Adjacent Cancer Cells Research Articles

Abstract 5089: Extracellular Survivin uptake by cancer cells via receptor-mediated endocytosis

Abstract The tumor microenvironment is a crucial player in tumorigenesis, proliferation, and survival. Communication between its various components through microvesicular trafficking is central to the interaction between tumor cells and the surrounding milieu. Exosomes, nanovesicles released by all cell types, carry various proteins and nucleic acids. Once released into the extracellular environment, exosomes are taken up by surrounding cells or the exosomes travel in various bodily fluids to reach distant parts of the body. Those exosomes derived from tumor cells contain proteins and nucleic acids essential to the development and maintenance of tumors. One such protein, Survivin, a member of the Inhibitor of Apoptosis (IAP) protein family, has been identified in virtually all cancer cells. Survivin function is closely linked with subcellular location. Nuclear Survivin plays a key role in cell division and cell cycle regulation as a member of the chromosome passenger complex. Cytosolic and mitochondrial Survivin primarily facilitate the inhibition of apoptosis. Recent research has identified a unique pool of Survivin in the extracellular environment. Functions tied to this location are as yet undefined, however, evidence suggests that this population may contribute to increased proliferation, invasiveness and therapeutic resistance when taken into recipient cancer cells. Using tandem affinity purification we have identified a relationship between Survivin and TNF receptor 1 (TNFR1), as well as transferrin receptor 2 (TfR2). Both of these receptors play a role in endocytosis of extracellular proteins. Transferrin receptors are upregulated on cancer cells to facilitate the increased iron demand of proliferating cells. We therefore hypothesize that exosomal Survivin is taken up by adjacent cancer cells via receptor-mediated endocytosis. We have shown that in the presence of antibodies to these receptors, there is a decreased uptake of extracellular Survivin (from conditioned media). Results were similar when conditioned media was introduced to HeLa cells with siRNA knockdowns of each receptor, as well as to CHO cells deficient in these receptors. We conclude that uptake of extracellular Survivin into cancer cells is dependent upon these two endocytosis receptors. The relationship of Survivin and exosomes in the extracellular environment is indicative that exosomes transport Survivin out of tumor cells and into the surroundings. These results suggest that exosomal Survivin enters cancer cells by way of receptor-mediated endocytosis. Understanding this mechanism of signaling between cancer cells shows the potential intercellular trafficking of oncoproteins may play in tumor maintenance and progression. It also provides us with a novel means to combat the resulting enhanced aggressive nature of the disease. Citation Format: Amber Gonda, Salma Khan, Heather R. Ferguson Bennit, Ron B. Moyron, Nathan R. Wall. Extracellular Survivin uptake by cancer cells via receptor-mediated endocytosis. [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 5089.

Abstract 4105: Dissecting the mechanisms of hepatocyte replacement in breast cancer liver metastases

Abstract The liver is a common site of metastasis in metastatic breast cancer and is associated with significant mortality. Histopathological examination of human breast cancer liver metastases (BCLMs) reveals that the majority of these tumours present with a ‘replacement growth pattern.’ In this growth pattern, the breast cancer cells freely invade the liver parenchyma and replace the resident hepatocytes. In advanced BCLMs, breast cancer cells can eventually replace a significant volume of the liver parenchyma which, ultimately, leads to organ failure and death. However, the mechanism through which breast cancer cells replace the resident hepatocytes is unknown. Here we set out to establish how this replacement of hepatocytes by breast cancer cells occurs in BCLMs. To do so, we established two in vivo models of advanced BCLM: (a) an ER+PR+HER2- model using the MCF7 cell line, and (b) a triple negative patient-derived xenograft model using breast cancer cells isolated from a pleural effusion. We have also established an in vitro co-culture system, where MCF7 cells are co-cultured with HepG2 cells, which is designed to mimic the breast cancer cell-hepatocyte interaction observed in vivo. Both in vivo models exhibit a histology that closely mimics the replacement growth pattern of human BCLMs, where breast cancer cells replace hepatocytes at the tumour-liver interface. To address whether hepatocytes are killed by adjacent breast cancer cells in vivo, we evaluated hepatocyte death by apoptosis using appropriate markers (cleaved-PARP and cleaved-caspase-3). Surprisingly, although extensive apoptotic death of breast cancer cells in the tumour mass could be observed, no evidence for apoptotic death of hepatocytes could be found. However, we observed extensive cell-in-cell invasion at the tumour-liver interface, where live breast cancer cells enter the cytoplasm of adjacent hepatocytes. We also observe a similar process of cell-in-cell invasion within in vitro cultures of MCF-7 cells with HepG2 cells. Although cell-in-cell invasion has been described as a mechanism of non-apoptotic cell death in breast cancer cells, the role of cell-in-cell invasion between breast cancer cells and hepatocytes in BCLMs has not been investigated. Our current studies are focused in two areas: (a) we are using intra-vital microscopy and time-lapse microscopy to investigate whether cell-in-cell invasion between breast cancer cells and hepatocytes mediates non-apoptotic death of hepatocytes both in vivo and in vitro, and (b) we are investigating the molecular mechanisms that mediate cell-in-cell invasion between breast cancer cells and hepatocytes. In conclusion, we present preliminary evidence for a novel mechanism via which breast cancer cells could replace hepatocytes in BCLMs. Further studies aimed at elucidating the molecular basis of this mechanism may reveal novel targets for preventing the replacement of the liver parenchyma by metastatic breast cancer cells. Citation Format: Mark R. Nathan, Lefteris Kostaras, Victoria Bridgeman, Shane Foo, Peter Vermeulen, Andrew Reynolds. Dissecting the mechanisms of hepatocyte replacement in breast cancer liver metastases. [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 4105.

MP62-17 A NEW INSIGHT OF CELL-CELL INTERACTIONS IN TUMOR STROMA OF PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I1 Apr 2016MP62-17 A NEW INSIGHT OF CELL-CELL INTERACTIONS IN TUMOR STROMA OF PROSTATE CANCER Kenichiro Ishii, Takeshi Sasaki, Manabu Miki, Manabu Kato, Hideki Kanda, Kiminobu Arima, Taizo Shiraishi, and Yoshiki Sugimura Kenichiro IshiiKenichiro Ishii More articles by this author , Takeshi SasakiTakeshi Sasaki More articles by this author , Manabu MikiManabu Miki More articles by this author , Manabu KatoManabu Kato More articles by this author , Hideki KandaHideki Kanda More articles by this author , Kiminobu ArimaKiminobu Arima More articles by this author , Taizo ShiraishiTaizo Shiraishi More articles by this author , and Yoshiki SugimuraYoshiki Sugimura More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.925AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Activation of tumor-stroma interactions plays a critical role in the promotion of tumorigenesis. During primary cancer progression, cancer cells generate the carcinoma-associated fibroblasts (CAFs) in adjacent normal fibroblasts, and then CAFs support the proliferation and invasion of adjacent cancer cells in a paracrine fashion. In the tumor microenvironment, CAFs communicate not only with cancer cells but also surrounding stromal cells. The tumor stroma consists not only of CAFs but also normal fibroblasts, myofibroblasts, or smooth muscle cells. Unfortunately, we know very little about cell-cell interactions in the tumor stroma. To investigate stromal cell-cell interactions, we performed in vitro co-culture experiments using primary cultured human prostate cancer (PCa) CAFs PCaSC-8 cells, and commercialized normal human prostate stromal cells PrSC and PrSMC. METHODS PCaSC-8 cells were isolated from needle biopsy samples of a PCa patient. PrSC and PrSMC were purchased from Lonza Group Ltd. Co-culture experiments were performed using cell culture inserts. RESULTS Cell growth of PrSC and PrSMC was significantly increased by co-culturing with PCaSC-8 cells, while that of PCaSC-8 cells was not affected by co-culturing neither with PrSC or PrSMC. In the condition of co-cultures with PCaSC-8 cells, mRNA expression of TNC, ACTA2 and FGF2 was significantly increased in PrSC. In contrast, expression of EGF, FGF7, HGF, and IGF1 mRNA was significantly increased in PrSMC. In PCaSC-8 cells, mRNA expression of ACTA2, HGF, and IGF1 was significantly increased by co-culturing with PrSC. In both PrSC and PrSMC, protein production of IL-6 was significantly increased by co-culturing with PCaSC-8 cells. In addition, VEGF in PrSC was significantly increased by co-culturing with PCaSC-8 cells. In PCaSC-8 cells, IL-6 and VEGF were significantly increased by co-culturing with PrSC. CONCLUSIONS We previously reported that PCa cells induced CAF-like differentiation in normal fibroblasts, i.e. PCa cells generate the CAFs. Here our results have demonstrated that normal fibroblasts co-cultured with CAFs become activated and exhibit the biochemical characteristics consistent with CAFs, i.e. CAFs also induce CAF-like differentiation in normal fibroblasts. Of interest, normal fibroblasts boost the biochemical characteristics of CAFs. Those cell-cell interactions in the tumor stroma suggest that CAFs may spread autonomously. In this cell competition, CAFs must be winner in the tumor stroma. Identifying mechanisms underlying the cell competition in the tumor stroma is an initial step toward designing CAF-targeted therapy for treatment of PCa. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e819 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Kenichiro Ishii More articles by this author Takeshi Sasaki More articles by this author Manabu Miki More articles by this author Manabu Kato More articles by this author Hideki Kanda More articles by this author Kiminobu Arima More articles by this author Taizo Shiraishi More articles by this author Yoshiki Sugimura More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Direct Interaction between Carcinoma Cells and Cancer Associated Fibroblasts for the Regulation of Cancer Invasion.

The tumor stroma acts as an essential microenvironment of the cancer cells, which includes many different types of non-cancerous cells and the extracellular matrix (ECM). Stromal fibroblasts (SFs) are the major cellular constituents of the tumor stroma and are often called cancer-associated fibroblasts (CAFs). They are often characterized by α-smooth muscle actin (αSMA) expression, which is indicative of the myofibroblast phenotype and strong contractility. These characteristics contribute to the remodeling and stiffening of the stromal ECM, thereby offering an appropriate field for cancer cell invasion. Importance of the tumor stroma in cancer progression has recently been highlighted. Moreover, several reports suggest that stromal fibroblasts interact with adjacent cancer cells through soluble factors, exosomes, or direct cell-cell adhesion to promote cancer cell invasion. In this review, current models of the regulation of cancer cell invasion by surrounding fibroblasts are summarized, including our recent work on the interaction between stromal fibroblasts and scirrhous gastric carcinoma (SGC) cells by using a three-dimensional (3D) culture system. Further mechanistic insights into the roles of the interaction between cancer cells and stromal fibroblasts in cancer invasion will be required to identify novel molecular targets for preventing cancer cell invasion.

Targeting the tumor-stromal-immune cell axis.

Targeting the tumor-stromal-immune cell axis.

Abstract 3372: Molecular characteristics of cancer-associated adipocytes in breast cancer

Abstract Adipose tissue in the tumor microenvironment is now regarded to be an active player in cancer development and progression. Recent studies have suggested critical roles of the cancer-associated adipocytes in breast cancer proliferation and invasion. However, unlike the well-known cancer-associated fibroblasts, the molecular profiles of cancer-associated adipocytes are largely unknown. We studied the histo-morphologic changes in adipose tissue adjacent to the breast cancer cells in human tissues. Adipocytes had smaller fat droplets and often associated with immune cell infiltration. We investigated the changes of mRNA expression profiles of differentiated adipocytes when they were cultured with breast cancer cells (MCF-7 and MDA-MB-231) by using the NIH-3T3-L1 cell line. Indirect coculture with cancer cells resulted in significantly altered expression of genes involved in immune response, extracellular matrix remodeling, angiogenesis, metabolism, and apoptosis. IL6 and its downstream genes, transcription factors such as RUNX1, as well as PTX3 and TIMP1 were most unregulated genes. Increased secretion of IL6 was further validated in the culture media by ELISA. Also, the gene expression data showed that adipocytes cocultured with cancer cells showed decreased adipocyte-differentiation genes suggesting the cells underwent de-differentiation process. Microscopic examination of co-cultured adipocytes showed smaller fat droplets similar to human specimens. The co-cultured adipocytes showed different effect on cancer cells according to the molecular subtypes of the breast cancer cells. The conditioned media from co-cultured adipocytes resulted in increased proliferation of ER+ breast cancer cells, suggesting a subtype-dependent effect of co-cultured adipocytes in breast cancer. Our results suggest that cancer-associated adipocytes show increased expression of genes involved in immune response, extracellular matrix remodeling, angiogenesis, metabolism, and apoptosis. Futhermore, these cancer-associated adipocytes show features toward the preadipocyte state suggesting the possibility of de-differentiation of adipocyte under the influence of adjacent cancer cells. The functional impact of the cancer-associated adipocyte seem to vary according to the molecular subtype of epithelial cancer cells. Citation Format: Hyeong-Gon Moon, Jiwoo Lee, Han-Byoel Lee, Han Suk Ryu, Wonshik Han, Dong-Young Noh. Molecular characteristics of cancer-associated adipocytes in breast cancer. [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 3372. doi:10.1158/1538-7445.AM2015-3372

Chemotherapy induces the cancer-associated fibroblast phenotype, activating paracrine Hedgehog-GLI signalling in breast cancer cells.

Cancer cells recruit normal cells such as fibroblasts to establish reactive microenvironments. Via metabolic stress, catabolism and inflammation, these cancer-associated fibroblasts set up a synergistic relationship with tumour cells, that contributes to their malignancy and resistance to therapy. Given that chemotherapy is a systemic treatment, the possibility that healthy cell damage affects the metastatic risk or the prospect of developing a second malignancy becomes relevant. Here, we demonstrate that standard chemotherapies phenotypically and metabolically transform stromal fibroblasts into cancer-associated fibroblasts, leading to the emergence of a highly glycolytic, autophagic and pro-inflammatory microenvironment. This catabolic microenvironment, in turn, activates stemness (Sonic hedgehog/GLI signalling), antioxidant response and interferon-mediated signalling, in adjacent breast cancer cells. Thus, we propose a model by which chemotherapy-induced catabolism in healthy fibroblasts constitutes a source of energy-rich nutrients and inflammatory cytokines that would activate stemness in adjacent epithelial cells, possibly triggering new tumorigenic processes. In this context, immune cell recruitment would be also stimulated to further support malignancy.

Enhanced radiation therapy with multilayer microdisks containing radiosensitizing gold nanoparticles.

A challenge of X-ray radiation therapy is that high dose X-rays at therapeutic conditions damage normal cells. This paper describes the use of gold nanoparticle-loaded multilayer microdisks to enhance X-ray radiation therapy, where each microdisk contains over 10(5) radiosensitizing nanoparticles. The microdisks are attached on cell membranes through electrostatic interaction. Upon X-ray irradiation, more photoelectrons and Auger electrons are generated in the vicinity of the nanoparticles, which cause water ionization and lead to the formation of free radicals that damage the DNA of adjacent cancer cells. By attaching a large amount of gold nanoparticles on cancer cells, the total X-ray dose required for DNA damage and cell killing can be reduced. Due to their controllable structure and composition, multilayer microdisks can be a viable choice for enhanced radiation therapy with nanoparticles.

Radiofluorinated probe for PET imaging of fatty acid binding protein 4 in cancer

IntroductionCancer-associated adipocytes metabolically interact with adjacent cancer cells to promote tumor proliferation and metastasis. Fatty acid binding protein 4 (FABP4) participates in this interaction, and is gathering attention as a therapeutic and diagnostic target. Positron emission tomography (PET) is a useful diagnostic method that enables noninvasive in vivo quantitative imaging of biofunctional molecules with probes labeled with positron-emitting radioisotopes. Here a novel 18F labeled probe for PET FABP4 imaging developed through dedicated drug design from a radioiodinated probe we recently reported is evaluated in vitro and in vivo. MethodsWe designed the [18F]-labeled FTAP1 and FTAP3 probe, composed of a single or triple oxyethylene linker and a triazolopyrimidine scaffold derived from an FABP4 inhibitor. FABP4 binding affinities for chemically synthesized FTAP1 and FTAP3 were measured using FABP4 and 8-anilino-1-naphthalene sulfonic acid. Cell membrane permeability was measured using a commercially available plate assay system. After radiosynthesis, [18F]FTAP1 affinity and selectivity were evaluated using immobilized FABP3, FABP4, and FABP5. Cell uptake was investigated using differentiated adipocytes expressing FABP4 with inhibitor treatment. Following biodistribution studies in C6 glioblastoma-bearing mice, ex vivo autoradiography and immunohistochemistry were performed using thin sliced tumor sections. PET/CT imaging was then performed on C6 tumor bearing mice. ResultsFTAP1 showed high FABP4 affinity (Ki=68±8.9 nM) and adequate cell permeability. [18F]FTAP1 with ≥98% radiochemical purity was shown to selectively bind to FABP4 (16.3- and 9.3-fold higher than for FABP3 and FABP5, respectively). [18F]FTAP1 was taken up by FABP4 expressing cells, and this uptake could be blocked by an inhibitor, indicating very low non-specific cell binding. [18F]FTAP1 showed high tumor accumulation, which demonstrates its potential use for in vivo tumor PET imaging, and the intratumoral radioactivity distribution corresponded to the FABP4 expression profile. Conclusion[18F]FTAP1 is a promising PET probe to target FABP4.

Acidic pH derived from cancer cells may induce failed reprogramming of normal differentiated cells adjacent tumor cells and turn them into cancer cells

BackgroundEelier studies demonstrated the up-regulation of some transcriptional factors such as Oct4, Nanog, Sox2 in undifferentiated cancer cells. These transcriptional regulators are up-regulated in pluripotent cells, as well and are responsible for cell reprogramming in normal cells. It might be said that normal cells adjacent tumor site are undergone of failed cell reprogramming. Presentation of the hypothesisExtracellular pH of cancer cell is acidic and recent studies reveal the role of acidic environment in cell reprogramming of normal cells. This hypothesis deals with the potential role of acidic pH in malignant tumor development through normal cells adjacent cancer cells. It seems that cancer cells are more intelligent and acid release from these cells is not just a by-product but also and more important reason, is a tool to up-regulate cell reprogramming markers, induce epigenetic modification and tumor progress in normal cells adjacent cancer cells. If this is correct, then it could be expected that with alkaline targeting of tumor environment, failed cell reprogramming, aberrant epigenetic modification will decrease in normal cells adjacent cancer cells and afterward metastasis will decrease. Testing the hypothesisIt is proposed to investigate altered genetic and epigenetic modification (DNA methylation, histone modification) in cancer, early cancer and cells in vicinity of cancer cells at different pH range of 5.8–7.8. This study is performed to determine whether acidic pH induces reprogramming, global hypomethylation and promoter hypermethylation in cancer cells and cells in vicinity of cancer cells at different pH values. Implications of the hypothesisThis hypothesis deal with the ability of acidic pH to convert normal cells adjacent cancer cells to cancerous cells and its inductive potential on genetic and epigenetic modification of normal cells adjacent cancer cells and will further emphasize the important of extracellular acidic targeting in cancer therapy.

The Reprogramming of Tumor Stroma by HSF1 Is a Potent Enabler of Malignancy

Stromal cells within the tumor microenvironment areessential for tumor progression and metastasis. Surprisingly little is known about the factors that drive the transcriptional reprogramming of stromal cells within tumors. We report that the transcriptional regulator heat shock factor 1 (HSF1) is frequently activated in cancer-associated fibroblasts (CAFs), where it is a potent enabler of malignancy. HSF1 drives a transcriptional program in CAFs that complements, yet is completely different from, the program it drives in adjacent cancer cells. This CAF program is uniquely structured to support malignancy in a non-cell-autonomous way. Two central stromal signaling molecules-TGF-β and SDF1-play a critical role. In early-stage breast and lung cancer, high stromal HSF1 activation is strongly associated with poor patient outcome. Thus, tumors co-opt the ancient survival functions of HSF1 to orchestrate malignancy in both cell-autonomous and non-cell-autonomous ways, with far-reaching therapeutic implications.

1334 PROSTATE CANCER-ASSOCIATED FIBROBLASTS EXHIBIT DIFFERENCES IN GENE EXPRESSION PROFILES COMPARED TO NORMAL FIBROBLASTS IN THE SAME PATIENT

INTRODUCTION AND OBJECTIVES: There is strong evidence that the tumor microenvironment contributes to the normal prostate development as well as to proliferation, invasion and migration in prostatic carcinoma. Especially fibroblasts surrounding epithelial tumor cells seem to play a pivotal role during EMT and tumor invasion. Several strategies were undertaken to describe reliable biomarkers for cancer associated fibroblasts (CAF). Using fibroblasts from patients undergoing radical prostatectomy the expression profile of CAF was analysed using microarrays and compared to non-cancer associated fibroblasts of the same patient (NAF). To monitor the stability of gene expression profile during cell culture we quantified the expression levels of some CAFs specific genes like FAP, SMA, PDGFR and TGFbetween p1 to p7. METHODS: Tissue pieces from cancerous and cancer free areas of 7 prostates were cultivated for selection of CAF and NAF, respectively. Total RNA was isolated using RNeasy Kit (Qiagen) according to manufactures recommendation. For microarray expression analysis we used Illumina HT-12v4.0 Expression BeadChips. All primers and kits used for the qRT-PCR expression analysis of CAF-related genes during cell culture were purchased from Applied Biosystems. RESULTS: Using microarray analysis we found a significant overexpression of the following genes in fibroblasts associated to PCa tissue: MAP3K1 (a MAP kinase associated to stress and NFkB-dependent pathways), NBPF11 (neuroblastoma breakpoint family member 11), SCARB1 (regulates transport of HDL thus increasing the intracellular Cholesterol level). The expression of the plasminogen receptor S100A1 and SNAPC2 was decreased. Our qRT-PCR analysis offers a constant expression of SDF1, PDGFR , TGFand SMA during culture whereas the expression levels of the FAP gene decreased dramatically from p1 to p7. CONCLUSIONS: This is the first comprehensive expression analysis of NAF/CAF in PCa. Enhanced expression (MAP3K1, SCARB1) may influence cell movement and the level of cholesterol for androgen synthesis in fibroblasts. Since macrophage migration into tumor cells is controlled by S100A10 CAFs are able to control this process, too. Thus, our results provide strong evidence that these changes in gene expression in CAFs can contribute to functional alteration of adjacent prostate cancer cells. It is very important to note that during serial culture of fibroblast the expression profile of CAFrelated genes undergoes changes as a consequence to the particular in vitro culture.

Ketone body utilization drives tumor growth and metastasis

We have previously proposed that catabolic fibroblasts generate mitochondrial fuels (such as ketone bodies) to promote the anabolic growth of human cancer cells and their metastasic dissemination. We have termed this new paradigm “two-compartment tumor metabolism.” Here, we further tested this hypothesis by using a genetic approach. For this purpose, we generated hTERT-immortalized fibroblasts overexpressing the rate-limiting enzymes that promote ketone body production, namely BDH1 and HMGCS2. Similarly, we generated MDA-MB-231 human breast cancer cells overexpressing the key enzyme(s) that allow ketone body re-utilization, OXCT1/2 and ACAT1/2. Interestingly, our results directly show that ketogenic fibroblasts are catabolic and undergo autophagy, with a loss of caveolin-1 (Cav-1) protein expression. Moreover, ketogenic fibroblasts increase the mitochondrial mass and growth of adjacent breast cancer cells. However, most importantly, ketogenic fibroblasts also effectively promote tumor growth, without a significant increase in tumor angiogenesis. Finally, MDA-MB-231 cells overexpressing the enzyme(s) required for ketone re-utilization show dramatic increases in tumor growth and metastatic capacity. Our data provide the necessary genetic evidence that ketone body production and re-utilization drive tumor progression and metastasis. As such, ketone inhibitors should be designed as novel therapeutics to effectively treat advanced cancer patients, with tumor recurrence and metastatic disease. In summary, ketone bodies behave as onco-metabolites, and we directly show that the enzymes HMGCS2, ACAT1/2 and OXCT1/2 are bona fide metabolic oncogenes.

Metabolic reprogramming of cancer-associated fibroblasts by TGF-β drives tumor growth: Connecting TGF-β signaling with “Warburg-like” cancer metabolism and L-lactate production

We have previously shown that a loss of stromal Cav-1 is a biomarker of poor prognosis in breast cancers. Mechanistically, a loss of Cav-1 induces the metabolic reprogramming of stromal cells, with increased autophagy/mitophagy, mitochondrial dysfunction and aerobic glycolysis. As a consequence, Cav-1-low CAFs generate nutrients (such as L-lactate) and chemical building blocks that fuel mitochondrial metabolism and the anabolic growth of adjacent breast cancer cells. It is also known that a loss of Cav-1 is associated with hyperactive TGF-β signaling. However, it remains unknown whether hyperactivation of the TGF-β signaling pathway contributes to the metabolic reprogramming of Cav-1-low CAFs. To address these issues, we overexpressed TGF-β ligands and the TGF-β receptor I (TGFβ-RI) in stromal fibroblasts and breast cancer cells. Here, we show that the role of TGF-β in tumorigenesis is compartment-specific, and that TGF-β promotes tumorigenesis by shifting cancer-associated fibroblasts toward catabolic metabolism. Importantly, the tumor-promoting effects of TGF-β are independent of the cell type generating TGF-β. Thus, stromal-derived TGF-β activates signaling in stromal cells in an autocrine fashion, leading to fibroblast activation, as judged by increased expression of myofibroblast markers, and metabolic reprogramming, with a shift toward catabolic metabolism and oxidative stress. We also show that TGF-β-activated fibroblasts promote the mitochondrial activity of adjacent cancer cells, and in a xenograft model, enhancing the growth of breast cancer cells, independently of angiogenesis. Conversely, activation of the TGF-β pathway in cancer cells does not influence tumor growth, but cancer cell-derived-TGF-β ligands affect stromal cells in a paracrine fashion, leading to fibroblast activation and enhanced tumor growth. In conclusion, ligand-dependent or cell-autonomous activation of the TGF-β pathway in stromal cells induces their metabolic reprogramming, with increased oxidative stress, autophagy/mitophagy and glycolysis, and downregulation of Cav-1. These metabolic alterations can spread among neighboring fibroblasts and greatly sustain the growth of breast cancer cells. Our data provide novel insights into the role of the TGF-β pathway in breast tumorigenesis, and establish a clear causative link between the tumor-promoting effects of TGF-β signaling and the metabolic reprogramming of the tumor microenvironment.

Chemical Molecule-Induced Light-Activated System for Anticancer and Antifungal Activities

Except for chemotherapy, surgery, and radiotherapy, photodynamic therapy (PDT) as new therapy modality is already in wide clinic use for the treatment of various diseases. The major bottleneck of this technique is the requirement of outer light source, which always limits effective application of PDT to the lesions in deeper tissue. Here, we first report a new modality for treating cancer and microbial infections, which is activated by chemical molecules instead of outer light irradiation. In this system, in situ bioluminescence of luminol can be absorbed by a cationic oligo(p-phenylene vinylene) (OPV) that acts as the photosensitizer through bioluminescence resonance energy transfer (BRET) process. The excited OPV sensitizes oxygen molecule in the surroundings to produce reactive oxygen species (ROS) that kill the adjacent cancer cells in vitro and in vivo, and pathogenic microbes. By avoiding the use of light irradiation, this work opens a new therapy modality to tumor and pathogen infections.

Autophagy and senescence in cancer-associated fibroblasts metabolically supports tumor growth and metastasis, via glycolysis and ketone production

Senescent fibroblasts are known to promote tumor growth. However, the exact mechanism remains largely unknown. An important clue comes from recent studies linking autophagy with the onset of senescence. Thus, autophagy and senescence may be part of the same physiological process, known as the autophagy-senescence transition (AST). To test this hypothesis, human fibroblasts immortalized with telomerase (hTERT-BJ1) were stably transfected with autophagy genes (BNIP3, CTSB or ATG16L1). Their overexpression was sufficient to induce a constitutive autophagic phenotype, with features of mitophagy, mitochondrial dysfunction and a shift toward aerobic glycolysis, resulting in L-lactate and ketone body production. Autophagic fibroblasts also showed features of senescence, with increased p21(WAF1/CIP1), a CDK inhibitor, cellular hypertrophy and increased β-galactosidase activity. Thus, we genetically validated the existence of the autophagy-senescence transition. Importantly, autophagic-senescent fibroblasts promoted tumor growth and metastasis, when co-injected with human breast cancer cells, independently of angiogenesis. Autophagic-senescent fibroblasts stimulated mitochondrial metabolism in adjacent cancer cells, when the two cell types were co-cultured, as visualized by MitoTracker staining. In particular, autophagic ATG16L1 fibroblasts, which produced large amounts of ketone bodies (3-hydroxy-butyrate), had the strongest effects and promoted metastasis by up to 11-fold. Conversely, expression of ATG16L1 in epithelial cancer cells inhibited tumor growth, indicating that the effects of autophagy are compartment-specific. Thus, autophagic-senescent fibroblasts metabolically promote tumor growth and metastasis, by paracrine production of high-energy mitochondrial fuels. Our current studies provide genetic support for the importance of “two-compartment tumor metabolism” in driving tumor growth and metastasis via a simple energy transfer mechanism. Finally, β-galactosidase, a known lysosomal enzyme and biomarker of senescence, was localized to the tumor stroma in human breast cancer tissues, providing in vivo support for our hypothesis. Bioinformatic analysis of genome-wide transcriptional profiles from tumor stroma, isolated from human breast cancers, also validated the onset of an autophagy-senescence transition. Taken together, these studies establish a new functional link between host aging, autophagy, the tumor microenvironment and cancer metabolism.

788 ABERRANT ACTIVATION OF PARACRINE LOOP BETWEEN PROSTATE CANCER CELLS AND CARCINOMA-ASSOCIATED FIBROBLASTS BY OVEREXPRESSION OF STROMAL INTERLEUKIN-6

You have accessJournal of UrologyProstate Cancer: Basic Research IV1 Apr 2012788 ABERRANT ACTIVATION OF PARACRINE LOOP BETWEEN PROSTATE CANCER CELLS AND CARCINOMA-ASSOCIATED FIBROBLASTS BY OVEREXPRESSION OF STROMAL INTERLEUKIN-6 Kenichiro Ishii, Yoichi Iwamoto, Manabu Kato, Kazuhiro Iguchi, Atsushi Mizokami, Kiminobu Arima, Taizo Shiraishi, and Yoshiki Sugimura Kenichiro IshiiKenichiro Ishii Tsu, Japan More articles by this author , Yoichi IwamotoYoichi Iwamoto Tsu, Japan More articles by this author , Manabu KatoManabu Kato Tsu, Japan More articles by this author , Kazuhiro IguchiKazuhiro Iguchi Gifu, Japan More articles by this author , Atsushi MizokamiAtsushi Mizokami Ishikawa, Japan More articles by this author , Kiminobu ArimaKiminobu Arima Tsu, Japan More articles by this author , Taizo ShiraishiTaizo Shiraishi Tsu, Japan More articles by this author , and Yoshiki SugimuraYoshiki Sugimura Tsu, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.876AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES In the tumor microenvironment, activation of tumor-stromal interactions is considered to play a critical role in the promotion of tumorigenesis. During primary cancer progression, cancer cells generate the carcinoma-associated fibroblasts (CAFs) in adjacent normal fibroblasts, and then CAFs support the proliferation and invasion of adjacent cancer cells in a paracrine fashion. The serum level of interleukin-6 (IL-6) is a significant prognostic factor for prostate cancer (PCa) patients with disseminated diseases, suggesting positive roles of IL-6 in the aberrant activation of paracrine loop between PCa cells and CAFs. In this study, we investigated the relationship between responsiveness of PCa cells to stromal IL-6 and cellular AR signaling. METHODS The androgen-low-sensitive E9 cells were obtained from androgen-sensitive LNCaP cell population by limiting dilution method in regular culture condition. In contrast, the androgen-insensitive AIDL cells were established from LNCaP cells by continuous passaging in hormone-depleted condition. PCaSC-8 and PCaSC-9 cells were primary cultured from different human PCa specimens. RESULTS IL-6 and VEGF protein levels in both PCaSC-8 and PCaSC-9 cells were generally higher than normal human prostate stromal cells PrSC. In contrast to stromal cells, no IL-6 secretion from LNCaP, E9, and AIDL was detected. IL-6 treatment (10 ng/mL) suppressed cell proliferation of LNCaP and E9 accompanied with changes of cell morphology, i.e. induction of neuroendocrine-like differentiation. Induction of PSA secretion was observed only in IL-6-treated LNCaP. VEGF secretion was significantly increased in both IL-6-treated LNCaP and AIDL. A selective alpha1-adrenoceptor antagonist naftopidil (10 μM) but not tamsulosin suppressed IL-6-induced VEGF secretion in AIDL. CONCLUSIONS In this study, we have shown the evidence that secretions of IL-6 and VEGF from primary cultured CAFs derived PCa patients are generally higher than normal fibroblasts. Our results suggest that overexpression of stromal IL-6 might increase VEGF secretion from adjacent PCa cells in a paracrine fashion. Using LNCaP sublines, E9 and AIDL, differing in androgen-sensitivity, we demonstrated here that IL-6-induced VEGF secretion from PCa cells might be through cellular AR signaling-independent pathway. Identifying mechanisms underlying the aberrant activation of paracrine loop between PCa cells and CAFs is an initial step toward designing CAF-targeted therapy for treatment of PCa. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e322-e323 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kenichiro Ishii Tsu, Japan More articles by this author Yoichi Iwamoto Tsu, Japan More articles by this author Manabu Kato Tsu, Japan More articles by this author Kazuhiro Iguchi Gifu, Japan More articles by this author Atsushi Mizokami Ishikawa, Japan More articles by this author Kiminobu Arima Tsu, Japan More articles by this author Taizo Shiraishi Tsu, Japan More articles by this author Yoshiki Sugimura Tsu, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract A27: MicroRNA 21 in adipocyte-derived microvesicles confers taxol-resistance of ovarian cancer cells through down-regulation of Apoptotic Protease Activating Factor 1

Abstract Over the past 30 years, there has been a dramatic increase in the incidence of obesity worldwide. As of 2010, approximately one-third of U.S. adults are obese, while another one-third of adults are overweight. Obesity is one of the risk factors for ovarian cancer. Greater than 75% of ovarian cancer patients present with advanced stage disease. Tumors preferentially spread to the omentum, a major site of abdominal fat, where ovarian cancer cells infiltrate omental adipose tissue. Omental adipocytes may interact directly with cancer cells to create a microenvironment niche that supports cancer growth. Recent studies have shown that cells can communicate through the exchange of bioactive molecules via microvesicles (exosomes and ectosomes). Microvesicles fuse with target cell membranes and transfer proteins, lipids, mRNAs and non-coding RNAs from donor to recipient cells. We hypothesize that adipocytes secrete microvesicles containing tumor-supporting factors that promote ovarian cancer progression. Ion Torrent RNA sequencing and subsequent qRT-PCR analyses were used to identify non-coding microvesicle RNAs secreted by primary cultures of cancer associated adipocytes (CAA), cancer associated fibroblasts (CAF), normal adipocytes, normal fibroblasts and ovarian cancer cells. Of the non-coding microvesicle RNAs identified, microRNA 21 (miR21) showed significantly higher expression in microvesicles secreted from CAA when compared to those from CAF and normal omental adipocytes, fibroblasts, and ovarian cancer cells. Using qRT-PCR of RNA isolated from microdissected frozen tissue and In-situ hybridization on FFPE tissue sections, CAA demonstrated significant higher miR21 expression levels compared to the adjacent cancer cells and normal adipocytes. MiR21 was up-regulated in metastatic ovarian cancer cells in the omentum as compared to those in the primary tumor site. Moreover, strong miR21 staining was observed in omental cancer cells particularly at the invasion front, suggesting that miR21 may be transferred from CAA to their neighboring ovarian cancer cells. To test this hypothesis, SKOV3 ovarian cancer cells were co-cultured with adipocytes transfected with miR21-FAM or incubated with fluorescently labeled adipocyte-derived microvesicles. Confocal microscopy confirmed the delivery of miR21 by adipocyte-derived microvesicles to cancer cells. Since miR21 expression in tumor cells has been linked with resistance to a variety of chemotherapeutic agents, we further delineated the direct effect of miR21 on the chemoresistance of ovarian cancer cell. The ovarian cancer cell lines, OVCA432 and SKOV3, transfected with miR21 precursor showed an increase in cell survival and a decrease in apoptosis in the presence of taxol. Furthermore, miR21 was significantly up-regulated in taxol-resistant HeyA8-MDR and SKOV3-TR cells as compared to the parental cell lines, HeyA8 and SKOV3, respectively. These data strongly suggest that miR21 may confer taxol resistance to ovarian cancer cells. Using microarray analysis of RNA isolated from SKOV3 cells transfected with the miR21 precursor, we identified a set of chemoresistance-related genes associated with miR21 expression. One of the most significantly down-regulated genes is Apoptotic Protease Activating Factor 1 (APAF1). By qRT-PCR, we confirmed that miR21 over-expression decreased APAF1 mRNA expression levels in OVCA432 and SKOV3 cells. A strong miR21 binding site is predicted on the 3′-UTR of APAF1, suggesting APAF1 is the direct target of miR21. In summary, up-regulation of miR21 in omental ovarian cancer cells is mediated through transport of miR21 in adipocyte-derived microvesicles from CAA in the omentum. Further, miR21 confers taxol-resistance in ovarian cancer cells by down-regulating APAF1 expression in ovarian cancer cells to decrease taxol-induced apoptosis. Citation Format: Ngai Na Co, Rosemarie Schmandt, Karen Lu, Samuel Mok. MicroRNA 21 in adipocyte-derived microvesicles confers taxol-resistance of ovarian cancer cells through down-regulation of Apoptotic Protease Activating Factor 1 [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A27.

Mitochondrial oxidative stress in cancer-associated fibroblasts drives lactate production, promoting breast cancer tumor growth

Increasing chronological age is the most significant risk factor for cancer. Recently, we proposed a new paradigm for understanding the role of the aging and the tumor microenvironment in cancer onset. In this model, cancer cells induce oxidative stress in adjacent stromal fibroblasts. This, in turn, causes several changes in the phenotype of the fibroblast including mitochondrial dysfunction, hydrogen peroxide production, and aerobic glycolysis, resulting in high levels of L-lactate production. L-lactate is then transferred from these glycolytic fibroblasts to adjacent epithelial cancer cells and used as “fuel” for oxidative mitochondrial metabolism. Here, we created a new pre-clinical model system to directly test this hypothesis experimentally. To synthetically generate glycolytic fibroblasts, we genetically-induced mitochondrial dysfunction by knocking down TFAM using an sh-RNA approach. TFAM is mitochondrial transcription factor A, which is important in functionally maintaining the mitochondrial respiratory chain. Interestingly, TFAM-deficient fibroblasts showed evidence of mitochondrial dysfunction and oxidative stress, with the loss of certain mitochondrial respiratory chain components, and the over-production of hydrogen peroxide and L-lactate. Thus, TFAM-deficient fibroblasts underwent metabolic reprogramming towards aerobic glycolysis. Most importantly, TFAM-deficient fibroblasts significantly promoted tumor growth, as assayed using a human breast cancer (MDA-MB-231) xenograft model. These increases in glycolytic fibroblast driven tumor growth were independent of tumor angiogenesis. Mechanistically, TFAM-deficient fibroblasts increased the mitochondrial activity of adjacent epithelial cancer cells in a co-culture system, as seen using MitoTracker. Finally, TFAM-deficient fibroblasts also showed a loss of caveolin-1 (Cav-1), a known breast cancer stromal biomarker. Loss of stromal fibroblast Cav-1 is associated with early tumor recurrence, metastasis, and treatment failure, resulting in poor clinical outcome in breast cancer patients. Thus, this new experimental model system, employing glycolytic fibroblasts, may be highly clinically relevant. These studies also have implications for understanding the role of hydrogen peroxide production in oxidative damage and “host cell aging,” in providing a permissive metabolic microenvironment for promoting and sustaining tumor growth.

Abstract B216: A soil-to-seeds anticancer strategy: Targeting stroma to kill cancer cells.

Abstract Rationale: We propose a dual targeting anticancer theragnostic strategy called OncoCidia, with which we intend to destroy and radioactively contaminate tumor stroma (soil) in order to eradicate all cancer cells (seeds) using vascular disrupting agents (VDAs) followed by radiolabeled necrosis-avid compounds (NACs), both of which are small molecular, naturally or synthetically derivable, systemically injectable, and of unique and complementary targetabilities. Methods: Combretastatin A4 phosphate (CA4P) and Hypericin, as a representative of VDAs and NACs respectively, were tested among liver and subcutaneous tumor models (Ø > 0.8 cm) in rats, mice and rabbits (n > 100). We first injected CA4P intravenously at a dose of 10 mg/kg to cause tumor necrosis that became the next target. Hypericin was labeled with iodine-131 to form 131I-Hypericin (131I-Hyp), which was then iv injected at 300 MBq/kg 24h after administration of CA4P. 131I-Hyp accumulated in necrotic tumor, killing adjacent residual cancer cells by cross-fire irradiation and preventing tumor re-growth. We studied OncoCidia by comparing vehicle-control, single-targeting (either CA4P or 131I-Hyp) and dual-targeting (CA4P plus 131I-Hyp) groups using in vivo MRI and scintiscans, and ex vivo gamma-counting, autoradiography and histology. Results: Eight days after only one dosage of each drug, typically the tumor size in vehicle-controls doubled that in single-targeting groups (p < 0.001), and was 5-times that in dual-targeting group (p < 0.0001), without treatment-related animal death. Tumors in OncoCidia groups appeared as hot-spots on scintiscans even weeks after treatment, corresponding to > 3.34% ID/g of 131I-Hyp and a target-to-liver ratio > 20. Significantly prolonged survival and reduced lung metastases occurred in tumor-bearing mice and rabbits respectively. Such encouraging results with histomorphological proofs were independent of animal species and tumor types. Conclusion: Our preclinical experiments suggest that OncoCidia may present a simple, workable, affordable and general solution for treatment, detection and therapeutic monitoring of diverse solid malignant tumors, and deserve further exploitation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B216.