment, drive metastasis and promote immune cell evasion.

Signaling by human hepatocyte growth factor (hHGF) via its cell surface receptor (MET) drives mitogenesis, motogenesis and morphogenesis in a wide spectrum of target cell types and embryologic, developmental and homeostatic contexts. Oncogenic pathway activation also contributes to tumorigenesis and cancer progression, including tumor angiogenesis and metastasis, in several prevalent malignancies. The HGF gene encodes full-length hHGF and two truncated isoforms known as NK1 and NK2. NK1 induces all three HGF activities at modestly reduced potency, whereas NK2 stimulates only motogenesis and enhances HGF-driven tumor metastasis in transgenic mice. Prior studies have shown that mouse HGF (mHGF) also binds with high affinity to human MET. Here we show that, like NK2, mHGF stimulates cell motility, invasion and spontaneous metastasis of PC3M human prostate adenocarcinoma cells in mice through human MET. To identify target genes and signaling pathways associated with motogenic and metastatic HGF signaling, i.e., the HGF invasive program, gene expression profiling was performed using PC3M cells treated with hHGF, NK2 or mHGF. Results obtained using Ingenuity Pathway Analysis software showed significant overlap with networks and pathways involved in cell movement and metastasis. Interrogating The Cancer Genome Atlas project also identified a subset of 23 gene expression changes in PC3M with a strong tendency for co-occurrence in prostate cancer patients that were associated with significantly decreased disease-free survival.

The surrounding microenvironment has been implicated in the progression of breast tumors to metastasis. However, the degree to which metastatic breast tumors locally reprogram stromal cells as they disrupt tissue boundaries is not well understood. We used species-specific RNA sequencing in a mouse xenograft model to determine how the metastasis suppressor RKIP influences transcription in a panel of paired tumor and stroma tissues. We find that gene expression in metastatic breast tumors is pervasively correlated with gene expression in local stroma of both mouse xenografts and human patients. Changes in stromal gene expression elicited by tumors better predicts subtype and patient survival than tumor gene expression, and genes with coordinated expression in both tissues predict metastasis-free survival. These observations support the use of stroma-based strategies for the diagnosis and prognosis of breast cancer.

Objective To measure Met protein content in prostate biopsies guided by fused magnetic resonance and ultrasound imaging, and to measure soluble Met (sMet) protein concentration in plasma samples from patients presenting evidence of prostate cancer. Patients and Methods 345 patients had plasma samples drawn prior to image-guided biopsy of the prostate. Of these, 32% had benign biopsies. Of the 236 that were positive for prostate adenocarcinoma (PCa), 132 treated by total prostatectomy had Gleason scores of 6 (17%), 7, (55%), 8 (16%), or 9–10 (12%). 23% had evidence of local invasion. Plasma samples were also obtained from 80 healthy volunteers. Tissue Met and plasma sMet were measured by two-site immunoassay; values were compared among clinically defined groups using non-parametric statistical tests to determine significant differences or correlations. Results PCa tumor Met correlated significantly with plasma sMet, but median values were similar among benign and malignant groups. Median plasma sMet values were also similar among those groups, although both medians were significantly above normal. Median Met content in primary PCa tumors and sMet concentrations were independent of Gleason score, final pathologic stage and age. Conclusion Plasma sMet is not predictive of PCa or its severity in patients with organ-confined or locally invasive disease. Quantitative analysis of Met protein content and activation state in PCa tumor biopsy samples was highly feasible and may have value in follow-up to genomic and/or transcriptomic-based screens that show evidence of oncogenically relevant MET gene features that occur at relatively low frequency in non-metastatic PCa.

SummaryHepatocyte growth factor (HGF) and vascular endothelial cell growth factor (VEGF) regulate normal development and homeostasis and drive disease progression in many forms of cancer. Both proteins signal by binding to receptor tyrosine kinases and heparan sulfate (HS) proteoglycans on target cell surfaces. Basic residues comprising the primary HS binding sites on HGF and VEGF provide similar surface charge distributions without underlying structural similarity. Combining three acidic amino acid substitutions in these sites in the HGF isoform NK1 or the VEGF isoform VEGF165 transformed each into potent, selective competitive antagonists of their respective normal and oncogenic signaling pathways. Our findings illustrate the importance of HS in growth factor driven cancer progression and reveal an efficient strategy for therapeutic antagonist development.

Metastatic progression of tumors is the major cause of death in patients with triple-negative breast cancer (TNBC). However, since metastasis is a multistep process, unraveling its complexity is a major challenge. One effective way of tackling this question is to study natural blockers of the metastatic process, metastasis suppressors, and identify the mechanisms by which they regulate metastasis. Raf kinase inhibitory protein (RKIP), a protein that regulates kinase activity, is a suppressor of TNBC metastasis. Although RKIP inhibits the activity of key kinases such as Raf-1, GRK2, NIK/IKK in cultured cells, the kinase targets of RKIP in tumors are not known. To address this question, we used a mass spectrometry approach involving inhibitor-conjugated beads to identify kinases that are downregulated by RKIP in human TNBC xenograft tumors. Our results identified a network of stress kinases targeted by RKIP, including kinases that have not been previously reported as RKIP targets. In order to unravel the effect of this stress network on metastatic gene expression, we investigated genes that correlate with RKIP expression in TCGA breast cancer patient data set. We identified prometastatic genes such as APC and DOCK4 as novel RKIP targets in in vitro and in vivo models of TNBC. We also demonstrated these genes are downstream of the RKIP-stress network. Finally, by using a high-throughput invasion assay, we developed a low-dose multidrug cocktail of small-molecule kinase inhibitors that mimic RKIP's antimetastatic role in TNBCs. Elucidating RKIP function at a systems level reveals the interplay between key metastatic signaling cascades, particularly in relation to cell motility and invasion. Our findings suggest that the low-dose multidrug combination that targets a network of stress kinases is a viable antimetastatic therapy for TNBC patients. Citation Format: Ali Ekrem Yesilkanal, Daniel C. Rabe, Payal Tiwari, Casey Frankenberger, Gary L. Johnson, Marsha Rosner. A novel approach for antimetastatic therapies against TNBC utilizing a physiologic suppressor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4179.

Triple-negative breast cancers (TNBC) are highly infiltrated by tumor-associated macrophages (TAMs) that promote tumor growth, survival, metastasis and therapeutic resistance. Although cytokines such as CCL5 have been implicated in TAM recruitment to TNBC tumors, the mechanism by which tumor cells educate TAMs is not understood. Here we show that tumor extracellular vesicles (EVs) are both necessary and sufficient for programming TAMs toward a pro-metastatic phenotype. The mechanism involves CCL5 regulation of tumor EVs, which activate TLR2 and TLR3, leading to secretion of a common set of cytokines that further stimulate tumor cell invasion and metastasis as well as alter the tumor microenvironment. Cytokine expression is significantly correlated to CCL5 expression and up-regulated in TNBC patient tumors. These results demonstrate for the first time that tumor EVs are key mediators of TAM education, phenocopy the pro-metastatic and drug resistant state of the tumors to TAMs, and illustrate the potential clinical relevance of these findings to TNBC patients. Citation Format: Daniel C. Rabe, Jiyoung Lee, Felicia Rustandy, Marsha R. Rosner. Tumor extracellular vesicles are required for tumor-associated macrophage programming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1739.

Oxidative phosphorylation is an attractive target for cancer therapy. Reprogramming metabolic pathways by promoting oxidative phosphorylation could improve the ability of metabolic inhibitors to suppress cancers with limited treatment options like triple negative breast cancer (TNBC). Here we show that BACH1, a heme-binding transcription factor whose expression is enriched in patients with TNBC, inhibits oxidative phosphorylation through direct transcriptional regulation of electron transport chain (ETC) gene expression. Treatment of cells with hemin, which induces BACH1 degradation, mimics BACH1 depletion with shRNA. Pretreatment of TNBC tumors with BACH1 shRNA or hemin overcame resistance to metformin, an anti-diabetic drug, and abolished the growth of both cell line and patient-derived tumor xenografts. BACH1 gene expression inversely correlated with ETC gene expression in breast cancer patients as well as other tumor types, highlighting the clinical relevance. This study demonstrates that oxidative phosphorylation represents an Achilles heel that can be exploited through targeting BACH1 to sensitize breast cancer and potentially other tumor tissues to mitochondrial inhibitors. Citation Format: Jiyoung Lee, Ali Yesilkanal, Casey Frankenberger, Mohamad Elbaz, Daniel Rabe, Jielin Yan, Felicia Rustandy, Peter Hart, Christie Kang, Elizabeth Grossman, Jason Locasale, Daniel Nomura, Marcelo Bonini, Marsha Rosner. Effective combination therapy for breast cancer targeting BACH1 and mitochondrial metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5497.

Triple-negative breast cancer (TNBC) patients have the highest risk of recurrence and metastasis. Because they cannot be treated with targeted therapies, and many do not respond to chemotherapy, they represent a clinically underserved group. TNBC is characterized by reduced expression of metastasis suppressors such as Raf kinase inhibitory protein (RKIP), which inhibits tumor invasiveness. Mechanisms by which metastasis suppressors alter tumor cells are well characterized; however, their ability to regulate the tumor microenvironment and the importance of such regulation to metastasis suppression are incompletely understood. Here, we use species-specific RNA sequencing to show that RKIP expression in tumors markedly reduces the number and metastatic potential of infiltrating tumor-associated macrophages (TAM). TAMs isolated from nonmetastatic RKIP+ tumors, relative to metastatic RKIP− tumors, exhibit a reduced ability to drive tumor cell invasion and decreased secretion of prometastatic factors, including PRGN, and shed TNFR2. RKIP regulates TAM recruitment by blocking HMGA2, resulting in reduced expression of numerous macrophage chemotactic factors, including CCL5. CCL5 overexpression in RKIP+ tumors restores recruitment of prometastatic TAMs and intravasation, whereas treatment with the CCL5 receptor antagonist Maraviroc reduces TAM infiltration. These results highlight the importance of RKIP as a regulator of TAM recruitment through chemokines such as CCL5. The clinical significance of these interactions is underscored by our demonstration that a signature comprised of RKIP signaling and prometastatic TAM factors strikingly separates TNBC patients based on survival outcome. Collectively, our findings identify TAMs as a previously unsuspected mechanism by which the metastasis-suppressor RKIP regulates tumor invasiveness, and further suggest that TNBC patients with decreased RKIP activity and increased TAM infiltration may respond to macrophage-based therapeutics. Cancer Res; 75(19); 4063–73. ©2015 AACR.

The Department of Defense (DoD) has identified that one of the main complaints of personnel exposed to JP-8 jet fuel is irritant dermatitis. The purpose of this investigation is to describe the JP-8-induced inflammatory cytokine response in skin. JP-8 jet fuel or acetone control (300 microl) was applied to the denuded skin of rats once a day for 7 days. Skin samples from the exposed area were collected 2 and 24 h after the final exposure. Histological examination of skin biopsies showed neutrophilic inflammatory infiltrate. Reverse transcription-polymerase chain reaction (RT-PCR) was performed utilizing skin total RNA to examine the expression of various inflammatory cytokines. The CXC chemokine GROalpha was significantly upregulated at both time points, whereas GRObeta was only increased 2 h post final exposure. The CC chemokines MCP-1, Mip-1alpha, and eotaxin were induced at both time points, whereas Mip-1beta was induced only 24 h post exposure. Interleukins-1beta and -6 (IL-1beta and IL-6) mRNAs were significantly induced at both time points, while TNFalpha was not significantly different from control. Enzyme-linked immunosorbent assay (ELISA) of skin protein confirmed that MCP-1, TNFalpha, and IL-1beta were modulated as indicated by PCR analysis. However, skin IL-6 protein content was not increased 2 h post exposure, whereas it was significantly upregulated by jet fuel after 24 h. Data from the present study indicate that repeated (7 days) JP-8 exposure induces numerous proinflammatory cytokines in skin. The increased expression of these cytokines and chemokines may lead to increased inflammatory infiltrate in exposed skin, resulting in JP-8-induced irritant dermatitis.

Kidney cancer was the cause of almost 13,000 deaths in the United States in 2009. Loss of function of the VHL tumor suppressor gene (von Hippel-Lindau disease) dramatically increases the risk of developing clear cell kidney cancer. The VHL protein is best understood for its regulation of hypoxia inducible factor (HIF). HIF responds to changes in oxygen levels in the cell and is responsible for mediating the transcriptional response to hypoxia. Of the three known HIFα gene products, HIF-2α appears to play a fundamental role in renal carcinoma. A high throughput screen was developed to identify small molecule inhibitors of HIF-2 gene expression. The screen was performed and yielded 153 confirmed active natural product extracts. Three of the active extracts were from marine soft corals of the order Alcyonacea: Sarcophyton sp., Lobophytum sarcophytoides and Asterospicularia laurae. Bioassay-guided fractionation led to the isolation of two new cembrane diterpenes, (4Z,8S*,9R*,12E,14E)-9-hydroxy-1-(prop-1-en-2-yl)-8,12-dimethyl-oxabicyclo[9.3.2]-hexadeca-4,12,14-trien-18-one (1), and (1E,3E,7R*,8R*,11E)-1-(2-methoxypropan-2-yl)-4,8,12-trimethyloxabicyclo[12.1.0]-pentadeca-1,3,11-triene (7), as well as eight known compounds, 2-6 and 8-10.

Under normal conditions, hepatocyte growth factor (HGF)-induced activation of its cell surface receptor, the Met tyrosine kinase (TK), is tightly regulated by paracrine ligand delivery, ligand activation at the target cell surface, and ligand activated receptor internalization and degradation. Despite these controls, HGF/Met signaling contributes to oncogenesis and tumor progression in several cancers and promotes aggressive cellular invasiveness that is strongly linked to tumor metastasis. The prevalence of HGF/Met pathway activation in human malignancies has driven rapid growth in cancer drug development programs. Pathway inhibitors can be divided broadly into biologicals and low molecular weight synthetic TK inhibitors; of these, the latter now outnumber all other inhibitor types. We review here Met structure and function, the basic properties of HGF/Met pathway antagonists now in preclinical and clinical development, as well as the latest clinical trial results. The main challenges facing the effective use of HGF/Met-targeted antagonists for cancer treatment include optimal patient selection, diagnostic and pharmacodynamic biomarker development, and the identification and testing of optimal therapy combinations. The wealth of basic information, analytical reagents and model systems available concerning HGF/Met oncogenic signaling will continue to be invaluable in meeting these challenges and moving expeditiously toward more effective disease control.

Purpose The receptors for hepatocyte and vascular endothelial cell growth factors (MET and VEGFR2, respectively) are critical oncogenic mediators in gastric adenocarcinoma. The purpose is to examine the safety and efficacy of foretinib, an oral multikinase inhibitor targeting MET, RON, AXL, TIE-2, and VEGFR2 receptors, for the treatment of metastatic gastric adenocarcinoma. Patients and Methods Foretinib safety and tolerability, and objective response rate (ORR) were evaluated in patients using intermittent (240 mg/day, for 5 days every 2 weeks) or daily (80 mg/day) dosing schedules. Thirty evaluable patients were required to achieve alpha = 0.10 and beta = 0.2 to test the alternative hypothesis that single-agent foretinib would result in an ORR of ≥25%. Up to 10 additional patients could be enrolled to ensure at least eight with MET amplification. Correlative studies included tumor MET amplification, MET signaling, pharmacokinetics and plasma biomarkers of foretinib activity. Results From March 2007 until October 2009, 74 patients were enrolled; 74% male; median age, 61 years (range, 25–88); 93% had received prior therapy. Best response was stable disease (SD) in 10 (23%) patients receiving intermittent dosing and five (20%) receiving daily dosing; SD duration was 1.9–7.2 months (median 3.2 months). Of 67 patients with tumor samples, 3 had MET amplification, one of whom had SD. Treatment-related adverse events occurred in 91% of patients. Rates of hypertension (35% vs. 15%) and elevated aspartate aminotransferase (23% vs. 8%) were higher with intermittent dosing. In both patients with high baseline tumor phospho-MET (pMET), the pMET:total MET protein ratio decreased with foretinib treatment. Conclusion These results indicate that few gastric carcinomas are driven solely by MET and VEGFR2, and underscore the diverse molecular oncogenesis of this disease. Despite evidence of MET inhibition by foretinib, single-agent foretinib lacked efficacy in unselected patients with metastatic gastric cancer. Trial Registration ClinicalTrials.gov NCT00725712

Metastatic progression of tumors is the major cause of death in patients with triple negative breast cancer (TNBC), the most aggressive subtype of breast cancer. However, since metastasis is a multi-step process, unraveling its complexity is a major challenge. One effective way of tackling this question is to study natural blockers of the metastatic process, metastasis suppressors, and identify the mechanisms by which they regulate metastasis. Raf kinase inhibitory protein (RKIP), a protein that regulates kinase activity, is a suppressor of TNBC metastasis. Although RKIP inhibits the activity of key kinases such as Raf-1, GRK2, NIK/IKK in cultured cells, the kinase targets of RKIP in tumors are not known. To address this question, we used a mass spectrometry approach involving inhibitor-conjugated beads to identify kinases that are down-regulated by RKIP in human TNBC xenograft tumors. Our results identified novel targets of RKIP whose kinase activity is either down-regulated or up-regulated by RKIP. We used bioinformatics analysis to build RKIP-regulated signaling networks based upon RKIP-induced changes in kinase activity and related gene expression. Elucidating RKIP function at a systems level reveals the interplay between key metastatic signaling cascades, particularly in relation to cell migration and invasion, and can potentially identify novel anti-metastatic target combinations in TNBCs. Citation Format: Ali E. Yesilkanal, Casey Frankenberger, Daniel C. Rabe, Gary L. Johnson, Marsha Rosner. Elucidating metastatic signaling networks in TNBC by investigating RKIP-regulated kinome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1620.

Triple-negative breast cancer (TNBC) patients have the highest risk of recurrence and metastasis. Because they cannot be treated with targeted therapies, and many do not respond to chemotherapy, they represent a clinically underserved group. While physiological inhibitors of metastasis (metastasis suppressors) play key roles in regulating tumor growth, invasion and metastasis, their role in regulating the tumor microenvironment and immune system is unknown. We hypothesized that the metastasis suppressor Raf Kinase Inhibitory Protein (RKIP) regulates stromal cells, which then affect tumor invasiveness. Using species-specific RNAseq we determined that expression of RKIP in tumors markedly reduces the number and metastatic potential of infiltrating TAMs. While TAMs isolated from TNBC xenografts drive in vitro invasion, RKIP+ derived TAMs did not drive invasion and had decreased secretion of pro-metastatic factors including SLPI, OPN, MMP-12, Galectin-3, VEGF-A, VEGF-D, TNFR2, and PGRN. We determined that RKIP regulates TAM recruitment by blocking HMGA2, which activates CCL5 expression. CCL5 rescued pro-metastatic TAM infiltration as well as tumor intravasation. We additionally showed that factors decreased in RKIP-derived TAMs were restored in CCL5-derived TAMs. CCL5 derived TAMs were also able to promote metastasis when co-injected with MDA-MB-231 tumors. These tumor cells demonstrated permanent increases in both growth and invasive potential after co-injection with highly pro-metastatic CCL5 derived TAMs. To determine the clinical utility of these TAM genes we combined their expression with RKIP signaling in the tumor to create a signature that strikingly separates TNBC patients based on outcome. Our results demonstrate for the first time that metastasis suppressors can regulate the microenvironment, regulating invasion through TAMs. Our results also suggest aggressive triple negative breast cancers could be controlled by attacking CCL5 derived TAMs crucial for promoting metastasis. Funded by: GM087630, CA184494, and CA192780 Citation Format: Daniel C. Rabe, Casey Frankenberger, Russell Bainer, Devipriya Sankarasharma, Kiran Chada, Thomas Krausz, Yoav Gilad, Lev Becker, Marsha R. Rosner. Metastasis suppressors regulate the tumor microenvironment by blocking recruitment of prometastatic TAMs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1557.

This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section (PR02) of the Conference Proceedings. Citation Format: Daniel C. Rabe, Casey Frankenberger, Russell Bainer, Devipriya Sankarasharma, Kiran Chada, Thomas Krausz, Yoav Gilad, Lev Becker, Marsha Rich Rosner. Metastasis suppressors regulate the tumor microenvironment by blocking recruitment of pro-metastatic tumor-associated macrophages. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A01.

s: AACR Special Conference on Tumor Metastasis; November 30-December 3, 2015; Austin, TX Metastatic progression of tumors is the major cause of death in patients with triple negative breast cancer (TNBC), the most aggressive subtype of breast cancer. However, since metastasis is a multi-step process, unraveling its complexity is a major challenge. One effective way of tackling this question is to study natural blockers of the metastatic process, metastasis suppressors, and identify the mechanisms by which they regulate metastasis. Raf kinase inhibitory protein (RKIP), a protein that regulates kinase activity, is a suppressor of TNBC metastasis. Although RKIP inhibits the activity of key kinases such as Raf-1, GRK2, NIK/IKK in cultured cells, the kinase targets of RKIP in tumors are not known. To address this question, we used a mass spectrometry approach involving inhibitor-conjugated beads to identify kinases that are down-regulated by RKIP in human TNBC xenograft tumors. Our results identified novel targets of RKIP whose kinase activity is either down-regulated or up-regulated by RKIP. We used bioinformatics analysis to build RKIP-regulated signaling networks based upon RKIP-induced changes in kinase activity and related gene expression. Elucidating RKIP function at a systems level reveals the interplay between key metastatic signaling cascades, particularly in relation to cell migration and invasion, and can potentially identify novel anti-metastatic target combinations in TNBCs. Citation Format: Ali E. Yesilkanal, Casey Frankenberger, Daniel C. Rabe, Gary L. Johnson, Marsha R. Rosner. Elucidating metastatic signaling networks in TNBC by investigating RKIP-regulated kinome. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B23.

Triple-negative breast cancer (TNBC) patients have the highest risk of recurrence and metastasis. Because they cannot be treated with targeted therapies, and many do not respond to chemotherapy, they represent a clinically underserved group. While physiological inhibitors of metastasis (metastasis suppressors) play key roles in regulating tumor growth, invasion and metastasis, their role in regulating the tumor microenvironment and immune system is unknown. We hypothesized that the metastasis suppressor Raf Kinase Inhibitory Protein (RKIP) regulates stromal cells, which then affect tumor invasiveness. Using species-specific RNAseq we determined that expression of RKIP in tumors markedly reduces the number and metastatic potential of infiltrating TAMs. While TAMs isolated from TNBC xenografts drive in vitro invasion, RKIP+ derived TAMs did not drive invasion and had decreased secretion of pro-metastatic factors including SLPI, OPN, MMP-12, Galectin-3, VEGF-A, VEGF-D, TNFR2, and PGRN. We determined that RKIP regulates TAM recruitment by blocking HMGA2, which activates CCL5 expression. CCL5 rescued pro-metastatic TAM infiltration as well as tumor intravasation. We additionally showed that factors decreased in RKIP-derived TAMs were restored in CCL5-derived TAMs. CCL5 derived TAMs were also able to promote metastasis when co-injected with MDA-MB-231 tumors. These tumor cells demonstrated permanent increases in both growth and invasive potential after co-injection with highly pro-metastatic CCL5 derived TAMs. To determine the clinical utility of these TAM genes we combined their expression with RKIP signaling in the tumor to create a signature that strikingly separates TNBC patients based on outcome. Our results demonstrate for the first time that metastasis suppressors can regulate the microenvironment, regulating invasion through TAMs. Our results also suggest aggressive triple negative breast cancers could be controlled by attacking CCL5 derived TAMs crucial for promoting metastasis. Citation Format: Daniel C. Rabe, Casey Frankenberger, Russell Bainer, Devipriya Sankarasharma, Kiran Chada, Thomas Krausz, Yoav Gilad, Lev Becker, Marsha Rich Rosner. Metastasis suppressors regulate the tumor microenvironment by blocking recruitment of pro-metastatic tumor-associated macrophages. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr PR02.

Renal or kidney cancer accounts for about 3% of all cancer cases reported each year in the U.S. Molecular signatures that define the cancer, such as the loss of functional VHL, are found in both sporadic and familial cases of cancer. In clear cell renal cancer, the transcription factor HIF-2α has been shown to have a distinct role in tumorigenesis. Our laboratories developed a cell-based screen to identify modulators of HIF-2α. Screening of the NCI's Natural Product Extract Repository resulted in the identification of 10 sponge extracts, from which 12 compounds were isolated. The biological evaluation of these compounds will be discussed including evaluation of HIF-1α vs HIF-2α selectivity and the isolated compounds' effects on mRNA from several pathways regulated by HIF.

Under normal conditions, hepatocyte growth factor (HGF)-induced activation of its cell surface receptor, the Met tyrosine kinase (TK), is tightly regulated by paracrine ligand delivery, ligand activation at the target cell surface, and ligand-activated receptor internalization and degradation. Despite these controls, HGF/Met signaling contributes to oncogenesis and tumor progression in several cancers and promotes aggressive cellular invasiveness that is strongly linked to tumor metastasis.The prevalence of HGF/Met pathway activation in human malignancies has driven rapid growth in cancer drug development programs. The authors review Met structure and function, the basic properties of HGF/Met pathway antagonists now in preclinical and clinical development, as well as the latest clinical trial results.Clinical trials with HGF/Met pathway antagonists show that as a class these agents are well tolerated. Although widespread efficacy was not seen in several completed Phase II studies, promising results have been reported in lung, gastric, prostate and papillary renal cancer patients treated with these agents. The main challenges facing the effective use of HGF/Met-targeted antagonists for cancer treatment are optimal patient selection, diagnostic and pharmacodynamic biomarker development, and the identification and testing of optimal therapy combinations. The wealth of basic information, analytical reagents, and model systems available concerning HGF/Met oncogenic signaling will continue to be invaluable in meeting these challenges and moving expeditiously toward more effective disease control.

A new trimethoxycinnamoyl-2-pyrrolidinone alkaloid, named langkamide (I), is isolated along with two known trimethoxycinnamoyl derivatives from roots and stems of the pepper shrub Piper sarmentosum.

The extraction of three active marine soft corals of the order Alcyonacea (Sarcophyton sp., L.

Under normal conditions, hepatocyte growth factor (HGF)-induced Met tyrosine kinase (TK) activation is tightly regulated by paracrine ligand delivery, ligand activation at the target cell surface, and ligand activated receptor internalisation and degradation. Despite these controls, HGF/Met signalling contributes to oncogenesis and tumour progression in several cancers and promotes aggressive cellular invasiveness that is strongly linked to tumour metastasis. The prevalence of HGF/Met pathway activation in human malignancies has driven rapid growth in cancer drug development programmes. Pathway inhibitors can be divided broadly into biologicals and low molecular weight synthetic TK inhibitors; of these, the latter now outnumber all other inhibitor types. We review here the basic properties of HGF/Met pathway antagonists now in preclinical and clinical development as well as the latest clinical trial results. The main challenges facing the effective use of HGF/Met-targeted antagonists for cancer treatment include optimal patient selection, diagnostic and pharmacodynamic biomarker development, and the identification and testing of optimal therapy combinations. The wealth of basic information, analytical reagents and model systems available concerning HGF/Met oncogenic signalling will continue to be invaluable in meeting these challenges and moving expeditiously toward more effective disease control.

The molecular interactions between cancer and stromal cells within the tumor microenvironment enable tumor invasion, intravasation, and metastasis at distant sites. However, the degree to which metastatic breast tumors reprogram stromal cells both locally and at distant mammary tissues is not well understood. To address this question, we used species-specific RNA sequencing in a mouse xenograft model to determine how the metastasis suppressor RKIP influences transcription in tumor and stroma tissues. Here we show that metastatic tumors prime mammary tissue at a distant site in a manner that reflects local stromal responses. In addition, gene expression in metastatic breast tumors is pervasively correlated with gene expression in local stroma of both mouse xenografts and human patients. Changes in local and distant stromal gene expression elicited by metastatic tumors are better predictors of subtype and patient survival than tumor gene expression, supporting the use of stromal-based strategies for the diagnosis and prognosis of breast cancer. One mechanism by which changes at contralateral distal mammary breast occur is through exosomes secreted by tumor cells. These results indicate that tumors prime contralateral mammary tissue in a manner that reflects local stromal changes and predicts metastatic disease. This study has future application to our understanding of contralateral breast cancer. Citation Format: Jiyoung Lee, Russell Bainer, Casey Frankenberger, Daniel Rabe, sadiq Saleh, Morag Park, Gary An, Yoav Gilad, Marsha Rich Rosner. Metastatic breast tumors regulate gene expression at distal mammary sites that predicts patient outcome. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B15.

Foretinib is an oral multi-kinase inhibitor targeting MET, vascular endothelial growth factor receptor (VEGFR)-2, RON, KIT, and AXL kinases. In this Phase 1, open-label, non-randomized study, foretinib was administered once daily at doses of 60 mg, 80 mg, 100 mg, or 120 mg for 28 days. The primary objectives were to determine the maximum tolerated dose (MTD) and assess the safety and tolerability of the daily oral administration schedule. Secondary objectives included pharmacokinetics, pharmacodynamics, and assessment of tumor response. Patients had histologically confirmed metastatic or unresectable solid tumors for which no standard treatments existed and all received oral foretinib once daily. Dose escalation was planned as a conventional "3+3" design with an expansion at the MTD for collection of additional safety and pharmacokinetic information. Thirty-seven patients were treated across four dose levels. The MTD was established as 80 mg foretinib. Dose-limiting toxicities were hypertension, dehydration, and diarrhea. The most common adverse events included fatigue, hypertension, nausea, and diarrhea. Twenty-three of 31 patients (74 %) had a best response of stable disease. No patient had a confirmed partial or complete response. At the MTD, steady state was achieved by approximately 2 weeks, with average post-dose time to maximum concentration, peak concentration, and trough concentration of 4 h, 46 ng/mL, and 24 ng/mL, respectively. In patients treated at the MTD, soluble MET and VEGF-A plasma levels significantly increased (P

Triple-negative breast cancer (TNBC) patients have the highest risk of recurrence and metastasis. Because they cannot be treated with targeted therapies, and many do not respond to chemotherapy, they represent a clinically underserved group. While physiological inhibitors of metastasis (metastasis suppressors) play key roles in regulating tumor growth, invasion and metastasis, their role in regulating the tumor microenvironment and immune system is unknown. We hypothesized that the metastasis suppressor Raf Kinase Inhibitory Protein (RKIP) regulates stromal cells, which then affect tumor invasiveness. Using species-specific RNAseq we determined that expression of RKIP in tumors markedly reduces the number and metastatic potential of infiltrating TAMs. While TAMs isolated from TNBC xenografts drive in vitro invasion, RKIP+ derived TAMs did not drive invasion and had decreased secretion of pro-metastatic factors including SLPI, OPN, MMP-12, Galectin-3, VEGF-A, VEGF-D, TNFR2, and PGRN. We determined that RKIP regulates TAM recruitment by blocking HMGA2, which activates CCL5 expression. CCL5 rescued TAM infiltration as well as tumor intravasation. We additionally showed that factors decreased in RKIP-derived TAMs were restored in CCL5-derived TAMs. Finally, we demonstrated the TAM secreted factor PGRN is both necessary and sufficient for TAM driven invasiveness. To determine the clinical utility of these TAM genes we combined their expression with RKIP signaling in the tumor to create a signature that strikingly separates TNBC patients based on outcome. Our results demonstrate for the first time that metastasis suppressors can regulate the microenvironment, regulating invasion through TAMs. Our results also suggest TNBC patients with decreased RKIP activity and increased TAM infiltration may respond to therapies targeting CCL5 or PGRN. Citation Format: Daniel C. Rabe, Casey Frankenberger, Russell Bainer, Devipriya Sankarasharma, Kiran Chada, Thomas Krausz, Yoav Gilad, Lev Becker, Marsha R. Rosner. Metastasis suppressors regulate the tumor microenvironment by blocking recruitment of pro-metastatic TAMs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5077. doi:10.1158/1538-7445.AM2015-5077

Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with very low survival rates. Metastatic progression of tumors is the major cause of death in TNBC patients. Metastatic spread of tumor cells is generally associated with resistance to therapy and poor prognosis. Therefore, there is a clinical need for understanding the molecular and cellular basis of metastasis and developing effective therapies against metastatic progression. However, since metastasis is a multi-step process, unraveling its complexity is a major challenge. One effective way of tackling this question is to study natural blockers of the metastatic process, metastasis suppressors, and identify the mechanisms by which they regulate metastasis. Raf kinase inhibitory protein (RKIP) is a metastasis suppressor in TNBC, which acts as a homeostatic factor that regulates kinase activity. RKIP blocks invasion in vitro and experimental metastasis in vivo. Therefore, RKIP constitutes a powerful tool for studying metastatic signaling pathways in cancer cells. RKIP is known to regulate the activity of certain kinases such as Raf-1, GRK2, NIK, and GSK3β, affecting major pathways such as MAPK, NFκB, and WNT. However, our preliminary data suggests that RKIP affects a much larger part of the TNBC kinome. Using proteomics approaches, we have identified putative novel kinase targets of RKIP, and used bioinformatics analysis to build RKIP-regulated signaling networks. Elucidating RKIP's function at a systems level will reveal the interplay between key metastatic signaling cascades and potentially identify novel anti-metastatic target combinations in TNBCs. Citation Format: Ali E. Yesilkanal, Casey Frankenberger, Daniel Rabe, Gary L. Johnson, Marsha R. Rosner. Regulation of TNBC kinome by the metastasis suppressor Raf kinase inhibitory protein (RKIP). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3251. doi:10.1158/1538-7445.AM2015-3251

Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer. While five-year survival rates have reached 98% in patients treated with anti-ER or anti-HER2 therapies, patients with TNBC have a five-year survival rate of only 24%. Currently, the only form of therapy for these patients is surgery and platinum based chemotherapy. However, patient outcome is generally poor. Additionally, this disease disproportionately affects African-American women and lower income women, with rates seen approximately three times higher in African-American women compared to the rest of the population. An alternative strategy for treating TNBC patients involves targeting the tumor stroma. To better understand interaction between the tumor and stroma necessary for metastasis and invasion, we employed a triple-negative breast cancer (TNBC) model in which the metastasis suppressor Raf Kinase Inhibitory Protein (RKIP) controls primary tumor invasiveness. RKIP expression, which converts invasive tumors to non-invasive tumors, dramatically inhibits macrophage infiltration. The mechanism, which is dependent on Let-7 suppression of HMGA2, involves decreased expression of the chemokine CCL5. Furthermore, overexpression of CCL5 partially rescued the infiltration of macrophages into the tumor and intravasation of tumor cells into the blood stream. The relationships of the genes in the RKIP, HMGA2, CCL5, and macrophage pathways were observed in multiple sets of expression array data from breast cancer patients. Regulation of macrophage infiltration was observed in tumors from an HMGA2 knockout mouse model. These results show that RKIP regulates macrophage recruitment by tumors and demonstrate for the first time that metastasis suppressor genes can regulate the tumor microenvironment. Citation Format: Daniel C. Rabe, Casey A. Frankenberger, Russell Bainer, Devipriya Sankarasharma, Kiran Chada, Thomas Krausz, Yoav Gilad, Marsha Rich Rosner. The role of tumor associated macrophages (TAMs) in triple-negative breast cancer (TNBC) invasion revealed by species-specific RNA sequencing. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B35. doi:10.1158/1538-7445.CHTME14-B35

Cancer progression is critically dependent on specific molecular interactions between tumor cells and their microenvironment, but little is known about the global dynamics of this crosstalk within and across individuals. Here, we used an RNA sequencing approach with species-of-origin information in a xenograft mouse model to computationally resolve and compare tumor versus stromal transcriptional changes. Our study employed a triple-negative breast cancer (TNBC) model in which primary tumor invasiveness is controlled by the metastasis suppressor Raf Kinase Inhibitory Protein (RKIP) allowing systemic observation of stromal response to invasive or noninvasive breast tumours at both proximal and distal sites. Initially, we observed that expression levels of gene homologs in human tumor and mouse stroma are highly synchronized. Surprisingly, the set of genes most prognostic for metastasis-free survival in multiple clinical data sets were those whose mRNA expression was inversely correlated between tumor and stroma. These results were confirmed in an independent set of microarray data from tumor and stroma micro-dissected from primary human breast cancer patient tissue. Additionally, we found broad-based and invasion-specific gene expression changes in stromal tissues far from the primary lesion, with these results being confirmed in an independent set of samples. Of note, genes differentially expressed in the tumor-associated stroma were better classifiers of tumor phenotype than genes differentially expressed in the tumor alone. As well, stromal genes differentially expressed both locally and distally, are significant predictors of patient prognosis. These findings demonstrate the potential for using patient tissue remote from the primary tumor as indicators of disease aggressiveness. Furthermore, these results suggest that stromal gene expression, acting to compensate for changes in the tumor, may have both prognostic and therapeutic implications. Citation Format: Casey A. Frankenberger, Russell O. Bainer, Daniel C. Rabe, Sadiq Saleh, Morag Park, Yoav Gilad, Marsha R. Rosner. Systemic tumor-stroma interactions are prognostic indicators of breast tumor invasiveness. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A43. doi:10.1158/1538-7445.CHTME14-A43

A new trimethoxycinnamoyl-2-pyrrolinone alkaloid, langkamide (1), along with the known compounds piplartine (2) and 3,4,5-trimethoxycinnamic acid (3) were isolated from the roots and stems of the shrub Piper sarmentosum Roxb. The structures were established by spectroscopic analyses and comparison of their spectral data with values reported in the literature. The compounds were tested for their ability to modulate hypoxia inducible factor-2 (HIF-2) transcription activity and all three showed HIF-2 inhibitory activity with EC50 values of 14.0, 4.8, and 60.6 μM, respectively, for compounds 1, 2, and 3.

Signal Transduction via the hepatocyte growth factor (HGF)/Met receptor kinase pathway is critical for normal embryogenesis and adult homeostasis, and aberrant HGF/Met signaling contributes to tumorigenesis and metastasis in many prevalent forms of cancer. By defining the basic molecular mechanisms of pathway activation at the target cell surface, we hope to develop novel strategies to antagonize oncogenic signaling as well as methods to assess pathway status in cancer patients. These methods may help identify those most likely to benefit from HGF/Met targeted therapies and help monitor treatment response. Distinct domains of HGF bind to the Met receptor tyrosine kinase and to cell-surface heparan sulfate (HS) proteoglycans. The latter are widely expressed, structurally diverse biopolymers that modulate many important protein-protein interactions, but their importance in eliciting HGF biological responses is controversial. We have identified basic (positively charged) amino acid residues in HGF that form the primary binding site for (negatively charged) HS proteoglycans. HS binding by these residues increases the affinity of ligand-receptor binding and, with HS polymers of sufficient length, facilitates ligand oligomerization. Ligand oligomerization, in turn, controls Met kinase activation, cell motility and proliferation under normal conditions. Opposite (negative) charge amino acid substitutions at these critical HS binding residues in HGF can mimic bound HS in promoting high affinity receptor binding, but antagonize the binding of long HS polymers on the cell surface, thereby antagonizing ligand oligomerization and its consequences. Engineering these substitutions in the context of a short HGF isoform containing only the primary HS and Met binding domains yields a potent and selective competitive inhibitor of oncogenic HGF signaling in vivo. These findings further define the role of HS in growth factor signaling and reveal a novel strategy for antagonist development that may be applicable to other HS-binding growth factors. The Met receptor kinase is among many transmembrane proteins that are proteolytically released from the cell surface by a process known as ectodomain shedding. Consistent with the concept that Met ectodomain shedding is a normal physiological process, we found that healthy human volunteers (n > 100) display soluble Met (sMet) plasma concentrations in the range of 100 - 300 ng/ml and urinary sMet concentrations that are approximately 1000-fold lower. We hypothesized that aberrantly high Met expression, a frequent occurrence in many forms of cancer, and/or dysregulated proteolytic activity, another common feature of aggressive malignancies, could lead to increased ectodomain shedding and that sMet levels could be a useful biomarker of tumorigenesis, tumor progression and overall tumor burden. In prior studies we found that increased Met shedding correlated with malignancy in several cell lines, and that urinary sMet levels were significantly elevated in metastatic prostate cancer patients relative to those with organ-confined cancer or no evidence of disease. In an ongoing investigation of sMet as a diagnostic and/or prognostic biomarker of bladder cancer, the mean urinary sMet level among 150 patients with pathologically confirmed disease was significantly higher than that obtained from a group of 50 individuals with no evidence of disease, whether mean values were normalized to urine creatinine concentration or not (two-tailed Mann-Whitney t-test p-value We have also measured plasma sMet levels in patients participating in a phase II clinical trial of Foretinib (formerly GSK1363089/XL880), a small molecule multikinase inhibitor targeting Met and vascular endothelial cell growth factor-2 (VEGFR-2) Axl and Ron, designed to evaluate the safety and efficacy of 2 dosing schedules (continuous daily dosing and intermittent 5 days on/9 days off dosing) of foretinib as a single therapeutic agent in patients with metastatic gastric carcinoma (GC). Plasma sMet concentrations, as well as plasma VEGF-A, HGF and soluble VEGFR-2 (sVEGFR-2) levels were measured before and during treatment with foretinib to measure potential pharmacodynamic (PD) effects and/or the modulation by foretinib of Met and VEGFR-2 pathways. Marker changes from baseline were analyzed at days 5, 15, 29 and 47 using analysis of variance and their relationships with plasma concentrations of foretinib (PK) and clinical outcome (sum of longest diameter [SLD] of tumor as a surrogate for tumor shrinkage, progression-free survival [PFS], and RECIST) were also examined. In GC patients on the intermittent dosing schedule, plasma sMet and VEGF-A concentrations tended to increase during the treatment periods compared with the drug holidays, suggestive of a direct short-term drug effect on sMet and VEGF-A levels. Although mean tumor burden did not change significantly from baseline to week 8, plasma levels of sMet and VEGF-A correlated positively with the change in SLD over this period, suggesting that the increases of sMet and VEGF-A may be reflective of increased GC tumor burden. Citation Format: Fabiola Cecchi, Deborah Pajalunga, Brian McNeil, Daniel Rabe, Andrew Fowler, Yuan Liu, C. Robert Gagnon, Howard Kallender, Manish A. Shah, Robert Getzenberg, Mark Schoenberg, Anne-Marie Martin, Ramaprasad Srinivasan, W. Marston Linehan, R Andrew Byrd, Donald P. Bottaro. Oncogenic signal transduction via the hepatocyte growth factor/Met receptor kinase pathway [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY23-03

Clear cell renal cell carcinoma (RCC) is the most prevalent form of kidney cancer and is frequently associated with loss of von Hippel Lindau (VHL) gene function, resulting in the aberrant transcriptional activation of hypoxia response genes that contribute to tumor growth, angiogenesis and metastasis. Prior studies have shown that normoxic stabilization of hypoxia inducible factor (HIF)-1-alpha alone, while capable of mimicking some aspects of VHL loss, are not sufficient to reproduce tumorigenesis, indicating that it is not the most critical oncogenic substrate of VHL protein. To isolate compounds that selectively modulate HIF2-alpha for use as research tools and drug development leads, a cell-based high throughput screen of the NCI Natural Products Repository was performed. Stably transformed clones of the human renal clear cell carcinoma cell line 786-0 expressing a luciferase reporter construct under the control of the human vascular endothelial growth factor (VEGF) promoter-enhancer were used to identify natural product samples with inhibitory activity and minimal cellular toxicity. Leads from initial high throughput screen were chromatographically separated into component structures yielding several pure compounds with micromolar or submicromolar IC50 values, >80% inhibition and 90% inhibition of VEGF secretion at micromolar doses under normoxic conditions. Studies to establish HIF selectivity and effects on RCC cell proliferation, survival, motility and anchorage independent growth are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 773.

Hepatocyte growth factor (HGF) stimulates cell proliferation, motility, and morphogenesis upon binding to the receptor tyrosine kinase Met and cell surface heparan sulfate (HS) glycans. NK1 is a truncated HGF isoform consisting of the N-terminal (N) and first kringle (K1) domains of full-length HGF that stimulates all major HGF biological activities. Within NK1, the N domain contains the HS binding site, while K1 contains the primary determinants of Met binding. To further define the role of HS in ligand-receptor interaction and biological signaling, we identified the primary HS binding residues in N domain (K60, K62, R73) and generated opposite charge mutations at these positions in NK1. Nuclear magnetic resonance analysis of recombinantly expressed mutant and wild type (WT) proteins revealed that both were folded normally. Mutant proteins displayed diminished binding to immobilized HS relative to WT, and the triple mutant protein (3M) also lacked intrinsic motogenic and mitogenic activity. Interestingly, 3M NK1 maintained Met binding and competitively inhibited DNA synthesis stimulated by WT NK1 as well as full-length HGF in normal cells. Expression plasmids encoding 3M and WT NK1 proteins were transfected into the glioblastoma cell U87 MG, which is dependent on HGF/Met autocrine signaling for tumorigenicity in mice. 3M/U87 MG cells displayed a substantially lower rate of growth relative to WT/U87 MG or the parental cell line, suggestive of growth antagonism by 3M NK1. As anticipated, expression of WT NK1 significantly accelerated the growth of U87 MG tumor xenografts in mice, which reached maximal volume (1 ml) by 22 days post-implantation, compared to 45 days required by the parental cell line. In contrast, tumor growth in animals injected with 3M/U87 MG was dramatically impaired: at 60 days post implantation, average tumor volume for this group was < 0.3 ml. Because both WT and 3M transfectants expressed equal amounts of their respective recombinant proteins, the HS binding site mutations delayed measurable tumor growth from one week to two months. Thus HS binding is critical for HGF-driven mitogenesis and tumor growth. We now show that HGF signaling requires specific HS binding interactions and that targeted disruption of these HS binding sites in HGF yields a potent and selective competitive inhibitor of normal and oncogenic HGF signaling. Our findings further define the role of HS in growth factor signaling and reveal a novel strategy for antagonist development which may be applicable to other HS-binding growth factors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 342.

Foretinib (formerly GSK1363089) is a small molecule multikinase inhibitor which includes inhibition of MET and VEGFR2. The ongoing phase II study, MET111643, is evaluating the safety and efficacy of 2 dosing schedules (continuous daily dosing and intermittent 5 days on/9 days off dosing) of foretinib as a single agent in pts with metastatic GC. An additional component of the study is to collect patient plasma samples before and during treatment with foretinib, to explore the potential pharmacodynamic effect and/or the modulation by foretinib of MET and VEGFR2. Plasma samples were collected from 42 pts on the intermittent 5 days on/9 off foretinib schedule at baseline and prior to dosing on days 5, 15, 29, and 47. The plasma levels of sMET, HGF, sVEGFR2, and VEGFA were measured using the Meso Scale Discovery platform (MSD). These marker changes from baseline were analyzed at each time point using analysis of variance and their relationships with plasma concentrations of foretinib (PK) and clinical outcome (progression free survival [PFS] and RECIST response) were also examined. Statistically significant increases from baseline of sMET at days 5, 15, 29, and 47 (p Changes in sMET, VEGFA, and sVEGFR2 levels were observed for both intermittent and daily dosing schedules in the gastric cancer study. Although SLD at 8 weeks did not change significantly from baseline in the GC study, plasma levels of sMET and VEGFA correlated positively with the week 8 magnitude of SLD. Hence plasma levels of sMET and VEGFA may reflect biological changes following foretinib treatment. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B210.

Foretinib is a potent, orally available, small-molecule inhibitor of MET and VEGFR2. Significant tumor cell growth inhibition was observed preclinically following treatment in multiple tumor models. Antitumor activity was observed in a previous phase I study in which foretinib was dosed intermittently on days 1–5 every 14 days. This phase I dose-escalation study to evaluate daily oral administration of foretinib was conducted in adults with solid tumors. Foretinib was orally administered until disease progression or toxicity mandated removal from the study. Dose-limiting toxicities (DLTs) that occurred during the first 28 days of treatment were used to determine the maximum tolerated dose (MTD). Safety, tolerability, pharmacodynamics (PD), and pharmacokinetics (PK) were evaluated. Tumor volume was assessed every 8 weeks by RECIST. A total of 37 patients (pts) were treated across 4 dose levels in the following order: 60 mg/d (6 pts), 80 mg/d (12 pts), 120 mg/d (3 pts), 100 mg/d (3 pts), and 80 mg/d (13 pts, PK expansion). Reported DLTs were hypertension and dehydration at 120 mg/d and diarrhea at 100 mg/d. The MTD of foretinib was determined to be 80 mg. With chronic dosing, 12 of 25 pts (48%) on 80 mg required a dose reduction to 60 mg between 21 days and 4 months on treatment. Frequent AEs associated with foretinib were hypertension (62%), fatigue (51%), nausea (43%), diarrhea (41%), proteinuria (30%), increased lactate dehydrogenase (27%), vomiting (24%), anorexia (22%), increased aspartate transaminase (19%), rash (16%), increased GGT (16%), and increased lipase (16%), primarily CTCAE grades 1 and 2. The foretinib plasma PK (at MTD) was characterized by a tmax of ≈4 hours, a steady-state oral clearance (CL/F) of ≈83 L/h, and ≈3.8-fold accumulation at steady state (achieved by ≈day 15). At steady state, values of Cmax and AUC0–24 were ≈45.7 ng/mL and ≈805 h · ng/mL, respectively, and the Cmin was ≈52% of Cmax. Plasma PD markers were measured during cycle 1 in 19 pts treated at the MTD. Shed Met (sMET) and VEGF showed increases, whereas sVEGFR2 showed a decrease. No pts had confirmed partial response or complete response, but stable disease (SD) (range, 2.1–18.1 months) was seen in 23 pts (74.2%). 11 pts had tumor regression from baseline, with tumor shrinkage ranging from 1%–21%. Disease progression was seen in 8 pts (25.8%). Overall, 35.5% of pts were event-free at 6 months, and 12.9% of pts (diagnoses included medullary thyroid, hepatocellular, and papillary renal cell carcinomas, and alveolar soft part sarcoma) were event-free at 12 months. 62% percent of pts withdrew due to progressive disease, 8% due to AEs, 5% due to death, and 24% due to consent withdrawal/investigator discretion/other. Foretinib can be safely administered at the oral daily dose of 80 mg (MTD); however, with chronic administration, dose reductions were not uncommon. The safety profile observed with daily dosing of foretinib is similar to that seen with intermittent dosing. Plasma PK results indicate that exposure to foretinib was well maintained over the daily dosing interval. PD results are similar to that seen with intermittent dosing and indicate that sMET, VEGF, and sVEGFR2 are promising markers for monitoring biological activity of foretinib. Prolonged SD and tumor regression in pts with various cancers suggest that foretinib has promise as an anticancer therapy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A8.

Hepatocyte growth factor (HGF) stimulates cell proliferation, motility, and morphogenesis upon binding to the receptor tyrosine kinase Met and cell surface heparan sulfate (HS) glycans. NK1 is a truncated HGF isoform consisting of the N-terminal (N) and first kringle (K1) domains of full-length HGF that stimulates all major HGF biological activities. Within NK1, the N domain contains the HS binding site, while K1 contains the primary determinants of Met binding. To further define the role of HS in ligand-receptor interaction and biological signaling, we identified the primary HS binding residues in N domain (K60, K62, R73) and generated opposite charge mutations at these positions in NK1. Nuclear magnetic resonance analysis of recombinantly expressed mutant and wild type (WT) proteins revealed that both were folded normally. Mutant proteins displayed diminished binding to immobilized HS relative to WT, and the triple mutant protein (3M) also lacked intrinsic motogenic and mitogenic activity. Interestingly, 3M NK1 maintained Met binding and competitively inhibited DNA synthesis stimulated by WT NK1 as well as full-length HGF in normal cells. Expression plasmids encoding 3M and WT NK1 proteins were transfected into the glioblastoma cell U87 MG, which is dependent on HGF/Met autocrine signaling for tumorigenicity in mice. 3M/U87 MG cells displayed a substantially lower rate of growth relative to WT/U87 MG or the parental cell line, suggestive of growth antagonism by 3M NK1. As anticipated, expression of WT NK1 significantly accelerated the growth of U87 MG tumor xenografts in mice, which reached maximal volume (1 ml) by 22 days post-implantation, compared to 45 days required by the parental cell line. In contrast, tumor growth in animals injected with 3M/U87 MG was dramatically impaired: at 60 days post implantation, average tumor volume for this group was < 0.3 ml. Because both WT and 3M transfectants expressed equal amounts of their respective recombinant proteins, the HS binding site mutations delayed measurable tumor growth from one week to two months. Thus HS binding is critical for HGF-driven mitogenesis and tumor growth, and inhibition of these activities by 3M NK1 validates a novel strategy for developing antagonists of the HGF pathway and potentially those of other HS-binding ligands Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B228.