Survival Of Metastatic Cancer Cells Research Articles

Abstract 3144: Pleiotrophin drives a pro-metastatic immune niche within breast tumor microenvironment

Abstract Immune cells in the tumor microenvironment (TME) can impact cancer cell invasion, seeding and colonization of secondary metastatic sites. Our findings show that Pleiotrophin (PTN), a unique and previously under-studied heparin binding cytokine, contributes to inflammation within the TME. PTN appears to be particularly efficient in recruiting neutrophils, which have been reported to cause immune suppression. Consequently, inhibition of PTN pharmacologically or genetically results in lower breast cancer metastasis to lungs and better survival in multiple mouse models of breast cancer. Corroborating our mouse studies, we have found that higher PTN expression in breast cancer patient plasma correlates with poor prognosis. Concurrently, stage IV breast cancer patients that have lower PTN expression have a highly significant survival advantage (median survival 78.27 months) over patients expressing high levels of PTN (median survival 27.5 months). Additionally, we have also found PTN positive cancer cells are enriched in metastatic lesions suggesting that PTN-high cancer cells are more successful at colonizing and surviving at the secondary site. Uncovering the function of PTN in these unique cancer cells might have a major impact in the clinical setting. Overall, our data suggests that PTN is important in driving a pro-metastatic immune niche within the TME that promotes tumor cell escape from the primary tumor and survival of metastatic cancer cells in secondary sites. Additionally, our studies highlight that inhibition of PTN has potential to reduce metastatic burden in breast cancer and suggest that future studies testing the combination of PTN inhibition with standard chemotherapy or immune therapy are warranted. Citation Format: Debolina Ganguly, Tuong V. Ngo, Morgan Coleman, Noah Sorrelle, Adrian Dominguez, Jason Toombs, Marcel Schmidt, Fa V. Mora, David G. Ortega, Anton Wellstein, Rolf A. Brekken. Pleiotrophin drives a pro-metastatic immune niche within breast tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3144.

Different Membrane Environments Generate Multiple Functions of P-type Ion Pumps

P-type ion pumps (P-type ATPases) are involved in various fundamental biological processes. For example, the gastric proton pump (H+,K+-ATPase) and sodium pump (Na+,K+-ATPase) are responsible for secretion of gastric acid and maintenance of cell membrane potential, respectively. In this review, we summarize three topics of our studies. The first topic is gastric H+,K+-ATPase associated with Cl--transporting proteins (Cl-/H+ exchanger ClC-5 and K+-Cl- cotransporter KCC4). In gastric parietal cells, we found that ClC-5 is predominantly expressed in intracellular tubulovesicles and that KCC4 is predominantly expressed in the apical membrane. Gastric acid (HCl) secretion may be accomplished by the two different complexes of H+,K+-ATPase and Cl--transporting protein. The second topic focuses on the Na+,K+-ATPase α1-isoform (α1NaK) associated with the volume-regulated anion channel (VRAC). In the cholesterol-enriched membrane microdomains of human cancer cells, we found that α1NaK has a receptor-like (non-pumping) function and that binding of low concentrations (nM level) of cardiac glycosides to α1NaK activates VRAC and exerts anti-cancer effects without affecting the pumping function of α1NaK. The third topic is the Na+,K+-ATPase α3-isoform (α3NaK) in human cancer cells. We found that α3NaK is abnormally expressed in the intracellular vesicles of attached cancer cells and that the plasma membrane translocation of α3NaK upon cell detachment contributes to the survival of metastatic cancer cells. Our results indicate that multiple functions of P-type ion pumps are generated by different membrane environments and their associated proteins.

Anoikis resistant gastric cancer cells promote angiogenesis and peritoneal metastasis through C/EBPβ-mediated PDGFB autocrine and paracrine signaling

Anoikis is a type of programmed cell death induced by loss of anchorage to the extracellular matrix (ECM). Anoikis resistance (AR) is crucial for the survival of metastatic cancer cells in blood, lymphatic circulation and distant organs. Compared to ordinary cancer cells, anoikis resistant cancer cells undergo various cellular and molecular alterations, probably characterizing the cells with unique features not limited to anoikis resistance. However, the molecular mechanisms connecting anoikis resistance to other metastatic properties are still poorly understood. Here, the biological interaction between anoikis resistance and angiogenesis as well as their involvement into peritoneal metastasis of gastric cancer (GC) were investigated in vitro and in vivo. The prognostic value of key components involved in this interaction was evaluated in the GC cohort. Compared to ordinary GC cells, GCAR cells exhibited stronger metastatic and pro-angiogenic traits corresponding to elevated PDGFB secretion. Mechanistically, transcription factor C/EBPβ facilitated PDGFB transcription by directly binding to and interacting with PDGFB promoter elements, subsequently increasing PDGFB secretion. Secreted PDGFB promoted the survival of detached GC cells through a C/EBPβ-dependent self-feedback loop. Moreover, secreted PDGFB promoted angiogenesis in metastases via activation of the MAPK/ERK signaling pathway in vascular endothelial cells. Both C/EBPβ activation level and PDGFB expression were significantly elevated in GC and correlated with metastatic progression and poor prognosis of patients with GC. Overall, interaction between GCAR cells and vascular endothelial cells promotes angiogenesis and peritoneal metastasis of GC based on C/EBPβ-mediated PDGFB autocrine and paracrine signaling. C/EBPβ-PDGFB-PDGFRβ-MAPK axis promises to be potential prognostic biomarkers and therapeutic targets for peritoneal metastasis of GC.

Abstract PO021: Carcinoma-associated mesenchymal stem/stromal cells enhance ovarian cancer metastasis and increase cancer cell clonal heterogeneity through direct mitochondrial transfer

Abstract Ovarian cancer is the most deadly gynecologic cancer largely due to early, diffuse metastatic spread. The ovarian cancer tumor microenvironment (TME) significantly impacts the metastatic capacity of ovarian cancer. We recently demonstrated that a critical stromal progenitor cell within the TME, termed carcinoma-associated mesenchymal stem/stromal cells (CA-MSCs), dramatically enhance the metastasis of ovarian cancer cells. CA-MSCs directly bind to cancer cells forming heterocellular units which co-metastasize. The goal of this work is to understand the mechanism driving CA-MSC-mediated ovarian cancer metastasis. We hypothesize that CA-MSCs increase the metastatic potential of ovarian cancer cells during the process of co-metastasis. Ovarian cancer uses both hematogenous and transcoelomic routes of metastasis therefore we used two ovarian cancer murine models to evaluate both modes of metastasis. We used a genomic barcode system to assess the clonal patterns of metastasis. Ovarian cancer cells with one unique barcode per cell were grown with or without CA-MSCs. Sites of metastasis were quantified and cancer cells were harvested from each site. Barcodes were sequenced to determined clonal representation at each metastatic site. We found CA-MSCs increased the number of metastatic sites in both the hematogenous and transcoelomic mouse models. Interestingly, CA-MSCs also increased the clonal heterogeneity at all metastatic sites (2.5 - 5 fold increase in blood, lung and liver). To investigate the mechanism enabling CA-MSC-mediated enhancement of clonal heterogeneity, we studied primary patient derived CA-MSCs and ovarian cancer cells. The process of metastasis poses unique metabolic stresses on cancer cells and normal mesenchymal stem cells are known to donate mitochondria to damaged epithelial cells. We therefore assessed if CA-MSCs use a similar mechanism to enhance survival of metastatic cancer cells. We stably labeled CA-MSC mitochondria with COX8-GFP via lentiviral transduction to enable the visualization and quantification of potential mitochondrial transfer. We demonstrate that CA-MSCs actively transfer mitochondria to cancer cells. This transfer is dependent on the physical interaction of CA-MSCs with cancer cells and is increased 2-4 fold when CA-MSCs and cancer cells form metastatic heterocellular units under non-adherent conditions. We tested the functional consequences of CA-MSC to cancer cell mitochondrial transfer and demonstrate cancer cells which receive mitochondria have increased chemotherapy resistance, increased sphere forming capacity and stem-like properties. Collectively, our results indicate CA-MSCs are a significant driver of ovarian cancer metastasis by forming heterocellular metastatic units which enable CA-MSC to cancer cell mitochondrial transfer. This leads to increased cancer cell survival and tumor initiation properties and represents a novel method of maintaining cancer cell heterogeneity during metastasis. Citation Format: Catherine A. Pressimone, Leonard G. Frisbie, Alexander Pearson, Lan G. Coffman. Carcinoma-associated mesenchymal stem/stromal cells enhance ovarian cancer metastasis and increase cancer cell clonal heterogeneity through direct mitochondrial transfer [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO021.

Abstract LT010: Pleiotrophin drives pro-metastatic immune niche within breast tumor microenvironment

Abstract Background: Pleiotrophin (PTN), a heparin binding cytokine important during embryonic development, is expressed at elevated levels in several cancers, including breast cancer. High PTN expression in serum and tumor tissues of breast cancer patients correlates with worse outcome. Previous studies indicate PTN contributes to angiogenesis, hematopoiesis, and inflammation; however, the function of PTN in breast cancer progression and metastasis has not been elucidated. Methods: The effect of anti-PTN therapy (mAb; 3B10) on breast tumor progression and pulmonary metastasis was evaluated in four preclinical models of breast cancer: 4T1, E0771, Met-1, MMTV-PyMT. In addition, the effect of global loss of PTN using Ptn-deficient mice was evaluated in two murine models of breast cancer. Tissues from these experiments were analyzed by RNAseq and histology for changes in angiogenesis, hematopoiesis, immune cell infiltration, and cytokine/chemokine expression. Findings: TCGA data shows high PTN expression correlates with poor survival of Stage IV breast cancer patients. Furthermore, our data indicate that: a) blocking PTN pharmacologically or genetically results in a significant reduction of lung metastasis in multiple preclinical mouse models of breast cancer, b) scRNAseq and dual FISH-IHC (fluorescence in-situ hybridization and immunohistochemistry) demonstrates that endothelial cells and select population of tumor cells express PTN in the tumor microenvironment (TME), c) inhibition of PTN results in a decrease in overall inflammation with a particular decrease in neutrophil populations at primary and secondary tumor sites, d) in a genetically engineered mouse model (GEMM) of breast cancer, MMTV-PyMT, PTN expression is enriched at the metastatic site. Conclusion: Our data suggests that PTN is important in driving a pro-metastatic immune niche within the TME that promotes tumor cell escape from the primary tumor and survival of metastatic cancer cells in secondary sites. Additionally, our studies highlight that inhibition of PTN has potential to reduce metastatic burden in breast cancer and suggest that future studies testing the combination of PTN inhibition with standard chemotherapy or immune therapy are warranted. Citation Format: Debolina Ganguly, Noah Sorrelle, Adrian Dominguez, Jason Toombs, Marcel Schmidt, Fa Valdes Mora, David Gallego Ortega, Anton Wellstein, Rolf A. Brekken. Pleiotrophin drives pro-metastatic immune niche within breast tumor microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT010.

Benzoxazine Dimer Analogue Targets Integrin β3 in Lung Cancer Cells and Suppresses Anoikis Resistance and Migration.

Certain integrins including integrin β3 facilitate movement and survival of metastatic cancer cells. We examined whether benzoxazine dimer analogue N,N-bis(5-ethyl-2-hydroxybenzyl) methylamine (HM) has anti-metastatic effects. Cell viability was examined by the 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. Wound healing and phalloidin-rhodamine assays were performed to evaluate the migration and filopodia formation, respectively. Anoikis resistance was studied by anchorage-independent growth assay. The expression of proteins regulating migration were examined by western blot. HM treatment significantly inhibited growth and survival of detached lung cancer cells as indicated by the reduced colony number and size of anchorage-independent growth analysis. HM inhibited cell migration and suppressed filopodia formation. Protein analysis indicated that the compound dramatically decreased integrin β3 and its related downstream proteins including active focal adhesion kinase (FAK) and active protein kinase B (AKT); however, integrin β1 and α5 were found to be unaltered. HM shows a potential in targeting integrin β3 and could be a good candidate for further developed as an anti-metastatic therapy.

MiR-137 promotes anoikis through modulating the AKT signaling pathways in Pancreatic Cancer.

Anoikis resistance is a fundamental feature of the survival of metastatic cancer cells during cancer progression. However, the mechanisms underlying anoikis resistance in pancreatic cancer (PC) are still unclear. MicroRNA-137 (miR-137) is a tumor suppressor that inhibits the proliferation and invasion of cancer cells through targeting multiple oncogenes. However, the effects and molecular mechanism of miR-137 on anoikis of PC are still unclear. Here we demonstrated that miR-137 was downregulated after the induction of anoikis model in time dependent. Function assays revealed that miR-137 promoted the pancreatic cancer cells anoikis in vitro and vivo. According to bioinformation analysis of clinical databases, we predicted that paxillin (PXN) was a target of miR-137. Further, TCGA analysis revealed that PXN was closely associated with the development of PC. Through loss-of-function studies, we demonstrated that PXN was a functional target of miR-137 on anoikis of PC cells. Moreover, we found that PXN promoted the activation of the AKT signaling pathways which was involving in the cancer cells anoikis. Together, our findings reveal that miR-137 plays a novel role during anoikis and may serve as a potential target for the detection and treatment of PC.

Abstract 805: Adaptation of mitochondrial organization to aggregation in serous ovarian cancer

Abstract During metastasis, ovarian cancer cells exfoliate from the primary tumor and disseminate throughout the peritoneal cavity mostly as aggregates with a higher survival rate and metastatic potential. To determine how the mitochondrial morphology and dynamic expression of fusion and fission-regulating proteins contribute to spheroid survival, in this study we investigated changes in mitochondrial morphology in order to identify phenotypic alterations that are associated with changes in mitochondrial function. We compared monolayers and spheroids of murine and human benign, late-stage, and tumor-initiating cells in hypoxia using fluorescent microscopy and western blot analysis. In addition, we investigated how the mitochondrial membrane potential (Δψm) and ROS production contributed to this phenotype using the InCell analyzer with TMRM and Amplex Red dyes respectively. The data from this study suggest that the morphology and dynamic protein expression are highly altered during tumorigenesis, aggregation, and in hypoxic conditions, indicating a direct correlation of mitochondrial morphology in regulating survival, apoptosis and proliferation. The identification of these molecular events and changes that allow for the survival of metastatic cancer cells in an environment low in oxygen and nutrients may provide specific targets for interventions that would suppress successful metastatic outgrowth of disseminating cells and could potentially be used as a biomarker for treatment efficacy. Citation Format: Joseph P. Grieco, Stephanie Edwards, Mitchell Allen, Yao Wang, Justin Perry, Yipei Song, Nathan Swami, David Brown, Eva Schmelz. Adaptation of mitochondrial organization to aggregation in serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 805.

Abstract 5863: RGX-202, a first-in-class small-molecule inhibitor of the creatine transporter SLC6a8, is a robust suppressor of cancer growth and metastatic progression

Abstract Background: Colorectal cancer (CRC) is one of the leading causes of cancer deaths worldwide with more than 140,000 patients diagnosed and nearly 50,000 deaths annually in the U.S. alone. Roughly 60% of patients present with locally advanced or distant metastatic disease, with the liver being a primary site of metastatic colonization. Creatine metabolism has been implicated in colon cancer progression and metastatic colonization of the liver. Metastatic colon cancer cells upregulate and release creatine kinase-B (CKB) into the extracellular space, where it phosphorylates creatine to generate the high-energy metabolite phosphocreatine. Phosphocreatine is imported via the creatine transporter SLC6a8. Intracellular phosphocreatine can be converted to ATP to fuel the survival of metastatic cancer cells within the hypoxic hepatic microenvironment. Consistent with this finding, genetic depletion of SLC6a8 in colon and pancreatic cancer cell lines significantly reduced liver colonization in mouse xenograft models. Results: We herein demonstrate that the novel small molecule RGX-202 is a robust inhibitor of creatine uptake in cancer cells, both in vitro and in vivo. Oral administration of RGX-202 induced apoptosis of colon cancer cells in vivo, and significantly suppressed colon cancer liver metastatic colonization and primary tumor growth, both in KRAS wild-type and KRAS mutant colon cancer cell lines as well as in human PDX mouse models. Using genetic studies, these effects were found to be dependent on tumoral expression of SLC6a8. In addition, combination treatment of the CT26 syngeneic colon cancer mouse model with 5-FU resulted in synergistic antitumor activity, with complete tumor regressions observed in more than 40% of treated mice. Similarly, combination treatment of the KPC syngeneic mouse model with gemcitabine significantly reduced the growth of primary pancreatic tumors. Definitive 28-day GLP toxicology and pharmacokinetics studies of RGX-202 are currently ongoing. Preliminary observations suggest good tolerability in several animal species with a favorable pharmacokinetic profile, including bioavailability. Conclusion: These results strongly support clinical development of RGX-202 in patients with gastrointestinal cancers, including colorectal and pancreatic cancer, both as monotherapy and in combination with standard-of-care treatment. Citation Format: Isabel Kurth, Celia Andreu, Shugaku Takeda, Helen Tian, Foster Gonsalves, Katya Leites, Subhasree Sridhar, Jia Min Loo, Rob Busby, Sohail Tavazoie, Masoud Tavazoie. RGX-202, a first-in-class small-molecule inhibitor of the creatine transporter SLC6a8, is a robust suppressor of cancer growth and metastatic progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5863.

Abstract 4344: Cd146 modulates the malignant phenotype in human prostate cancer

Abstract Prostate Cancer (PCa) is the most frequent cancer in males and the second leading cause of death from cancer in men. When PCa progress from androgen-responsive to castration resistance, the formation of incurable metastases, mainly in the bone, is almost inevitable. Therefore, understanding the factors that regulate homing and survival of metastatic cancer cells in the bone is important for the identification of new therapeutic targets. High CD146 expression has been measured in the stroma of lytic and blastic lesions in preclinical models of PCa bone metastasis. The objective of this study is to characterize the role of CD146 in the maintenance of the aggressive and invasive phenotype in human PCa. We used shRNAs to knockdown the expression of CD146 in the lytic PC-3M-Pro4Luc2dTomato and in the blastic C4-2BdTomato PCa cell lines. We validated the knockdown at protein level and tested the effect with functional assays such as migration, proliferation. We used RT-qPCR to test CD146 knockdown on EMT markers. We measured the effect of the knockdown on the maintenance of cancer stem/progenitor-like cells by ALDEFLUOR assay. CD146 knockdown reduced proliferation in PC-3M-Pro4Luc2dTomato PCa cells and resulted in increased E-Cadherin expression. Conversely, no effect on proliferation was measured on C4-2BdTomato cells. It has been described that metastatic human PCa cells target the hematopoietic stem cell (HSC) niche in the bone marrow at the level of an “endosteal/osteoblast” niche and a “vascular/perivascular” niche. We optimized an in vitro model of “osteoblast niche” to study the behavior of prostate cancer cells upon co-culture with osteoblasts and to measure the resulting effects on cancer stem/progenitor-like markers. Our results showed that CD146 is required for the osteoblast-mediated induction of ALDH activity on PCa cells and CD146 knockdown prevented the increase in the size of the ALDHhigh subpopulation in the tumor cells, mediated by human osteoblasts. Additionally, CD146 knockdown in PCa cells co-cultured with osteoblast, reduced the amount of CD146 expressed by osteoblasts compared to non-targeted control. Finally, we showed that CD146 is significantly increased in the highly metastatic ALDHhigh cells and identified a new subset of ALDHhigh / CD146high cells which could be depleted upon CD146 knockdown. In Conclusion, we detected a novel subset of ALDHhigh/CD146high cells and found that CD146 influences the maintenance of an aggressive-mesenchymal phenotype in human PCa. Therefore, CD146 represents a promising molecule to modulate the behavior of aggressive PCa cells. Note: This abstract was not presented at the meeting. Citation Format: Eugenio Zoni, Letizia Astrologo, Janine Melsen, Sofia Karkampouna, Irena Klima, Joël Grosjean, Peter C. Gray, Gabri van der Pluijm, Marco G. Cecchini, Marianna Kruithof-de Julio, George N. Thalmann. Cd146 modulates the malignant phenotype in human prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4344. doi:10.1158/1538-7445.AM2017-4344

Effect of MCAM on the aggressive phenotype in human prostate cancer.

e23135 Background: Prostate Cancer (PCa) is the most common cancer in males and the second leading cause of death from cancer in men. Understanding the factors that regulate homing and survival of metastatic cancer cells in the bone is important for the identification of new therapeutic targets. High MCAM expression has been detected in the stroma of lytic and blastic lesions in preclinical models of PCa bone metastasis. The objective of this study is to characterize the role of MCAM in the maintenance of the aggressive phenotype in human PCa. Methods: We knocked and down MCAM in the lytic PC-3M-Pro4Luc2_dTomato and in the blastic C4-2B_dTomato PCa cell lines. Validation was done at both protein and RNA level. We performed functional assays such as migration and proliferation. RT-qPCR was used to test MCAM knockdown on EMT markers. The effect of the knockdown on the maintenance of cancer stem/progenitor-like cells was measured by ALDEFLUOR. Results: MCAM knockdown reduced proliferation in PC-3M-Pro4Luc2_dTomato PCa cells and resulted in increased E-Cadherin expression. Metastatic human PCa cells target the hematopoietic stem cell (HSC) niche in the bone marrow at the level of an “endosteal/osteoblast” niche and a “vascular/perivascular” niche. We set-up an in vitro model of “osteoblast niche” to study the prostate cancer cells upon co-culture with osteoblasts and to determine the effects on cancer stem/progenitor-like markers. We found that MCAM is required for the osteoblast-mediated induction of ALDH activity on PCa cells and MCAM knockdown prevented the increase in the size of the ALDHhigh subpopulation in PC-3M-Pro4Luc2_dTomato, mediated by human osteoblasts. Additionally, MCAM knockdown in PCa cells co-culture with osteoblast, prevented the induction of MCAM expression by osteoblasts. Finally, MCAM is significantly increased in the ALDHhigh cells and identifies a new subset of ALDHhigh / MCAMhigh cells which could be depleted upon MCAM knockdown. Conclusions: We detected a new subset of ALDHhigh/MCAMhigh cells and demonstrated the MCAM influences the maintenance of an aggressive-mesenchymal phenotype in human PCa. Therefore, MCAM represent an interesting target molecule to modulate the behavior of aggressive PCa cells.

MiR-221 to modulate tumor growth in vivo and as a regulator of TGF.

e23008 Background: Despite the advances in cancer therapy, when Prostate Cancer (PCa) progress to castration resistant phase, patients develop incurable bone metastases. Understanding the processes that regulate homing and survival of metastatic cancer cells in the bone is crucial for the identification of new therapies. TGF-β signaling plays a major role in bone remodeling and according to the “vicious cycle hypothesis” is a master regulator of maintenance of prostate cancer cells in lytic bone lesions. microRNAs (miRs) are a class of small non-coding RNAs that regulates many biological process. miR-221 expression has been previously associated with prostate cancer progression. Here we studied the effect of miR-221 on TGF-β signaling and the impact of miR-221 on tumor growth in vivo Methods: miR-221 was overexpressed in PC3 and RWPE-1 cells and PMEPA was monitored by RT-qPCR and western blot. Expression of miR-221 in PC3 and RWPE-1 cells was assessed by RT-qPCR. Luciferase assay was used to investigate the interaction between miR-221 and PMEPA1. For zebrafish experiment, fluorescently labelled PC-3M-Pro4 cells overexpressing miR-221 were injected in the Duct of Cuvier (DC) of zebrafish embryos. Results: miR-221 overexpression resulted in decreased PMEPA1 mRNA and protein in PC-3 cells. Luciferase reporter assay indicated that miR-221 can directly interact with PMEPA1 3’ UTR. We show an enhancement of the proliferative effect of TGF-β in PC-3 cells, following miR-221 overexpression. In conclusion, we observed increased Smad2 activation upon TGF-β treatment in miR-221 overexpressing PC-3 cells. Inoculation of PC-3M-Pro4 cells overexpressing miR-221 in the DC of zebrafish embryos resulted reduced tumor burden compared to control. Finally, we observed inverse correlation between miR-221 and PMEPA1 expression in normal vs. tumor tissue collected from PCa patients. Conclusions: Our results indicate that miR-221 is a regulator of TGF-β signaling via modulation of PMEPA1 and miR-221 overexpression can reduce tumor growth in vivo.

Omentum and bone marrow: how adipocyte-rich organs create tumour microenvironments conducive for metastatic progression.

A number of clinical studies have linked adiposity with increased cancer incidence, progression and metastasis, and adipose tissue is now being credited with both systemic and local effects on tumour development and survival. Adipocytes, a major component of benign adipose tissue, represent a significant source of lipids, cytokines and adipokines, and their presence in the tumour microenvironment substantially affects cellular trafficking, signalling and metabolism. Cancers that have a high predisposition to metastasize to the adipocyte-rich host organs are likely to be particularly affected by the presence of adipocytes. Although our understanding of how adipocytes influence tumour progression has grown significantly over the last several years, the mechanisms by which adipocytes regulate the metastatic niche are not well-understood. In this review, we focus on the omentum, a visceral white adipose tissue depot, and the bone, a depot for marrow adipose tissue, as two distinct adipocyte-rich organs that share common characteristic: they are both sites of significant metastatic growth. We highlight major differences in origin and function of each of these adipose depots and reveal potential common characteristics that make them environments that are attractive and conducive to secondary tumour growth. Special attention is given to how omental and marrow adipocytes modulate the tumour microenvironment by promoting angiogenesis, affecting immune cells and altering metabolism to support growth and survival of metastatic cancer cells.

Abstract 3526: Novel function of NRP2 in therapy resistant metastatic cancer

Abstract Neuropilins (NRPs) are cell surface glycoproteins known for their role as co-receptors for plexins and VEGF family members in neuronal development and vascular and lymphatic growth. Neuropilin-2 (NRP2), a family member of NRPs has been reported to enhance the survival of cancer cells following therapeutic stress. In a recent study, our group has shown that the expression of NRP2 can be a poor prognostic factor for invasive bladder cancer patients treated with radiochemotherapy. Its expression correlated with an increased risk of an early cancer specific death and significantly decreased cancer specific survival among these patients supporting its role as a promoter of therapy resistance. This function of NRP2 is distinctly different from its known function as a co-receptor. In this study, we have provided the molecular mechanism of how NRP2 imparts therapy resistance in cancer cells. Our results indicate that depletion of NRP2 inhibits autophagy, a process which promotes survival of cancer cells during therapeutic stress. Further, we observed a role of NRP2 in the maturation of early to late endosomes. Since late endosomes are required for the fusion of autophagosomes with lysosomes, the decrease in the formation of late endosomes following NRP2 depletion can explain the defect in autophagy observed during inhibition of the NRP2 axis. We have provided a detailed molecular mechanism of how the recruitment and functions of several endocytic proteins such as EHD4, Rubicon, Mon1 and PIKFYVE are affected due to the depletion of NRP2, which resulted in the defect in endosomal trafficking. Recent evidences indicate that cancer cells maintain a high endosomal trafficking activity and the defect in trafficking promotes aberrant cellular signaling cascades either by interfering with receptor recycling to the cell surface or by inhibiting their lysosomal degradation. In this respect our data demonstrate that depletion of the NRP2 axis leads to an increased accumulation of active EGFR in early endosomes, which induces aberrant signaling cascade to promote apoptosis in cancer cells. We were also able to identify a novel downstream target of NRP2 axis known as WD Repeat and FYVE Domain Containing 1 (WDFY1). Our data indicate that depletion of NRP2 transcriptionally upregulates the expression of WDFY1. This is achieved due to an inhibition of the nuclear localization of a transcriptional inhibitor FAC1. Overall, our data suggest that NRP2 axis maintains the expression of WDFY1 in cancer cells to promote enhanced level of endocytic acitivity and autophagy. NRP2 is therefore important for the progression and survival of metastatic cancer cells and targeting this axis is crucial to enhance therapeutic efficacy of aggressive malignancies. Citation Format: Samikshan Dutta, Sohini Roy, Navatha S. Polavaram, Marissa Stanton, Surinder Batra, Michael H. Muders, Kaustubh Datta. Novel function of NRP2 in therapy resistant metastatic cancer. [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 3526.

Abstract 1134: Slug promotes survival during metastasis through suppression of Puma-mediated apoptosis

Abstract Tumor cells must overcome apoptosis to survive throughout metastatic dissemination and distal organ colonization. Here we show in the Polyoma Middle T mammary tumor model that N-cadherin causes Slug upregulation, which promotes carcinoma cell survival. Slug was dramatically upregulated in distant metastases relative to primary tumors. Consistent with a role in metastasis, Slug knockdown in metastatic carcinoma cells suppressed pulmonary colonization, due to reducing cell survival, but had no effect on tumor cell invasion or extravasation. In support of a role in cell survival, Slug inhibition by shRNA, sensitized tumor cells to apoptosis by genotoxic stress, resulting in caspase-3 activation and PARP cleavage. Furthermore, the effects of Slug on cell survival were due to direct repression of the pro-apoptotic gene, Puma. Importantly, the survival function of the SLUG-PUMA axis was confirmed in human breast cancer cells, where Slug knockdown increased Puma expression and inhibited lung colonization. Thus, our study demonstrates a pivotal role for Slug in cell survival, which is beyond its role in epithelial-to-mesenchymal transition, implying that disruption of the SLUG-PUMA axis may impinge on the survival of metastatic cancer cells. Citation Format: Rachel Hazan. Slug promotes survival during metastasis through suppression of Puma-mediated apoptosis. [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 1134. doi:10.1158/1538-7445.AM2014-1134

Enhanced Metastasis Suppression by Targeting TRAIL Receptor 2 in a Murine Model of Triple-Negative Breast Cancer

Metastatic breast cancer is a deadly disease which requires new therapeutic strategies. Endogenous TNF-related apoptosis-inducing ligand (TRAIL) functions as a metastasis suppressor by activating proapoptotic TRAIL receptors (TRAIL-R1/DR4 and/or TRAIL-R2/DR5) in transformed cells, making it an attractive pathway for antimetastatic therapies. However, it is unclear whether TRAIL-R1 or TRAIL-R2 is a better therapeutic target in metastatic breast cancer. Several metastatic, triple (estrogen receptor, progesterone receptor, and HER2)-negative cancer cell lines were treated with human agonistic monoclonal antibodies targeting TRAIL-R1 (mapatumumab) or TRAIL-R2 (lexatumumab). The effects on cell viability, apoptosis, and caspase-8 activation were determined. An orthotopic model of triple-negative breast cancer in which fluorescently labeled breast cancer cells metastasize from the mammary gland to lymph nodes and lung was utilized to evaluate the effects of mapatumumab, lexatumumab, or doxorubicin on primary and metastatic tumor burden in vivo. Lexatumumab was more effective than mapatumumab in activating caspase-8, inducing apoptosis and inhibiting long-term survival of metastatic cancer cells, which expressed both TRAIL-R1 and TRAIL-R2. Human mammary epithelial cells transformed by oncogenic Ras were more sensitive to lexatumumab than nontransformed cells. Lexatumumab inhibited lymph node and lung metastases more robustly than mapatumumab in an orthotopic model of triple-negative breast cancer; both agents inhibited mammary tumor growth. In addition, lexatumumab was more effective than doxorubicin at suppressing metastases at doses of doxorubicin that were associated with toxicity, even though doxorubicin reduced primary tumor burden more robustly than lexatumumab. Targeting TRAIL-R2 receptor may be an effective therapeutic strategy for metastatic breast cancer.