CXCL12γ induces human prostate and mammary gland development.

BACKGROUND: Epithelial stem cells (ESCs) demonstrate a capacity to maintain normal tissues homeostasis and ESCs with a deregulated behavior can contribute to cancer development. The ability to reprogram normal tissue epithelial cells into prostate or mammary stem-like cells holds great promise to help understand cell of origin and lineage plasticity in prostate and breast cancers in addition to understanding normal gland development. We previously showed that an intracellular chemokine, CXCL12γ induced cancer stem cells and neuroendocrine characteristics in both prostate and breast adenocarcinoma cell lines. However, its role in normal prostate or mammary epithelial cell fate and development remains unknown. Therefore, we sought to elucidate the functional role of CXCL12γ in the regulation of ESCs and tissue development. METHODS: Prostate epithelial cells (PNT2) or mammary epithelial cells (MCF10A) with overexpressed CXCL12γ was characterized by quantitative real-time polymerase chain reaction, Western blots, and immunofluorescence for lineage marker expression, and fluorescence activated cell sorting analyses and sphere formation assays to examine stem cell surface phenotype and function. Xenotransplantation animal models were used to evaluate gland or acini formation in vivo. RESULTS: Overexpression of CXCL12γ promotes the reprogramming of cells with a differentiated luminal phenotype to a nonluminal phenotype in both prostate (PNT2) and mammary (MCF10A) epithelial cells. The CXCL12γ-mediated nonluminal type cells results in an increase of epithelial stem-like phenotype including the subpopulation of EPCAMLo /CD49fHi /CD24Lo /CD44Hi cells capable of sphere formation. Critically, overexpression of CXCL12γ promotes the generation of robust gland-like structures from both prostate and mammary epithelial cells in in vivo xenograft animal models. CONCLUSIONS: CXCL12γ supports the reprogramming of epithelial cells into nonluminal cell-derived stem cells, which facilitates gland development.

Bone microenvironment signaling of cancer stem cells as a therapeutic target in metastatic prostate cancer.

Prostate cancer (PCa) is one of the most prevalent cancers and the second leading cause of cancer death among US males. When diagnosed in an early disease stage, primary tumors of PCa may be treated with surgical resection or radiation, sometimes combined with androgen deprivation therapy, with favorable outcomes. Unfortunately, the treatment efficacy of each approach decreases significantly in later stages of PCa that involve metastasis to soft tissues and bone. Metastatic PCa is a heterogeneous disease containing host cells, mature cancer cells, and subpopulation of cancer stem cells (CSC). CSCs are highly tumorigenic due to their self-renewing and differentiating potential, clinically resulting in recurrence and resistance to standard therapies. Therefore, there is a large unmet clinical need to develop therapies, which target CSC activity. In this review, we summarize the main signaling pathways that are implicated in the current pre-clinical and clinical studies of recurrent metastatic PCa within the bone microenvironment targeting CSCs and discuss the trajectory of therapeutics moving forward.

Detection and isolation of disseminated tumor cells in bone marrow of patients with clinically localized prostate cancer.

BACKGROUND: Disseminated tumor cells (DTCs) have been reported in the bone marrow (BM) of patients with localized prostate cancer (PCa). However, the existence of these cells continues to be questioned, and few methods exist for viable DTC isolation. Therefore, we sought to develop novel approaches to identify and, if detected, analyze localized PCa patient DTCs. METHODS: We used fluorescence-activated cell sorting (FACS) to isolate a putative DTC population, which was negative for CD45, CD235a, alkaline phosphatase, and CD34, and strongly expressed EPCAM. We examined tumor cell content by bulk cell RNA sequencing (RNA-Seq) and whole-exome sequencing after whole genome amplification. We also enriched for BM DTCs with α-EPCAM immunomagnetic beads and performed quantitative reverse trancriptase polymerase chain reaction (qRT-PCR) for PCa markers. RESULTS: At a threshold of 4 cells per million BM cells, the putative DTC population was present in 10 of 58 patients (17%) with localized PCa, 4 of 8 patients with metastatic PCa of varying disease control, and 1 of 8 patients with no known cancer, and was positively correlated with patients' plasma PSA values. RNA-Seq analysis of the putative DTC population collected from samples above (3 patients) and below (5 patients) the threshold of 4 putative DTCs per million showed increased expression of PCa marker genes in 4 of 8 patients with localized PCa, but not the one normal donor who had the putative DTC population present. Whole-exome sequencing also showed the presence of single nucleotide polymorphisms and structural variants in the gene characteristics of PCa in 2 of 3 localized PCa patients. To examine the likely contaminating cell types, we used a myeloid colony formation assay, differential counts of cell smears, and analysis of the RNA-Seq data using the CIBERSORT algorithm, which most strongly suggested the presence of B-cell lineages as a contaminant. Finally, we used EPCAM enrichment and qRT-PCR for PCa markers to estimate DTC prevalence and found evidence of DTCs in 21 of 44 samples (47%). CONCLUSION: These data support the presence of DTCs in the BM of a subset of patients with localized PCa and describe a novel FACS method for isolation and analysis of viable DTCs.

Reduction of two histone marks, H3k9me3 and H3k27me3 by epidrug induces neuroendocrine differentiation in prostate cancer.

Neuroendocrine prostate cancer (NE PCa) is an aggressive malignancy, often presenting with advanced metastasis. We previously reported that reduction of histone marks regulated by DNMT1 following epidrug (5-Azacitidine, 5-Aza) treatment controls induction of epithelial to mesenchymal (EMT) and a cancer stem cell (CSC) phenotype, which facilitates tumorigenesis in PCa cells. Here, we use the epidrug 5-Aza as a model for how histone marks may regulate the reprogramming of prostate adenocarcinoma into NE phenotypic cells. First, we observed that 5-Aza treatment of PCa cells in vitro induces a neuron-like phenotype. In addition, significant increases in the expression of the NE markers N-Myc downstream regulated gene 1 (NDRG1), enolase-2 (ENO2), and synaptophysin were observed. Critically, a high density of NE cells with synaptophysin expression was found in tumors generated by 5-Aza pretreatment of PCa cells. Importantly, induction of NE differentiation of PCa cells was associated with an enhancement of NDRG1 expression by reduction of two histone marks, H3K9me3 and H3K27me3. Further, more NDRG1 expression was detected in the subset of PCa cells with reduced expression of H3K9me3 or H3K27me3 in the tumors generated by 5-Aza pretreated PCa cells and critically, these biological differences are also observed in small cell carcinoma in advanced stage of human primary PCa tumors. Our results suggest that reduction of histone marks regulated by the epidrug 5-Aza may control induction of a NE phenotype, which facilitates PCa progression. These studies suggest a strong rationale for developing therapeutics, which target epigenetic regulation.

Integrin alpha V beta 3 targeted dendrimer-rapamycin conjugate reduces fibroblast-mediated prostate tumor progression and metastasis.

Therapeutic strategies targeting both cancer cells and associated cells in the tumor microenvironment offer significant promise in cancer therapy. We previously reported that generation 5 (G5) dendrimers conjugated with cyclic-RGD peptides target cells expressing integrin alpha V beta 3. In this study, we report a novel dendrimer conjugate modified to deliver the mammalian target of rapamycin (mTOR) inhibitor, rapamycin. In vitro analyses demonstrated that this drug conjugate, G5-FI-RGD-rapamycin, binds to prostate cancer (PCa) cells and fibroblasts to inhibit mTOR signaling and VEGF expression. In addition, G5-FI-RGD-rapamycin inhibits mTOR signaling in cancer cells more efficiently under proinflammatory conditions compared to free rapamycin. In vivo studies established that G5-FI-RGD-rapamycin significantly inhibits fibroblast-mediated PCa progression and metastasis. Thus, our results suggest the potential of new rapamycin-conjugated multifunctional nanoparticles for PCa therapy.

Minimal Residual Disease in Prostate Cancer.

Detection of minimal residual disease (MRD) in prostate cancer over several decades has greatly informed our understanding of dissemination and recurrence, but has not yet been routinely used in clinical care. Investigators have detected MRD by identification of prostate cancer cells in the bone marrow; termed disseminated tumor cells (DTCs) and blood; termed circulating tumor cells (CTCs). Various techniques including PSA-RT PCR, PSA immunocytochemistry, cytokeratin immunocytochemistry, and immune-magnetic depletion of hematopoietic cells followed by EPCAM based positive selection, have been used. Importantly, detection of DTCs correlates with recurrence. Research into prostate cancer CTCs has intensified recently, but their use in MRD evaluation has been more limited. Investigators are using semi-automated platforms to detect and begin to study prostate cancer CTCs in patients with no evidence of disease. PSA immunocytochemistry also detects CTCs and correlates with recurrence. Emerging technologies have the potential to greatly aid research in this exciting field.

Mer Tyrosine Kinase Regulates Disseminated Prostate Cancer Cellular Dormancy.

Many prostate cancer (PCa) recurrences are thought to be due to reactivation of disseminated tumor cells (DTCs). We previously found a role of the TAM family of receptor tyrosine kinases TYRO3, AXL, and MERTK in PCa dormancy regulation. However, the mechanism and contributions of the individual TAM receptors is largely unknown. Knockdown of MERTK, but not AXL or TYRO3 by shRNA in PCa cells induced a decreased ratio of P-Erk1/2 to P-p38, increased expression of p27, NR2F1, SOX2, and NANOG, induced higher levels of histone H3K9me3 and H3K27me3, and induced a G1/G0 arrest, all of which are associated with dormancy. Similar effects were also observed with siRNA. Most importantly, knockdown of MERTK in PCa cells increased metastasis free survival in an intra-cardiac injection mouse xenograft model. MERTK knockdown also failed to inhibit PCa growth in vitro and subcutaneous growth in vivo, which suggests that MERTK has specificity for dormancy regulation or requires a signal from the PCa microenvironment. The effects of MERTK on the cell cycle and histone methylation were reversed by p38 inhibitor SB203580, which indicates the importance of MAP kinases for MERTK dormancy regulation. Overall, this study shows that MERTK stimulates PCa dormancy escape through a MAP kinase dependent mechanism, also involving p27, pluripotency transcription factors, and histone methylation. J. Cell. Biochem. 118: 891-902, 2017. © 2016 Wiley Periodicals, Inc.

Growth Arrest-Specific 6 (GAS6) Promotes Prostate Cancer Survival by G1 Arrest/S Phase Delay and Inhibition of Apoptosis During Chemotherapy in Bone Marrow.

Prostate cancer (PCa) is known to develop resistance to chemotherapy. Growth arrest-specific 6 (GAS6), plays a role in tumor progression by regulating growth in many cancers. Here, we explored how GAS6 regulates the cell cycle and apoptosis of PCa cells in response to chemotherapy. We found that GAS6 is sufficient to significantly increase the fraction of cells in G1 and the duration of phase in PCa cells. Importantly, the effect of GAS6 on G1 is potentiated during docetaxel chemotherapy. GAS6 altered the levels of several key cell cycle regulators, including the downregulation of Cyclin B1 (G2 /M phase), CDC25A, Cyclin E1, and CDK2 (S phase entry), while the upregulation of cell cycle inhibitors p27 and p21, Cyclin D1, and CDK4. Importantly, these changes became further accentuated during docetaxel treatment in the presence of GAS6. Moreover, GAS6 alters the apoptotic response of PCa cells during docetaxel chemotherapy. Docetaxel induced PCa cell apoptosis is efficiently suppressed in PCa cell culture in the presence of GAS6 or GAS6 secreted from co-cultured osteoblasts. Similarly, the GAS6-expressing bone environment protects PCa cells from apoptosis within primary tumors in vivo studies. Docetaxel induced significant levels of Caspase-3 and PARP cleavage in PCa cells, while GAS6 protected PCa cells from docetaxel-induced apoptotic signaling. Together, these data suggest that GAS6, expressed by osteoblasts in the bone marrow, plays a significant role in the regulation of PCa cell survival during chemotherapy, which will have important implications for targeting metastatic disease. J. Cell. Biochem. 117: 2815-2824, 2016. © 2016 Wiley Periodicals, Inc.

The bone marrow endosteal niche: how far from the surface?

Hematopoietic stem cells (HSC) self-renewal takes place in the same microenvironment in which massive hematopoietic progenitor proliferation, commitment, and differentiation will occur. This is only made possible if the bone marrow microenvironment comprises different specific niches, composed by different stromal cells that work in harmony to regulate all the steps of the hematopoiesis cascade. Histological and functional assays indicated that HSC and multipotent progenitors preferentially colonize the endosteal and subendosteal regions, in close association with the bone surface. Conversely, committed progenitors and differentiated cells are distributed in the central and perisinusoidal regions, respectively. Over the last decade, many investigative teams sought to define which cell types regulate the HSC niche, how they are organized, and to what extent they interface with each other. System dynamics requires different stromal cells to operate distinct functions over similar HSC pools rather than a single stromal cell type controlling everything. Therefore, our focus herein is to depict the players in the endosteal and subendosteal regions, named the endosteal niche, a necessary step to better understand the interactions of the HSC within the niche and to identify potential targets to manipulate and/or modulate normal and malignant HSC behavior.

Endothelial interleukin-6 defines the tumorigenic potential of primary human cancer stem cells.

Head and neck squamous cell carcinomas (HNSCC) contain a small subpopulation of stem cells endowed with unique capacity to generate tumors. These cancer stem cells (CSC) are localized in perivascular niches and rely on crosstalk with endothelial cells for survival and self-renewal, but the mechanisms involved are unknown. Here, we report that stromal interleukin (IL)-6 defines the tumorigenic capacity of CSC sorted from primary human HNSCC and transplanted into mice. In search for the cellular source of Interleukin-6 (IL-6), we observed a direct correlation between IL-6 levels in tumor-associated endothelial cells and the tumorigenicity of CSC. In vitro, endothelial cell-IL-6 enhanced orosphere formation, p-STAT3 activation, survival, and self-renewal of human CSC. Notably, a humanized anti-IL-6R antibody (tocilizumab) inhibited primary human CSC-mediated tumor initiation. Collectively, these data demonstrate that endothelial cell-secreted IL-6 defines the tumorigenic potential of CSC, and suggest that HNSCC patients might benefit from therapeutic inhibition of IL-6/IL-6R signaling.

Niche inheritance: a cooperative pathway to enhance cancer cell fitness through ecosystem engineering.

Cancer cells can be described as an invasive species that is able to establish itself in a new environment. The concept of niche construction can be utilized to describe the process by which cancer cells terraform their environment, thereby engineering an ecosystem that promotes the genetic fitness of the species. Ecological dispersion theory can then be utilized to describe and model the steps and barriers involved in a successful diaspora as the cancer cells leave the original host organ and migrate to new host organs to successfully establish a new metastatic community. These ecological concepts can be further utilized to define new diagnostic and therapeutic areas for lethal cancers.

A novel method for monitoring tumor proliferation in vivo using fluorescent dye DiD.

Monitoring single cell proliferation in vivo is difficult, but optimizing this technique is essential in order to expand our knowledge of the regulation of tumor proliferation. In this study, we used a lipophilic fluorescent dye, DiD, that rapidly and stably integrates into the phospholipid cell membrane. We cultured DiD-stained prostate cancer cell lines for 10 days and isolated cells by flow cytometry based on expression levels of DiD. We found that a decrease in DiD intensity was correlated to the reduction of EdU, where the DiD-high population proliferated more slowly than the DiD-low population and the DiD-low population exhibited a higher mitotic index. We also found that DiD was detected after 3 weeks of implantation in an in vivo setting. Importantly, DiD dye did not have any effect on normal cell growth, whereas a gold standard fluorescent dye for measuring cell proliferation, CFSE, slowed cell proliferation. Although further study is indicated, DiD can be useful for identifying the molecular mechanisms underlying tumor proliferation in vivo.

Abscisic acid signaling through LANCL2 and PPARγ induces activation of p38MAPK resulting in dormancy of prostate cancer metastatic cells.

Prostate cancer (PCa) is one the most common malignancies in men. The high incidence of bone metastasis years after primary therapy suggests that disseminated tumor cells must become dormant, but maintain their ability to proliferate in the bone marrow. Abscisic acid (ABA) is a stress response molecule best known for its regulation of seed germination, stomal opening, root shoot growth and other stress responses in plants. ABA is also synthesized by mammalian cells and has been linked to human disease. The aim of the present study was to examine the role of ABA in regulating tumor dormancy via signaling through lanthionine synthetase C­like protein 2 (LANCL2) and peroxisome proliferator activated receptor γ (PPARγ) receptors. ABA signaling in human PCa cell lines was studied using targeted gene knockdown (KD), western blotting, quantitative PCR, cell proliferation, migration, invasion and soft agar assays, as well as co­culture assays with bone marrow stromal cells. The data demonstrated that ABA signaling increased the expression of p21, p27 and p16, while inhibiting viability, migration, invasion and colony size in a reversable manner without toxicity. ABA also induced p38MAPK activation and NR2F1 signaling. Targeted gene KD of LANCL2 and PPARγ abrogated the cellular responses to ABA. Taken together, these data demonstrate that ABA may induce dormancy in PCa cell lines through LANCL2 and PPARγ signaling, and suggest novel targets to manage metastatic PCa growth.

Cell cycle variants during Drosophila male accessory gland development.

The Drosophila melanogaster male accessory gland (AG) is a functional analog of the mammalian prostate and seminal vesicles containing two secretory epithelial cell types, termed main and secondary cells. This tissue is responsible for making and secreting seminal fluid proteins and other molecules that contribute to successful reproduction. The cells of this tissue are binucleate and polyploid, due to variant cell cycles that include endomitosis and endocycling during metamorphosis. Here, we provide evidence of additional cell cycle variants in this tissue. We show that main cells of the gland are connected by ring canals that form after the penultimate mitosis, and we describe an additional post-eclosion endocycle required for gland maturation that is dependent on juvenile hormone signaling. We present evidence that the main cells of the D. melanogaster AG undergo a unique cell cycle reprogramming throughout organ development that results in step-wise cell cycle truncations culminating in cells containing two octoploid nuclei with under-replicated heterochromatin in the mature gland. We propose this tissue as a model to study developmental and hormonal temporal control of cell cycle variants in terminally differentiating tissues.

The effects of zoledronic acid in the bone and vasculature support of hematopoietic stem cell niches.

Hematopoietic stem cells (HSC) are maintained in a tightly regulated bone microenvironment constituted by a rich milieu of cells. Bone cells such as osteoblasts are associated with niche maintenance as regulators of the endosteal microenvironment. Bone remodeling also plays a role in HSC mobilization although it is poorly defined. The effects of zoledronic acid (ZA), a potent bisphosphonate that inhibits bone resorption, were investigated on bone marrow cell populations focusing on HSCs, and the endosteal and vascular niches in bone. ZA treatment significantly increased bone volume and HSCs in both young and adult mice (4 week and 4 month old, respectively). ZA increased vessel numbers with no overall change in vascular volume in bones of young and had no effect on vasculature in adult mice. Since both young and adult mice had increased HSCs and bone mass with differing vasculature responses, this suggests that ZA indirectly supports HSCs via the osteoblastic niche and not the vascular niche. Additionally, gene expression in Lin- cells demonstrated increased expression of self-renewal-related genes Bmi1 and Ink4a suggesting a role of ZA in the modulation of cell commitment and differentiation toward a long-term self-renewing cell. Genes that support the osteoblastic niche, BMP2 and BMP6 were also augmented in ZA treated mice. In conclusion, ZA-induced HSC expansion occurs independent of the vascular niche via indirect modulation of the osteoblastic niche.

Erythropoietin supports the survival of prostate cancer, but not growth and bone metastasis.

Erythropoietin (Epo) is used in clinical settings to enhance hematopoietic function and to improve the quality of life for patients undergoing chemotherapy by reducing fatigue and the need for transfusions. However, several meta-analyses have revealed that Epo treatments are associated with an increased risk of mortality in cancer patients. In this study, we examined the role of Epo in prostate cancer (PCa) progression, using in vitro cell culture systems and in vivo bone metastatic assays. We found that Epo did not stimulate the proliferation of PCa cell lines, but did protect PCa cells from apoptosis. In animal models of PCa metastasis, no evidence was found to support the hypothesis that Epo enhances metastasis. Together, these findings suggest that Epo may be useful for treating severe anemia in PCa patients without increasing metastatic risk.

Molecular signature and in vivo behavior of bone marrow endosteal and subendosteal stromal cell populations and their relevance to hematopoiesis.

In the bone marrow cavity, hematopoietic stem cells (HSC) have been shown to reside in the endosteal and subendosteal perivascular niches, which play specific roles on HSC maintenance. Although cells with long-term ability to reconstitute full hematopoietic system can be isolated from both niches, several data support a heterogenous distribution regarding the cycling behavior of HSC. Whether this distinct behavior depends upon the role played by the stromal populations which distinctly create these two niches is a question that remains open. In the present report, we used our previously described in vivo assay to demonstrate that endosteal and subendosteal stromal populations are very distinct regarding skeletal lineage differentiation potential. This was further supported by a microarray-based analysis, which also demonstrated that these two stromal populations play distinct, albeit complementary, roles in HSC niche. Both stromal populations were preferentially isolated from the trabecular region and behave distinctly in vitro, as previously reported. Even though these two niches are organized in a very close range, in vivo assays and molecular analyses allowed us to identify endosteal stroma (F-OST) cells as fully committed osteoblasts and subendosteal stroma (F-RET) cells as uncommitted mesenchymal cells mainly represented by perivascular reticular cells expressing high levels of chemokine ligand, CXCL12. Interestingly, a number of cytokines and growth factors including interleukin-6 (IL-6), IL-7, IL-15, Hepatocyte growth factor (HGF) and stem cell factor (SCF) matrix metalloproteases (MMPs) were also found to be differentially expressed by F-OST and F-RET cells. Further microarray analyses indicated important mechanisms used by the two stromal compartments in order to create and coordinate the "quiescent" and "proliferative" niches in which hematopoietic stem cells and progenitors reside.

Mycoplasma hyorhinis infection promotes TNF-α signaling and SMAC mimetic-mediated apoptosis in human prostate cancer.

Growing evidence suggests an association between Mycoplasma infections and the development and progression of prostate cancer (PCa). In this study, we report that chronic and persistent M. hyorhinis infection induced robust TNF-α secretion from PCa cells. TNF-α secreted from M. hyorhinis-infected PCa cells subsequently led to activation of the NF-κB pathway. Chronic M. hyorhinis infection induced gene expression of pro-inflammatory cytokines and chemokines in a NF-κB-dependent manner and promoted cell proliferation, migration, and invasion in PCa cells. The elimination of M. hyorhinis in PCa cells significantly blocked TNF-α secretion, gene expression of cytokines and chemokines, migration, and invasion in PCa cells, suggesting M. hyorhinis-induced TNF-α plays an important role to promote malignant transformation of PCa. Furthermore, second mitochondria-derived activator of caspases (SMAC) mimetics potentiated caspase activation and cell death in M. hyorhinis-infected PCa by antagonizing inhibitor of apoptosis proteins (IAPs) activity. Tissue microarray analysis indicated that TNF-α is co-expressed in M. hyorhinis-infected human patient tissues. Findings from this study advance our understanding of the mycoplasma-oncogenesis process and suggest the potential for new approaches for preventions, diagnosis, and therapeutic approaches against prostate cancers.

HER2 as a potential therapeutic target on quiescent prostate cancer cells.

Quiescent prostate cancer (PCa) cells are common in tumors but are often resistant to chemotherapy. Quiescent PCa cells are also enriched for a stem-like tumor initiating population, and can lead to recurrence after dormancy. Unfortunately, quiescent PCa cells are difficult to identify and / or target with treatment in part because the relevant markers are intracellular and regulated by protein stability. We addressed this problem by utilizing PCa cells expressing fluorescent markers for CDKN1B (p27) and CDT1, which can separate viable PCa cells into G0, G1, or combined S/G2/M populations. We used FACS to collect G1 and G0 PC3 PCa cells, isolated membrane proteins, and analyzed protein abundance in G0 vs G1 cells by gas chromatography mass spectrometry. Enrichment analysis identified nucleocytoplasmic transport as the most significantly different pathway. To identify cell surface proteins potentially identifying quiescent PCa cells for future patient samples or for antibody based therapeutic research, we focused on differentially abundant plasma membrane proteins, and identified ERBB2 (HER2) as a cell surface protein enriched on G0 PCa cells. High HER2 on the cell membrane is associated with quiescence in PCa cells and likely induced by the bone microenvironment. Using a drug conjugated anti-HER2 antibody (trastuzumab emtansine) in a mouse PCa xenograft model delayed metastatic tumor growth, suggesting approaches that target HER2-high cells may be beneficial in treating PCa. We propose that HER2 is deserving of further study in PCa as a target on quiescent cells to prevent recurrence, decrease chemotherapy resistance, or eradicate minimal residual disease.