Kerui Wu

Roles of the Cyclooxygenase 2 Matrix Metalloproteinase 1 Pathway in Brain Metastasis of Breast Cancer

Background: Mechanism of brain metastasis of breast cancer is poorly understood. Results: Inflammatory factors secreted from cancer cells degrade tight junction of BBB and stimulate the tumor-microenvironment cross-talk. Conclusion: COX2-prostaglandins-MMP1 pathway promotes brain metastasis by tampering with BBB and up-regulating CCL7 in astrocytes for the growth of tumor initiating cells. Significance: The COX2-prostaglandins-MMP1 pathway may serve as a novel therapeutic target for brain metastasis. Brain is one of the major sites of metastasis in breast cancer; however, the pathological mechanism of brain metastasis is poorly understood. One of the critical rate-limiting steps of brain metastasis is the breaching of blood-brain barrier, which acts as a selective interface between the circulation and the central nervous system, and this process is considered to involve tumor-secreted proteinases. We analyzed clinical significance of 21 matrix metalloproteinases on brain metastasis-free survival of breast cancer followed by verification in brain metastatic cell lines and found that only matrix metalloproteinase 1 (MMP1) is significantly correlated with brain metastasis. We have shown that MMP1 is highly expressed in brain metastatic cells and is capable of degrading Claudin and Occludin but not Zo-1, which are key components of blood-brain barrier. Knockdown of MMP1 in brain metastatic cells significantly suppressed their ability of brain metastasis in vivo, whereas ectopic expression of MMP1 significantly increased the brain metastatic ability of the cells that are not brain metastatic. We also found that COX2 was highly up-regulated in brain metastatic cells and that COX2-induced prostaglandins were directly able to promote the expression of MMP1 followed by augmenting brain metastasis. Furthermore, we found that COX2 and prostaglandin were able to activate astrocytes to release chemokine (C-C motif) ligand 7 (CCL7), which in turn promoted self-renewal of tumor-initiating cells in the brain and that knockdown of COX2 significantly reduced the brain metastatic ability of tumor cells. Our results suggest the COX2-MMP1/CCL7 axis as a novel therapeutic target for brain metastasis.

miR-509 suppresses brain metastasis of breast cancer cells by modulating RhoC and TNF α

The median survival time of breast cancer patients with brain metastasis is less than 6 months, and even a small metastatic lesion often causes severe neurological disabilities. Because of the location of metastatic lesions, a surgical approach is limited and most chemotherapeutic drugs are ineffective owing to the blood brain barrier (BBB). Despite this clinical importance, the molecular basis of the brain metastasis is poorly understood. In this study, we have isolated RNA from samples obtained from primary breast tumors and also from brain metastatic lesions followed by microRNA profiling analysis. Our results revealed that the miR-509 is highly expressed in the primary tumors, whereas the expression of this microRNA is significantly decreased in the brain metastatic lesions. MicroRNA target prediction and the analysis of cytokine array for the cells ectopically expressed with miR-509 demonstrated that this microRNA was capable of modulating the two genes essential for brain invasion, RhoC and TNF-α that affect the invasion of cancer cells and permeability of BBB, respectively. Importantly, high levels of TNF-α and RhoC-induced MMP9 were significantly correlated with brain metastasis-free survival of breast cancer patients. Furthermore, the results of our in vivo experiments indicate that miR-509 significantly suppressed the ability of cancer cells to metastasize to the brain. These findings suggest that miR-509 has a critical role in brain metastasis of breast cancer by modulating the RhoC-TNF-α network and that this miR-509 axis may represent a potential therapeutic target or serve as a prognostic tool for brain metastasis.

Secreted Protein Acidic and Rich in Cysteine (SPARC) Mediates Metastatic Dormancy of Prostate Cancer in Bone

Prostate cancer is known to frequently recur in bone; however, how dormant cells switch its phenotype leading to recurrent tumor remains poorly understood. We have isolated two syngeneic cell lines (indolent and aggressive) through in vivo selection by implanting PC3mm stem-like cells into tibial bones. We found that indolent cells retained the dormant phenotype, whereas aggressive cells grew rapidly in bone in vivo, and the growth rates of both cells in culture were similar, suggesting a role of the tumor microenvironment in the regulation of dormancy and recurrence. Indolent cells were found to secrete a high level of secreted protein acidic and rich in cysteine (SPARC), which significantly stimulated the expression of BMP7 in bone marrow stromal cells. The secreted BMP7 then kept cancer cells in a dormant state by inducing senescence, reducing “stemness,” and activating dormancy-associated p38 MAPK signaling and p21 expression in cancer cells. Importantly, we found that SPARC was epigenetically silenced in aggressive cells by promoter methylation, but 5-azacytidine treatment reactivated the expression. Furthermore, high SPARC promoter methylation negatively correlated with disease-free survival of prostate cancer patients. We also found that the COX2 inhibitor NS398 down-regulated DNMTs and increased expression of SPARC, which led to tumor growth suppression in bone in vivo. These findings suggest that SPARC plays a key role in maintaining the dormancy of prostate cancer cells in the bone microenvironment.

Activation of the c-Met Pathway Mobilizes an Inflammatory Network in the Brain Microenvironment to Promote Brain Metastasis of Breast Cancer

Brain metastasis is one of the chief causes of mortality in breast cancer patients, but the mechanisms that drive this process remain poorly understood. Here, we report that brain metastatic cells expressing high levels of c-Met promote the metastatic process via inflammatory cytokine upregulation and vascular reprogramming. Activated c-Met signaling promoted adhesion of tumor cells to brain endothelial cells and enhanced neovascularization by inducing the secretion of IL8 and CXCL1. Additionally, stimulation of IL1β secretion by activation of c-Met induced tumor-associated astrocytes to secrete the c-Met ligand HGF. Thus, a feed-forward mechanism of cytokine release initiated and sustained by c-Met fed a vicious cycle that generated a favorable microenvironment for metastatic cells. Reinforcing our results, we found that pterostilbene, a compound that penetrates the blood-brain barrier, could suppress brain metastasis by targeting c-Met signaling. These findings suggest a potential utility of this natural compound for chemoprevention. Cancer Res; 76(17); 4970-80. ©2016 AACR.

Extracellular vesicles as emerging targets in cancer: Recent development from bench to bedside

Extracellular vesicles (EVs) have emerged as important players of cancer initiation and progression through cell-cell communication. They have been recognized as critical mediators of extracellular communications, which promote transformation, growth invasion, and drug-resistance of cancer cells. Interestingly, the secretion and uptake of EVs are regulated in a more controlled manner than previously anticipated. EVs are classified into three groups, (i) exosomes, (ii) microvesicles (MVs), and (iii) apoptotic bodies (ABs), based on their sizes and origins, and novel technologies to isolate and distinguish these EVs are evolving. The biologically functional molecules harbored in these EVs, including nucleic acids, lipids, and proteins, have been shown to induce key signaling pathways in both tumor and tumor microenvironment (TME) cells for exacerbating tumor development. While tumor cell-derived EVs are capable of reprogramming stromal cells to generate a proper tumor cell niche, stromal-derived EVs profoundly affect the growth, resistance, and stem cell properties of tumor cells. This review summarizes and discusses these reciprocal communications through EVs in different types of cancers. Further understanding of the pathophysiological roles of different EVs in tumor progression is expected to lead to the discovery of novel biomarkers in liquid biopsy and development of tumor specific therapeutics. This review will also discuss the translational aspects of EVs and therapeutic opportunities of utilizing EVs in different cancer types.

MicroRNAs in cancer stem cells: new regulators of stemness.

In the past decade, cancer stem cells (CSCs) have been isolated, characterized, and studied in a variety of cancers and they are believed to be responsible for tumor initiation and progression. Like normal stem cells, these cells retain self-renewal property and therefore, can differentiate into multiple tumor types. Despite this clinical importance, how CSCs are regulated and their exact pathological role are yet to be elucidated. Recent studies have shed light on the potential role of miRNAs in regulating CSCs. In this review, we summarize the current findings of miRNAs in the regulation of CSCs through different self-renewal pathways and the potential therapeutic implications of miRNAs in clinical settings by targeting CSCs.

Abstract 4098: SPARC expression stimulates paracrine inhibitory response from bone marrow stroma during dormancy of prostate cancer in the bone

Recurrent disease is the most daunting aspect of cancer treatment; however, how tumor cells become dormant and later recur even years after “successful” treatment is poorly understood. However, studies to decipher mechanisms responsible for dormancy have been hampered due to lack of appropriate model. In this study, we isolated two syngeneic cell lines (Indolent and Aggressive), which recapitulates dormancy and recurrence of prostate cancer in the bone microenvironment. We found that Indolent cells retained dormant phenotype whereas Aggressive cells grew rapidly in the tibial bone in vivo whereas the in vitro cell proliferation, invasion, migration and self-renewal properties of both cells in culture were not altered, suggesting the role of microenvironment in regulation of dormancy and recurrence. The expression profile by our microarray analysis revealed that SPARC and Noggin (a known inhibitor of BMP7) were significantly upregulated in Indolent and Aggressive cells, respectively. SPARC secreted by Indolent cells was found to stimulate BMP7 expression in bone stromal cells that in turn inhibited cancer cells by activating the dormancy-associated p38 MAPK pathway and its downstream cell cycle inhibitors, p21 and p18. In addition, BMP7 increased senescence and diminished stem-cell phenotype of cancer cells, which was rescued by addition of recombinant Noggin. We also found that BMPR2 plays a crucial role in SPARC-induced paracrine inhibition of tumor cells residing in the bone. Accordingly, BMPR2 knockdown rescued the BMP7-mediated decrease in stemness. Importantly, the BMPR2 correlative signature was enriched in patients who did not experience recurrence for a long period of time, which further verified the role of BMPR2 downstream signaling in dormancy. When primary tumor samples were examined by immunohistochemistry, both SPARC and BMPR2 were found to be significantly downregulated in patients with bone metastasis. Moreover, patients who did not experience bone metastasis were found to express high level of both SPARC and BMPR2. Importantly, we also observed elevated expression of DNA methylase genes, DNMT1 and -3B in Aggressive cells. Treatment of Aggressive cells with NS398, a COX-2 inhibitor downregulated DNMTs and concomitantly augmented SPARC expression in vitro. Therefore, recurrence of cancer cells in the bone microenvironment involves epigenetic regulation of SPARC and disruption of inhibitory crosstalk of cancer cells with the stroma. These findings suggest that SPARC plays a critical role in maintaining dormancy of prostate cancer cells in bone microenvironment. Citation Format: Sambad Sharma, Fei Xing, Kerui Wu, Yin Liu, Kounosuke Watabe. SPARC expression stimulates paracrine inhibitory response from bone marrow stroma during dormancy of prostate cancer in the bone. [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 4098.

Abstract B22: Loss of LncRNA XIST induces Epithelial to Mesenchymal Transition in Breast Cancer

Brain is one of the major sites of metastasis of breast cancer, and approximately 20% of patients with aggressive breast cancer eventually develop the metastatic disease in the brain. Long non-coding RNAs (lncRNA) have recently drawn much attention due to their wide functional variations and potential roles in tumor progression. By performing lncRNA array analysis comparing non-metastatic primary tumors with brain metastatic tumors from breast cancer patients, we identified that lncRNA XIST expression was significantly down-regulated in brain metastatic tumors. The result of Taqman PCR validated the results in tumor samples and also indicated that XIST expression was down-regulated in brain metastatic cell lines compared to non-brain metastatic cell lines. Moreover, we found that the XIST expression was negatively correlated with breast cancer stage and brain metastasis-free survival. XIST is located on the q arm of the X chromosome (Xq13-2) and plays a critical role in the X inactivation, thus providing dosage equivalence between males and females. Importantly, XIST has been found to be down-regulated in several types of tumors including breast cancer. Loss of XIST facilitates escape of some X chromosome genes from inactivation. To further study the effect of XIST down-regulation on tumor cells, we knocked down XIST expression in MCF7 by siRNA. We found that the knockdown of XIST drastically altered the morphology of MCF7 cell from epithelial to mesenchymal type. Furthermore, several epithelial-mesenchymal transition (EMT)-related genes including ZEB2 and SNAIL were upregulated in the XIST knockdown cells. In addition, the results of qPCR, western blot and immunocytochemical analysis showed that the epithelial marker E-cad was significantly down-regulated, while mesenchymal markers N-cad and Vimentin were upregulated in MCF7-siXIST, SKBR-siXIST and ZR751-siXIST cells compared to the control cells. To further examine the effect of XIST knockdown in vivo, we transplanted MCF7-siXIST, SKBR3-siXIST and their control cells by intra-cardiac injection into nude mice. We found that knockdown of XIST significantly enhanced tumor metastasis, especially in the brain. Our results strongly suggest that lncRNA XIST plays a critical role in tumor progression and metastasis by inducing EMT. Citation Format: Yin Liu, Fei Xing, Kerui Wu, Sambad Sharma, Kounosuke Watabe. Loss of LncRNA XIST induces Epithelial to Mesenchymal Transition in Breast Cancer. [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 B22.

Abstract A32: Targeting c-Met by Pterostilbene (PTER) suppresses breast cancer brain metastasis

At the late stage of breast cancer, most patients develop metastatic lesions and the brain is one of the major metastatic sites. Brain metastasis profoundly affects the cognitive and sensory function as well as morbidity of patients, and the one year survival rate among these patients remains less than 20%. The result of our newly developed gene set enrichment analysis (GSEA)-based pathway screening indicates that c-Met pathway is highly activated among the patients who developed early brain metastasis. To identify a novel target of c-Met pathway in brain metastasis, we performed the microarray analysis in two brain metastatic cell lines with the expression of doxycycline-inducible sh-cMet. Our results indicate that a group of inflammatory cytokines including IL1β, IL8 and CXCL1 were significantly induced by c-Met activation. Furthermore, we found that IL1β was able to induce the secretion of HGF (hepatocyte growth factor) from tumor associated astrocytes (TAA) which in turn activates c-Met pathway in cancer cells. Our results of endothelial cell tube formation assays also strongly suggest that c-Met-induced IL8 and CXCL1, promote tumor angiogenesis which is essential for the metastatic growth of cancer cells. Natural compounds (NC) have been extensively studied for their anti-tumor effects. However, much less studies have been done on NC for the treatment/prevention of brain tumors mainly due to the obstacle of Blood-brain-barrier (BBB) permeability. To identify the NCs that can be used for treating brain metastasis, we performed pathway analysis which only focused on BBB-permeable NC. The results of GSEA indicate that Resveratrol-targeting genes were significantly enriched among those patients who developed brain metastasis compared to metastasis-free patients. Furthermore, we found that the one of derivatives of Resveratrol, Pterostilbene (PTER), showed significantly more potent activity than Resveratrol and suppressed brain metastasis in vitro and in vivo by targeting the c-Met oncogene. These findings suggest that TAA-mediated c-Met activation plays a key role in brain metastasis and that PTER is a potential therapeutic agent to treat brain metastasis by suppressing the c-Met expression. Citation Format: Fei Xing, Sambad Sharma, Yin Liu, Kerui Wu, Kounosuke Watabe. Targeting c-Met by Pterostilbene (PTER) suppresses breast cancer brain metastasis. [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 A32.

Abstract 3206: SPARC in tumor microenvironment induces dormancy of prostate cancer in bone

Despite significant advancement in early detection and treatment of prostate cancer, approximately 35% of patients who had surgically resected primary tumor experience recurrent disease after years of the treatment, suggesting that tumor cells disseminated before surgery remain dormant in patients for a period of time and awaken as recurrent disease. Prostate cancer is known to frequently recur in the bone; however, how dormant cells switch its phenotype leading to recurrent tumor remains poorly understood. We have isolated two syngeneic cell lines (Indolent and Aggressive), derived from in vivo selection after injection of PC3mm stem-like cells as a model system, which recapitulates dormancy and recurrence in bone microenvironment. We found that Indolent cells retained dormant phenotype whereas Aggressive cells grew rapidly in the tibial bone in vivo while the growth rates of both cells in culture were similar, suggesting the role of microenvironment in regulation of dormancy and recurrence. The expression profile by our microarray analysis revealed that SPARC and Noggin (a known inhibitor of BMP7) were significantly upregulated in Indolent and Aggressive cells, respectively. Furthermore, SPARC secreted by Indolent cells was found to stimulate BMP7 and DKK1 in microenvironmental cells (bone stromal cells and bone mesenchymal stem cells, respectively) that in turn kept cancer cells in dormant state. We previously showed that BMP7 secreted by bone stromal cells induced senescence in prostate cancer stem-like cells leading to dormancy of tumor cells. On the other hand, DKK1, an inhibitor of the Wnt pathway, suppressed Wnt-induced growth and decreased self-renewal of prostate cancer stem-like cells. We also found that DKK1 inhibited prostate cancer growth by inducing G1 cell cycle arrest. In addition, Wnt was found to up-regulate Noggin expression which might be a possible switch toward recurrence. Importantly, we observed elevated expression of a DNA methylase gene, DNMT3B, in Aggressive cells. Treatment of Aggressive cells with 5-Azacytidine reversed the SPARC gene expression in vitro, suggesting that the SPARC gene is epigenetically controlled by microenvironment via downregulation of the DNMT3B methylase gene. Furthermore, when Aggressive cells were cultured in the conditioned medium (CM) prepared from Indolent CM-treated bone mesenchymal cells, cell proliferation was found to be significantly reduced. Therefore, Wnt potentially serves as a recurrent factor in the bone microenvironment by upregulating Noggin and by inhibiting the effect of BMP7 on cancer stem-like cells. These findings suggest that SPARC plays a key role in maintaining dormancy of prostate cancer cells in bone microenvironment. Citation Format: Sambad Sharma, Fei Xing, Yin Liu, Kerui Wu, Aya Kobayashi, Kounosuke Watabe. SPARC in tumor microenvironment induces dormancy of prostate cancer in bone. [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 3206. doi:10.1158/1538-7445.AM2015-3206