Irene M. Ogden

Circulating giant macrophages as a potential biomarker of solid tumors

Significance Using microfiltration as a liquid biopsy for the recovery of circulating tumor cells (CTCs) has revealed an accompanying macrophage subset that we use as a highly sensitive biomarker for solid tumors. We supply evidence that this circulating giant cell is a subset of disseminated tumor-associated macrophages capable of binding CTCs in peripheral blood of cancer patients. The presence of this cell expands the concept of using a liquid biopsy not only to indicate cancer presence but also to track cancer treatment effects sequentially using other circulating blood cells. Further, we supply observational evidence hypothesizing a metastasis pathway model in which CTCs migrate with pro-angiogenic macrophages, linking cancer cell intravasation, migration, and extravasation and the formation of metastatic microenvironments. Tumor-associated macrophages (TAMs) derived from primary tumors are believed to facilitate circulating tumor cell (CTC) seeding of distant metastases, but the mechanisms of these processes are poorly understood. Although many studies have focused on the migration of CTCs, less attention has been given to TAMs that, like CTCs, derive from tumor sites. Using precision microfilters under low-flow conditions, we isolated circulating cancer-associated macrophage-like cells (CAMLs) from the peripheral blood of patients with breast, pancreatic, or prostate cancer. CAMLs, which are not found in healthy individuals, were found to express epithelial, monocytic, and endothelial protein markers and were observed bound to CTCs in circulation. These data support the hypothesis that disseminated TAMs can be used as a biomarker of advanced disease and suggest that they have a participatory role in tumor cell migration.

Androgen Receptor-Independent Function of FoxA1 in Prostate Cancer Metastasis

FoxA1 (FOXA1) is a pioneering transcription factor of the androgen receptor (AR) that is indispensible for the lineage-specific gene expression of the prostate. To date, there have been conflicting reports on the role of FoxA1 in prostate cancer progression and prognosis. With recent discoveries of recurrent FoxA1 mutations in human prostate tumors, comprehensive understanding of FoxA1 function has become very important. Here, through genomic analysis, we reveal that FoxA1 regulates two distinct oncogenic processes via disparate mechanisms. FoxA1 induces cell growth requiring the AR pathway. On the other hand, FoxA1 inhibits cell motility and epithelial-to-mesenchymal transition (EMT) through AR-independent mechanism directly opposing the action of AR signaling. Using orthotopic mouse models, we further show that FoxA1 inhibits prostate tumor metastasis in vivo. Concordant with these contradictory effects on tumor progression, FoxA1 expression is slightly upregulated in localized prostate cancer wherein cell proliferation is the main feature, but is remarkably downregulated when the disease progresses to metastatic stage for which cell motility and EMT are essential. Importantly, recently identified FoxA1 mutants have drastically attenuated ability in suppressing cell motility. Taken together, our findings illustrate an AR-independent function of FoxA1 as a metastasis inhibitor and provide a mechanism by which recurrent FoxA1 mutations contribute to prostate cancer progression.

Mitogen-activated protein kinase kinase 4 (MAP2K4) promotes human prostate cancer metastasis

Prostate cancer (PCa) is the second leading cause of cancer death in the US. Death from PCa primarily results from metastasis. Mitogen-activated protein kinase kinase 4 (MAP2K4) is overexpressed in invasive PCa lesions in humans, and can be inhibited by small molecule therapeutics that demonstrate favorable activity in phase II studies. However, MAP2K4s role in regulating metastatic behavior is controversial and unknown. To investigate, we engineered human PCa cell lines which overexpress either wild type or constitutive active MAP2K4. Orthotopic implantation into mice demonstrated MAP2K4 increases formation of distant metastasis. Constitutive active MAP2K4, though not wild type, increases tumor size and circulating tumor cells in the blood and bone marrow. Complementary in vitro studies establish stable MAP2K4 overexpression promotes cell invasion, but does not affect cell growth or migration. MAP2K4 overexpression increases the expression of heat shock protein 27 (HSP27) protein and protease production, with the largest effect upon matrix metalloproteinase 2 (MMP-2), both in vitro and in mouse tumor samples. Further, MAP2K4-mediated increases in cell invasion are dependent upon heat shock protein 27 (HSP27) and MMP-2, but not upon MAP2K4s immediate downstream targets, p38 MAPK or JNK. We demonstrate that MAP2K4 increases human PCa metastasis, and prolonged over expression induces long term changes in cell signaling pathways leading to independence from p38 MAPK and JNK. These findings provide a mechanistic explanation for human studies linking increases in HSP27 and MMP-2 to progression to metastatic disease. MAP2K4 is validated as an important therapeutic target for inhibiting human PCa metastasis.

An orthotopic murine model of human prostate cancer metastasis.

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

Abstract 1448: Identifying and subtyping circulating tumor cells from breast, prostate, and pancreatic cancer patients based on distinct morphology.

Background: Isolation of circulating tumor cells (CTCs) from peripheral blood based on size exclusion is rapid and straight-forward using precision microfilters. We describe the use of CellSieve TM microfilters to isolate CTCs from the peripheral blood of breast, prostate, and pancreatic cancer patients. It is accepted that CTCs isolated from patient samples represent a highly heterogeneous population with varying degrees of epithelial mesenchymal differentiation. We hypothesized that the CTCs from three different epithelial malignancies can be identified and grouped into distinct subtypes by morphological characterization. Methods: Prostate, breast, and pancreatic patient blood samples were provided by Northwestern University, Fox Chase Cancer Center, University of Maryland, and Medical College of Wisconsin and analyzed by Creatv MicroTech. The CellSieve TM microfilters have 8 micron diameter pores in a uniform array, with 160,000 pores in a 9 mm diameter area. 7.5 mL of whole blood was diluted in fixative and drawn through a microfilter. CTCs collected by this size exclusion technique were post-fixed, permeabilized, and stained with DAPI, cytokeratin 8, 18 and 19 (FITC), EpCAM (PE), PSMA (Texas Red), and CD45 (Cy5). CTCs were CD45 negative cells identified by their morphology, nuclear profile, and expression of cytokeratin, PSMA, and EpCAM. Results: Each patient sample was found to have a number of phenotypic CTC subtypes. Distinct morphological patterns emerged in the three malignancies. CTCs from breast cancer patients demonstrated high expression of cytokeratin signal with web-like cytokeratin filamentation. Prostate cancer CTCs had less defined filamentation, but intense PSMA and cytokeratin signal and mottled cytokeratin morphology. Pancreatic CTCs had extremely fine filamentation, with spindle-like morphology and little or no EpCAM expression. Within each cancer, CTCs could be grouped into distinct subtypes. Additional markers, such as vimentin (PE), are used to further analyze the cells after bleaching the original PE. Conclusions: In addition to enumeration and identification, the phenotypic analysis of CTCs provides new information that can be used to characterize disease status for personalized treatment of cancer patients. We have shown that CTCs can have multiple distinct phenotypes. These phenotypic morphologies may implicate definable traits which can be exploited while tracking site directed treatment of metastatic cancer patients. Citation Format: Daniel Adams, R. Katherine Alpaugh, Massimo Cristofanilli, Stuart Martin, Saranya Chumsri, Monica Charpentier, Raymond C. Bergan, Irene May Ogden, Susan Tsai, Peixuan Zhu, Olga V. Makarova, Shuhong Li, Platte T. Amstutz, Cha-Mei Tang. Identifying and subtyping circulating tumor cells from breast, prostate, and pancreatic cancer patients based on distinct morphology. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1448. doi:10.1158/1538-7445.AM2013-1448

Abstract 5331: The effect of heat shock protein 27 (HSP27) on prostate cancer cell adhesion, invasion and metastasis

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Prostate cancer (PCa) is the most commonly diagnosed form of cancer among American men, and the second leading cause of all cancer-related deaths. PCa death is caused by the process of metastasis. In order to metastasize, tumor cells must acquire a series of phenotypic alterations, collectively known as the metastatic cascade. We have demonstrated that the small heat-shock protein, HSP27, affects key early steps of the metastatic cascade, and therefore may be a regulator of human PCa metastasis. HSP27 protein expression increases during PCa progression. It is phosphorylated in response to signaling from transforming growth factor-β (TGF-β), which in turn increases matrix metalloproteinase 2 (MMP-2) and cell invasion in vitro. We now demonstrate that HSP27 does not affect human PCa cell adhesion, cell detachment or cell migration, all of which contribute to the composite function of cell invasion. We went on to show that proteolysis by MMPs is necessary for HSP27-driven cell invasion by demonstrating that it was abrogated Marimastat, a broad-spectrum MMP inhibitor. By orthotopically implanting HSP27 variant human PCa cell lines, we demonstrated that HSP27 increases metastasis in a murine model. We also found that HSP27 increased tumor size. HSP27 increases human PCa metastasis and tumor growth in vivo. These findings provide a mechanistic explanation for the poor prognosis associated with increased HSP27 expression in human prostate tissue. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5331. doi:1538-7445.AM2012-5331

Abstract 3277: Increased mitogen-activated protein kinase kinase 4 (MEK4) induces metastasis in an orthotopic murine model of prostate cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Mitogen-activated protein kinase kinase 4 (MEK4) is a dual-specificity kinase that has been implicated in prostate cancer (PCa) progression. Increased MEK4 expression is observed in invasive cancer lesions in human prostate tissue. We have demonstrated the clinical importance of this in vitro, as MEK4 increases human PCa invasion. This phenotype is driven by increased protease production, and not via changes in cell migration. However, it is not known whether MEK4 regulates human PCa metastasis. This is of high importance as MEK4 has shown differential effects in various cancer models in a cell type specific fashion, and may therefore represent an important human PCa specific target. To emulate the situation of sustained altered MEK4 expression in humans, we engineered MEK4 clonal variant cell lines, generating multiple clones each for vector control (VC), increased wild type (WT), and increased constitutively-active mutant MEK4 (CA). Using a murine orthotopic implantation model designed to characterize in vivo behavior, we implanted 75 mice with these clones and demonstrated that both WT and CA MEK4 increase human PCa metastasis to the lung, a clinically relevant site in humans. In the primary tumor, interestingly, WT tumors were not increased compared to VC, while CA tumors were. Additionally, increased MMP-2 and MMP-10 were observed in both WT and CA tumors. However, MMP-9 was increased in WT tumors only, and MMP-13 in CA tumors only. Currently, we are expanding our efforts at characterizing additional molecular changes in primary tumor between these groups. Outside of the primary tumor, we isolated circulating tumor cells in the blood and bone marrow. We are currently characterizing their relevant cellular and molecular parameters. Using a clinically relevant murine model we have shown that increased MEK4 increases human PCa metastasis to the lung and protease production within the primary tumor. Further, a constitutively active phenotype drives tumor growth and circulating tumor cell formation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3277. doi:1538-7445.AM2012-3277

732 GENISTEIN INHIBITS PROSTATE CANCER METASTASIS BY PREVENTING CELL DETACHMENT

all regulated by high fat diet. 4 genes (GPx3, Crabp1, Cyp2b10 and Yipf5) were either up regulated and down regulated more than 2-fold in VP by the high fat diet. Although in most cases they showed the same trends, none of these genes showed statistically significant changes when DLP was analyzed by microarray analysis. In the validation, GPx3 mRNA levels were decreased by approximately 2-fold by high fat diet in not only in VP, but also in DLP and AP. In human nontransformed prostate cells (PrSC, PrEC and BPH-1), cholesterol loading decreased GPx3 expression, and increased H2O2 levels of culture medium. When GPx3 expression was reduced by a siRNA transfection, H2O2 was found to be increased in cell culture media of PrSC and BPH-1 cells, consistent with the known role of GPx3 in lowering H2O2 levels. Troglitazone increased GPx3 expression in 3 normal prostate cells, and decreased H2O2 levels. In addition, troglitazone attenuated cholesterol-induced H2O2 increase. Tissue from prostate cancer biopsies had decreased GPx3 mRNA and was inversely related to the Gleason score. CONCLUSIONS: High fat diet alters pathways related to many genes concerned with the increase of oxidative stress. GPx3, a gene identified by this analysis, was found to be down regulated by high fat diet and appears be decreased as well in human prostate cancers, suggesting that GPx3 may have a possible role in modulating carcinogenesis.

Abstract 1407: Mitogen-activated protein kinase kinase 4 (MEK4) is a key regulator of prostate cancer progression

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Mitogen-activated protein kinase kinase 4 (MEK4) is a dual-specificity kinase that has been implicated in cancer progression in multiple cancer types, including prostate cancer (PCa). MEK4 is upregulated in invasive PCa lesions in human tissue. We hypothesize that increased MEK4 promotes PCa invasion and metastasis. Our group has created PC3-M cells stably transfected with either constitutively-active or increased levels of wild-type MEK4. In a Matrigel Boyden chamber assay, constitutively-active or wild-type MEK4 was shown to increase PCa invasion. Cancer cell invasion requires the coupling of cell migration with increased production of proteases. We hypothesized that MEK4 promoted invasion via production of proteases, but not via cell migration. Using an uncoated boyden chamber, MEK4 was shown not to affect migration. Using quantitative real time polymerase chain reaction (qRT/PCR), we went on to demonstrate that MEK4 increased MMP-2 and MMP-9 transcript expression, but did not affect MMP-10 expression. In addition to these studies, we have also looked at the effect of increased or constitutively-active MEK4 on an orthotopic mouse model of PCa. In this model, PCa cells are injected into the prostates of nude mice and tumor size, circulating tumor cells, and lung metastasis are quantified. Preliminary data shows that constitutively-active MEK4 causes an increase in primary tumor size as well as an increase in circulating tumor cells in both the blood and the bone marrow. In summary, we demonstrated that chronic high expression of MEK4 increases human PCa invasion, that this is not due to increased migration, but is associated with increases in MMP-2 and MMP-9. Further, constitutively-active MEK4 appears to have different biological effects than high levels of wild type MEK4 in vivo. Ongoing studies are evaluating the effect of wild type MEK4 and of constitutively-active MEK4 on the formation of distant soft tissue metastasis in the above murine model. In related studies we are seeking to identify downstream proteins critical for MEK4s effects on cell invasion and metastasis both in vitro and in vivo. We are also seeking to examine the effects of sustained MEK4 knockdown on PCa invasion and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1407. doi:10.1158/1538-7445.AM2011-1407