James R. Hernandez

RB Loss Promotes Prostate Cancer Metastasis

RB loss occurs commonly in neoplasia but its contributions to advanced cancer have not been assessed directly. Here we show that RB loss in multiple murine models of cancer produces a prometastatic phenotype. Gene expression analyses showed that regulation of the cell motility receptor RHAMM by the RB/E2F pathway was critical for epithelial-mesenchymal transition, motility, and invasion by cancer cells. Genetic modulation or pharmacologic inhibition of RHAMM activity was sufficient and necessary for metastatic phenotypes induced by RB loss in prostate cancer. Mechanistic studies in this setting established that RHAMM stabilized F-actin polymerization by controlling ROCK signaling. Collectively, our findings show how RB loss drives metastatic capacity and highlight RHAMM as a candidate therapeutic target for treating advanced prostate cancer. Cancer Res; 77(4); 982-95. ©2016 AACR.

A phased strategy to differentiate human CD14+monocytes into classically and alternatively activated macrophages and dendritic cells

There are currently several in vitro strategies to differentiate human CD14(+) monocytes isolated from peripheral blood mononuclear cells (PBMCs) into the M1 or M2 macrophage cell types. Each cell type is then verified using flow cytometric analysis of cell-surface markers. Human CD14(+) monocytes have the potential to differentiate into M1 and M2 macrophages, both of which demonstrate varying degrees of cell-surface antigen overlap. Using multiple surface markers with current macrophage polarization protocols, our data reveal several limitations of currently used methods, such as highly ambiguous cell types that possess cell-surface marker overlap and functional similarities. Utilizing interleukin-6 (IL-6) and two phases of cytokine exposure, we have developed a protocol to differentiate human monocytes into M1, M2, or dendritic cells (DCs) with greater efficiency and fidelity relative to macrophages and DCs that are produced by commonly used methods. This is achieved via alterations in cytokine composition, dosing, and incubation times, as well as improvements in verification methodology. Our method reliably reproduces human in vitro monocyte-derived DCs and macrophage models that will aid in better defining and understanding innate and adaptive immunity, as well as pathologic states.

A bioinformatics approach reveals novel interactions of the OVOL transcription factors in the regulation of epithelial – mesenchymal cell reprogramming and cancer progression

BackgroundMesenchymal to Epithelial Transition (MET) plasticity is critical to cancer progression, and we recently showed that the OVOL transcription factors (TFs) are critical regulators of MET. Results of that work also posed the hypothesis that the OVOLs impact MET in a range of cancers. We now test this hypothesis by developing a model, OVOL Induced MET (OI-MET), and sub-model (OI-MET-TF), to characterize differential gene expression in MET common to prostate cancer (PC) and breast cancer (BC).ResultsIn the OI-MET model, we identified 739 genes differentially expressed in both the PC and BC models. For this gene set, we found significant enrichment of annotation for BC, PC, cancer, and MET, as well as regulation of gene expression by AP1, STAT1, STAT3, and NFKB1. Focusing on the target genes for these four TFs plus the OVOLs, we produced the OI-MET-TF sub-model, which shows even greater enrichment for these annotations, plus significant evidence of cooperation among these five TFs. Based on known gene/drug interactions, we prioritized targets in the OI-MET-TF network for follow-on analysis, emphasizing the clinical relevance of this work. Reflecting these results back to the OI-MET model, we found that binding motifs for the TF pair AP1/MYC are more frequent than expected and that the AP1/MYC pair is significantly enriched in binding in cancer models, relative to non-cancer models, in these promoters. This effect is seen in both MET models (solid tumors) and in non-MET models (leukemia). These results are consistent with our hypothesis that the OVOLs impact cancer susceptibility by regulating MET, and extend the hypothesis to include mechanisms not specific to MET.ConclusionsWe find significant evidence of the OVOL, AP1, STAT1, STAT3, and NFKB1 TFs having important roles in MET, and more broadly in cancer. We prioritize known gene/drug targets for follow-up in the clinic, and we show that the AP1/MYC TF pair is a strong candidate for intervention.

Alternative CD44 splicing identifies epithelial prostate cancer cells from the mesenchymal counterparts

An epithelial to mesenchymal transition (EMT) has been shown to be a necessary precursor to prostate cancer metastasis. Additionally, the differential expression and splicing of mRNAs has been identified as a key means to distinguish epithelial from mesenchymal cells by qPCR, western blotting and immunohistochemistry. However, few markers exist to differentiate between these cells by flow cytometry. We previously developed two cell lines, PC3-Epi (epithelial) and PC3-EMT (mesenchymal). RNAseq was used to determine the differential expression of membrane proteins on PC3-Epi/EMT. We used western blotting, qPCR and flow cytometry to validate the RNAseq results. CD44 was one of six membrane proteins found to be differentially spliced between epithelial and mesenchymal PC3 cells. Although total CD44 was positive in all PC3-Epi/EMT cells, PC3-Epi cells had a higher level of CD44v6 (CD44 variant exon 6). CD44v6 was able to differentiate epithelial from mesenchymal prostate cancer cells using either flow cytometry, western blotting or qPCR.

A simple selection-free method for detecting disseminated tumor cells (DTCs) in murine bone marrow

Bone metastasis is a lethal and incurable disease. It is the result of the dissemination of cancer cells to the bone marrow. Due to the difficulty in sampling and detection, few techniques exist to efficiently and consistently detect and quantify disseminated tumor cells (DTCs) in the bone marrow of cancer patients. Because mouse models represent a crucial tool with which to study cancer metastasis, we developed a novel method for the simple selection-free detection and quantification of bone marrow DTCs in mice. We have used this protocol to detect human and murine DTCs in xenograft, syngeneic, and genetically engineered mouse models. We are able to detect and quantify bone marrow DTCs in mice that do not have overt bone metastasis. This protocol is amenable not only for detection and quantification purposes but also to study the expression of markers of numerous biological processes or tissue-specificity.

Abstract 1592: Identifying novel drivers of the epithelial-to-mesenchymal transition across multiple cancer types: from bioinformatics to the bench

To emigrate from a primary tumor to a distant site, a proliferating cancer cell must acquire the phenotypic traits necessary for migration and invasion. This shift from “growing” to “going,” termed epithelial-to-mesenchymal transition (EMT), may be a genetic or epigenetic phenomenon, ultimately characterized by altered cellular function that promotes metastasis. In addition to its role in malignant progression, EMT is integral in development and for healthy wound healing. EMT has been described in all carcinomas, and, because it of its established physiologic role, we hypothesize that there are a set of central EMT regulators that are universal to all carcinoma types. To identify global regulators of EMT, we integrated data from 15 published gene expression microarray studies that include a total of 49 epithelial and 46 mesenchymal cell line samples across 6 malignant tissue types (breast, prostate, colon, esophageal, liver, retinal pigment) and with various in vitro EMT induction strategies. While accounting for batch effects and other technical variability inherent in gene expression data, we performed differential expression analysis to identify genes that vary significantly between epithelial and mesenchymal states. Importantly, we found differential expression of established EMT markers (e.g. CDH1, ZEB1) validating our approach. We also identified genes that had not previously been implicated in cancer progression, representing novel candidate drivers of EMT, for example SARG (C1orf116). We found that SARG expression is decreased in high-grade cancer and metastatic disease, and is negatively associated with chemotherapy resistance across multiple cancer types (Oncomine). In an in vitro model of prostate cancer EMT, we found that SARG had >5-fold increased RNA and protein expression in PC3-epithelial cells. Knockdown of SARG expression in PC3-epithelial cells resulted in decreased gene expression of the epithelial-marker CDH1 and elevated expression of the mesenchymal marker CDH2, suggesting a role as a driver of the epithelial phenotype. In parallel with functional in vitro experiments, we have applied a Bayesian network learning approach to identify differentially expressed genes that are likely to play regulatory roles in EMT with causal influence on other genes. Importantly, this analysis provides valuable information regarding novel downstream effectors of known key regulators of EMT such as CDH1 and ELF3. Moreover, we also identified new EMT regulators such as CEP170 that have not been previously described. Experiments are underway to further explore the functional implications of these gene networks. This integrative approach of global gene expression analysis, network learning, and functional validation may identify causal genes and regulatory interactions in EMT, both expanding upon current knowledge and identifying novel drivers of this key metastatic process. Citation Format: Sarah R. Amend, Princy Parsana, James Hernandez, Alexis Battle, Kenneth J. Pienta. Identifying novel drivers of the epithelial-to-mesenchymal transition across multiple cancer types: from bioinformatics to the bench. [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 1592.

Abstract 4109: Unbiased detection of disseminated tumor cells in murine bone marrow

Approximately 20-30% of prostate cancer patients develop disease recurrence and metastasis years after initial therapy. This is thought to be largely due to the presence of growth-arrested and chemoresistant disseminated tumor cells (DTCs) in secondary sites, such as bone. Bone metastasis is found in 90-100% of prostate cancer patients who succumb to the disease. There are still many gaps in knowledge about the biological mechanisms by which DTCs home to bone, resist chemotherapy, become dormant, and escape dormancy to grow into clinical metastases. As such, it is important to be able to detect, quantify, and study bone marrow DTCs. In particular, it must be possible to do this in metastatic cancer mouse models, which are critical to study the process of tumor dissemination. DTC detection techniques currently exist, usually as either a positive selection or negative selection methodology. Positive selection techniques use markers or cell size to isolate and purify tumor cells out of the bone marrow. Positive selection markers are generally epithelial-specific, such as EpCam, E-cadherin, or Cytokeratin, and therefore may miss cells that lose epithelial marker expression and may gain mesenchymal markers. DTCs can also be as small as or smaller than white blood cells, meaning that positive selection based on size may miss some DTCs. Negative selection enriches for DTCs by removing blood and bone marrow cells from the population, usually using cell-specific markers. A popular strategy is CD45-based depletion, which removes white blood cells, and theoretically leaves behind DTCs. In our hands, this strategy causes loss of DTCs in the depletion process. To capture these heterogeneous and rare DTCs, we have developed a strategy to detect DTCs in murine bone marrow in an un-biased manner. The procedure entails removal of the bone marrow via centrifugation from the long bones (femur and tibia) of mice that have been injected with cancer cells (the injection site may vary depending on the experimental setup). The bone marrow then undergoes red blood cell lysis, and further centrifugation. The white blood cells are then counted, and the bone marrow is spread onto glass slides. The cells on the slide are fixed, permeabilized, and stained (immunofluorescence and RNA fluorescent in situ hybridization can be used). The staining can include any type of marker, including epithelial, mesenchymal, disease-specific, species-specific, or other biologically interesting markers, such as cell cycle markers. The unbiased nature of this procedure is based on the lack of positive or negative selection based on cell size or protein expression. Some DTC loss is noted in this protocol, due to the centrifugation and staining steps, but the cell population on the slide should include all DTC types. Notably, this protocol can be used to detect human or mouse cells in the mouse bone marrow and can thus be used in immune-compromise and immune-competent mouse models of metastasis. Citation Format: Kenneth C. Valkenburg, James R. Hernandez, Sarah R. Amend, James E. Verdone, Michael A. Gorin, Kenneth J. Pienta. Unbiased detection of disseminated tumor cells in murine bone marrow. [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 4109.

Abstract 1394: CD44 isoform expression illuminates multiple CSC subpopulations in PC3

The concept of the cancer stem cell (CSC) is neither new nor without controversy. It is important to study these cells since they are often hypothesized to be androgen insensitive and resistant to chemotherapeutics. Therefore, finding reliable markers for identifying and targeting these cells has become increasingly attractive. The hyaluronic acid receptor, CD44, is associated with a wide variety of cell types, including CSCs. CD44 has many different isoforms which can be categorized as belonging to one of two groups; one expressing different combinations of 10 variant extracellular exons (CD44v), and the other only expressing standard exons found in all isoforms, but none of the 10 variant exons (CD44s). CD44s is typically associated with the mesenchymal stem cell niche, while the various CD44v forms are related to hematopoietic stem cells and increased cell adhesion. However, both CD44s and CD44v have been linked to the CSC niche and cancer progression. Similar to CD44, the aldehyde dehydrogenases (ALDH) superfamily expression is also associated with CSCs and cancer progression. Previously, our laboratory demonstrated that epithelial cancer cells undergo an EMT upon exposure to M2 macrophages. In order to obtain a purely epithelial PCa population, our laboratory isolated a single cell clone of PC3 that had high E-cadherin expression, denoted PC3-Epi. PC3-Epi cells were then incubated with M2 macrophages, which caused a stable EMT to occur after only a few days in culture, denoted PC3-EMT. Our finding demonstrate that these PC3 clones exhibit numerous CSC characteristics, such as high ALDH activity, CD44 expression and cell plasticity. FACS analysis of CD44 isoform expression show distinct CSC subpopulations within these PC3-Epi and PC3-EMT cell types. These subpopulations showed the ability to recapitulate the presorted parental population after being returned to culture. This data suggests not only that the PC3 cell line displays numerous CSC characteristics, but also that distinct CSC subpopulations may exist. Note: This abstract was not presented at the meeting. Citation Format: James R. Hernandez, Steven M. Mooney, Gonzalo Torga, James E. Verdone, Kenneth J. Pienta. CD44 isoform expression illuminates multiple CSC subpopulations in PC3. [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 1394. doi:10.1158/1538-7445.AM2015-1394

Abstract 1919: Profiling of cancer cell lines demonstrates a dynamic relationship between epithelial, mesenchymal and cancer stem states

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Our previous work has demonstrated that exposure of M2 - Tumor Associated Macrophages M2-TAMs) to epithelial cancer cells can induce an Epithelial to Mesenchymal Transition (EMT). The resulting mesenchymal cancer cells exhibit a more aggressive phenotype, which places patients at a higher risk of relapse and death. Although most patients are cured by radical prostatectomy or radiation, it has been demonstrated that the majority of patients have disseminated tumor cells in their bone marrow at the time of primary treatment and approximately 10-15% of these men will eventually suffer a relapse after a period of dormancy. Further characterization of these disseminated cells both at the time of primary treatment and over time, is needed. It is unclear if these cells, especially those destined to eventually proliferate, are “mesenchymal”, “epithelial”, or “cancer stem” cells (CSCs). In this work, we investigate the CSC molecular and gene expression profile across multiple cancer cell lines and cell phenotypes. In part, this was done by utilizing various culturing conditions, such as 3D growth in stem cell media in order to generate sphere-like cultures known as tumorspheres. Flow cytometric analysis was performed based on ALDH, CD44 variant and CD133 expression. This was followed by gene expression analysis and characterization of the resulting tumorspheres and standard 2D cultures. The results demonstrate that there may be a level of plasticity that allows these cancer cells to alter gene expression and shift towards a CSC state and/or that there is a common CSC progenitor that exists within the population which can give rise to both epithelial and mesenchymal cell types. Therefore, furthering our understanding of the dynamic relationship between CSCs, EMT, and the converse mesenchymal to epithelial transition, is critical in developing novel therapeutics and prognostic tools in the field of cancer medicine. Citation Format: James R. Hernandez, Steven M. Mooney, Takumi Shiraishi, James E. Verdone, Calvin A. Harberg, Donald Vindivich, Kenneth J. Pienta. Profiling of cancer cell lines demonstrates a dynamic relationship between epithelial, mesenchymal and cancer stem states. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1919. doi:10.1158/1538-7445.AM2014-1919

Abstract 1139: Extracellular matrix components direct chromatin texture and nuclear morphological changes in epithelial-mesenchymal transition

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Epithelial-mesenchymal transition (EMT) is associated with metastasis in human prostate cancer (PCa). As metastatic tumors are the major cause of death in most cancer patients, identifying instances of EMT in patients may serve as a good prognostic indicator of progressive disease. Abnormal morphologic deformation of the cell nucleus is strongly associated with an aggressive cancer phenotype and computer-aided quantitative nuclear morphometry (QNM) has been used to quantify this deformation. In the present study, we go further to investigate the association of nuclear morphologic features between an epithelial PCa phenotype and an EMT-induced mesenchymal PCa phenotype across a panel of extracellular matrix (ECM) substrates on which cells are grown in vitro. Methods: PC3 epithelial cells (PC3-Epi) are established human PCa cultures from bone metastatic lesions. M2 macrophages induced PC3-Epi cells to undergo EMT and become mesenchymal PC3 cells (PC3- EMT). PC3-Epi and PC3-EMT cells were cultured independently on glass tissue-culture slides and also on a panel of ECM component substrates, including collagen 1, poly-D-Lysine, and various molecular weight hyaluronic acid polymers. Slides were stained with Feulgen or hematoxylin and eosin (H&E). The images were digitized with a slide-scanning microscope. Computer aided image processing software measured a total of 41 non-redundant features. This included nuclear size, shape, and texture features. Features were statistically analyzed using both supervised and unsupervised machine learning algorithms. Results: Statistical classification between PC3-Epi and PC3-EMT was performed on a panel of ECM substrates. The PC3-Epi and PC3-EMT morphological features were characterized by the type of ECM substrate on which cells were cultured. Chromatin texture and shape features, such as Feret diameter, were among the most significant in contributing to separation of the PC3-Epi and PC3-EMT. In contrast, the morphological features could not be separated when cells were grown on glass surfaces. Conclusions: The PC3-Epi and PC3-EMT cells were statistically separated more effectively with extracellular matrix substrates than with glass. Furthermore, our findings suggest that ECM components affect chromatin texture, a function of chromatin organization, and nuclear morphology. Our results also indicate quantitative nuclear morphometric analysis may be a viable tool for demonstrating instances of EMT in clinical samples, potentially adding quantitative prognostic value to this technique. Nuclear morphometric factors, such as those used here, have also found applications to distinguish aggressive human prostate cancers in fixed biopsy samples (See review: Veltri, R.W., et al. “Nuclear morphometry, nucleomics and prostate cancer progression”. Asian Journal of Andrology (2012) 14, 375-384.) Citation Format: James E. Verdone, Robert W. Veltri, Steven M. Mooney, James R. Hernandez, Calvin A. Harberg, Donald S. Coffey, Kenneth J. Pienta. Extracellular matrix components direct chromatin texture and nuclear morphological changes in epithelial-mesenchymal transition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1139. doi:10.1158/1538-7445.AM2014-1139