Jake Lichterman

Stromal fibroblast-derived miR-409 promotes epithelial-to-mesenchymal transition and prostate tumorigenesis.

Tumor–stromal interaction is a dynamic process that promotes tumor growth and metastasis via cell–cell interaction and extracellular vesicles. Recent studies demonstrate that stromal fibroblast-derived molecular signatures can be used to predict disease progression and drug resistance. To identify the epigenetic role of stromal noncoding RNAs in tumor–stromal interactions in the tumor microenvironment, we performed microRNA profiling of patient cancer-associated prostate stromal fibroblasts isolated by laser capture dissection microscopy and in bone-associated stromal models. We found specific upregulation of miR-409-3p and miR-409-5p located within the embryonically and developmentally regulated DLK1-DIO3 (delta-like 1 homolog-deiodinase, iodothyronine 3) cluster on human chromosome 14. The findings in cell lines were further validated in human prostate cancer tissues. Strikingly, ectopic expression of miR-409 in normal prostate fibroblasts conferred a cancer-associated stroma-like phenotype and led to the release of miR-409 via extracellular vesicles to promote tumor induction and epithelial-to-mesenchymal transition in vitro and in vivo. miR-409 promoted tumorigenesis through repression of tumor suppressor genes such as Ras suppressor 1 and stromal antigen 2. Thus, stromal fibroblasts derived miR-409-induced tumorigenesis, epithelial-to-mesenchymal transition and stemness of the epithelial cancer cells in vivo. Therefore, miR-409 appears to be an attractive therapeutic target to block the vicious cycle of tumor–stromal interactions that plagues prostate cancer patients.

NanoVelcro Chip for CTC enumeration in prostate cancer patients

Circulating tumor cells (CTCs) are one of the most crucial topics in rare cell biology and have become the focus of a significant and emerging area of cancer research. While CTC enumeration is a valid biomarker in prostate cancer, the current FDA-approved CTC technology is unable to detect CTCs in a large portion of late stage prostate cancer patients. Here we introduce the NanoVelcro CTC Chip, a device composed of a patterned silicon nanowire substrate (SiNW) and an overlaid polydimethylsiloxane (PDMS) chaotic mixer. Validated by two institutions participating in the study, the NanoVelcro Chip assay exhibits very consistent efficiency in CTC-capture from patient samples. The utilized protocol can be easily replicated at different facilities. We demonstrate the clinical utility of the NanoVelcro Chip by performing serial enumerations of CTCs in prostate cancer patients after undergoing systemic therapy. Changes in CTC numbers after 4-10 weeks of therapy were compared with their clinical responses. We observed a statistically significant reduction in CTCs counts in the clinical responders. We performed long-term follow up with serial CTC collection and enumeration in one patient observing variations in counts correlating with treatment response. This study demonstrates the consistency of the NanoVelcro Chip assay over time for CTC enumeration and also shows that continuous monitoring of CTC numbers can be employed to follow responses to different treatments and monitor disease progression.

miR-409-3p/-5p promotes tumorigenesis, epithelial to mesenchymal transition and bone metastasis of human prostate cancer

Purpose: miR-409-3p/-5p is a miRNA expressed by embryonic stem cells, and its role in cancer biology and metastasis is unknown. Our pilot studies demonstrated elevated miR-409-3p/-5p expression in human prostate cancer bone metastatic cell lines; therefore, we defined the biologic impact of manipulation of miR-409-3p/-5p on prostate cancer progression and correlated the levels of its expression with clinical human prostate cancer bone metastatic specimens. Experimental Design: miRNA profiling of a prostate cancer bone metastatic epithelial-to-mesenchymal transition (EMT) cell line model was performed. A Gleason score human tissue array was probed for validation of specific miRNAs. In addition, genetic manipulation of miR-409-3p/-5p was performed to determine its role in tumor growth, EMT, and bone metastasis in mouse models. Results: Elevated expression of miR-409-3p/-5p was observed in bone metastatic prostate cancer cell lines and human prostate cancer tissues with higher Gleason scores. Elevated miR-409-3p expression levels correlated with progression-free survival of patients with prostate cancer. Orthotopic delivery of miR-409-3p/-5p in the murine prostate gland induced tumors where the tumors expressed EMT and stemness markers. Intracardiac inoculation (to mimic systemic dissemination) of miR-409-5p inhibitor–treated bone metastatic ARCaPM prostate cancer cells in mice led to decreased bone metastasis and increased survival compared with control vehicle–treated cells. Conclusion: miR-409-3p/-5p plays an important role in prostate cancer biology by facilitating tumor growth, EMT, and bone metastasis. This finding bears particular translational importance as miR-409-3p/-5p appears to be an attractive biomarker and/or possibly a therapeutic target to treat bone metastatic prostate cancer. Clin Cancer Res; 20(17); 4636–46. ©2014 AACR.

A comparison of isolated circulating tumor cells and tissue biopsies using whole-genome sequencing in prostate cancer

Previous studies have demonstrated focal but limited molecular similarities between circulating tumor cells (CTCs) and biopsies using isolated genetic assays. We hypothesized that molecular similarity between CTCs and tissue exists at the single cell level when characterized by whole genome sequencing (WGS). By combining the NanoVelcro CTC Chip with laser capture microdissection (LCM), we developed a platform for single-CTC WGS. We performed this procedure on CTCs and tissue samples from a patient with advanced prostate cancer who had serial biopsies over the course of his clinical history. We achieved 30X depth and ≥ 95% coverage. Twenty-nine percent of the somatic single nucleotide variations (SSNVs) identified were founder mutations that were also identified in CTCs. In addition, 86% of the clonal mutations identified in CTCs could be traced back to either the primary or metastatic tumors. In this patient, we identified structural variations (SVs) including an intrachromosomal rearrangement in chr3 and an interchromosomal rearrangement between chr13 and chr15. These rearrangements were shared between tumor tissues and CTCs. At the same time, highly heterogeneous short structural variants were discovered in PTEN, RB1, and BRCA2 in all tumor and CTC samples. Using high-quality WGS on single-CTCs, we identified the shared genomic alterations between CTCs and tumor tissues. This approach yielded insight into the heterogeneity of the mutational landscape of SSNVs and SVs. It may be possible to use this approach to study heterogeneity and characterize the biological evolution of a cancer during the course of its natural history.

miR-154* and miR-379 in the DLK1-DIO3 MicroRNA Mega-Cluster Regulate Epithelial to Mesenchymal Transition and Bone Metastasis of Prostate Cancer

Purpose: MicroRNAs in the delta-like 1 homolog–deiodinase, iodothyronine 3 (DLK1-DIO3) cluster have been shown to be critical for embryonic development and epithelial to mesenchymal transition (EMT). DLK1-DIO3 cluster miRNAs are elevated in the serum of patients with metastatic cancer. However, the biologic functions of these miRNAs in the EMT and metastasis of cancer cells are poorly understood. We previously demonstrated the oncogenic and metastatic role of miR-409-3p/5p, a member of this cluster, in prostate cancer. In this study, we defined the role of miR-154* and miR-379, two key members of this cluster, in prostate cancer progression and bone metastasis in both cell line models and clinical specimens. Experimental Design: Genetic manipulation of miR-154* and miR-379 was performed to determine their role in tumor growth, EMT, and bone metastasis in mouse models. We determined the expression of miR-154* in prostate cancer clinical samples and bone metastasis samples using in situ hybridization and quantum dot labeling. Results: Elevated expression of miR-154* and miR-379 was observed in bone metastatic prostate cancer cell lines and tissues, and miR-379 expression correlated with progression-free survival of patients with prostate cancer. Intracardiac inoculation (to mimic systemic dissemination) of miR-154* inhibitor-treated bone metastatic ARCaPM prostate cancer cells in mice led to decreased bone metastasis and increased survival. Conclusion: miR-154* and miR-379 play important roles in prostate cancer biology by facilitating tumor growth, EMT, and bone metastasis. This finding has particular translational importance because miRNAs in the DLK1-DIO3 cluster can be attractive biomarkers and possible therapeutic targets to treat bone metastatic prostate cancer. Clin Cancer Res; 20(24); 6559–69. ©2014 AACR.

Subclassification of prostate cancer circulating tumor cells by nuclear size reveals very small nuclear circulating tumor cells in patients with visceral metastases.

Although enumeration of circulating tumor cells (CTCs) has shown some clinical value, the pool of CTCs contains a mixture of cells that contains additional information that can be extracted. The authors subclassified CTCs by shape features focusing on nuclear size and related this with clinical information.

SRC family kinase FYN promotes the neuroendocrine phenotype and visceral metastasis in advanced prostate cancer

FYN is a SRC family kinase (SFK) that has been shown to be up-regulated in human prostate cancer (PCa) tissues and cell lines. In this study, we observed that FYN is strongly up-regulated in human neuroendocrine PCa (NEPC) tissues and xenografts, as well as cells derived from a NEPC transgenic mouse model. In silico analysis of FYN expression in prostate cancer cell line databases revealed an association with the expression of neuroendocrine (NE) markers such as CHGA, CD44, CD56, and SYP. The loss of FYN abrogated the invasion of PC3 and ARCaPM cells in response to MET receptor ligand HGF. FYN also contributed to the metastatic potential of NEPC cells in two mouse models of visceral metastasis with two different cell lines (PC3 and TRAMPC2-RANKL). The activation of MET appeared to regulate neuroendocrine (NE) features as evidenced by increased expression of NE markers in PC3 cells with HGF. Importantly, the overexpression of FYN protein in DU145 cells was directly correlated with the increase of CHGA. Thus, our data demonstrated that the neuroendocrine differentiation that occurs in PCa cells is, at least in part, regulated by FYN kinase. Understanding the role of FYN in the regulation of NE markers will provide further support for ongoing clinical trials of SFK and MET inhibitors in castration-resistant PCa patients.

Abstract 3473: Sub-classification of prostate cancer circulating tumor cells (CTCs) by nuclear size reveals very-small nuclear CTCs in patients with visceral metastases

Background Circulating tumor cells (CTCs) are an emerging biomarker in prostate cancer (PC). Most efforts have focused on enumeration, we and others have identified subsets within this pool of cells which may be clinically informative. Using the highly sensitive NanoVelcro technology for CTC isolation and fluorescence microscopy, our group sub-classified CTCs by nuclear size and focused on the correlation with sites of metastatic disease in advanced PC patients. Methods Blood samples were obtained from PC patients across the spectrum of metastatic states, i.e. no metastasis, non-visceral (osseous/lymph node) metastasis, and visceral metastasis. The patients were divided into training and validation cohorts. CTCs were captured from the blood and enumerated on NanoVelcro Chips. These were then subjected to pathologic review for cellular morphology and nuclear size. The distribution of nuclear sizes was analyzed using a Gaussian Mixture Model to sub-classify the CTCs. Correlations were made between CTC subpopulations and metastatic status. Individual CTCs were isolated by laser-capture microdissection for subsequent characterization focusing on cellular markers and transcriptomic signatures in comparison between vsnCTC and other CTC subpopulations. Results Statistical analysis and modeling of nuclear size distribution revealed 3 distinct subpopulations in the training cohort: large-nuclear CTCs (lnCTC), small-nuclear CTCs (snCTC), and very-small-nuclear CTCs (vsnCTCs). While the lnCTC subpopulation alone failed to distinguish metastatic disease from non-metastatic disease, snCTC + vsnCTC counts could make that distinction. Furthermore, vsnCTC counts were elevated in PC patients with visceral metastases when compared to those without visceral metastases (0.5 ± 0.5 versus 3.1 ± 5.2 cells/mL of blood, p = 0.005). This difference remained statistically significant in the validation cohort (0.6 ± 1.0 versus 3.5 ± 4.0 cells/mL of blood, p = 0.007). Serial enumerations for individual patients showed the emergence of vsnCTCs prior to detection of newly developed visceral lesions. The recurrence of vsnCTC in patients under treatment is also associated with radiographic progression of existing visceral lesions through therapy. Molecular characterization of vsnCTCs reveals differential expression of genes related to neuroendocrine, stem cell, and androgen biology. Conclusions Subsets of CTCs contain information on PC disease status when analyzed with pathologic approaches, such as nuclear size measurement. In particular, the detection of vsnCTCs correlated with the presence of visceral metastatic lesions and should be explored as a potential biomarker to identify patients at risk for developing this more aggressive form of PC. Citation Format: Jie-Fu Chen, Hao Ho, Jake Lichterman, Yi-Tsung Lu, Yang Zhang, Mitch A. Garcia, Shang-Fu Chen, An-Jou Liang, Haiyen E. Zhau, Shuang Hou, Rafi S. Ahmed, Daniel J. Luthringer, Jiaoti Huang, Ker-Chau Li, Leland WK Chung, Zunfu Ke, Hsian-Rong Tseng, Edwin M. Posadas. Sub-classification of prostate cancer circulating tumor cells (CTCs) by nuclear size reveals very-small nuclear CTCs in patients with visceral metastases. [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 3473. doi:10.1158/1538-7445.AM2015-3473

Sub-classification of prostate cancer circulating tumor cells (CTCs) by nuclear size reveals very-small nuclear CTCs in patients with visceral metastases

Although enumeration of circulating tumor cells (CTCs) has shown some clinical value, the pool of CTCs contains a mixture of cells that contains additional information that can be extracted. The authors subclassified CTCs by shape features focusing on nuclear size and related this with clinical information.

Subclassification of prostate cancer circulating tumor cells by nuclear size reveals very small nuclear circulating tumor cells in patients with visceral metastases: vsnCTCs and Visceral Metastases in PC

Although enumeration of circulating tumor cells (CTCs) has shown some clinical value, the pool of CTCs contains a mixture of cells that contains additional information that can be extracted. The authors subclassified CTCs by shape features focusing on nuclear size and related this with clinical information.