Cancer Cells In Nude Mice Research Articles

Citron kinase (CIT-K) promotes aggressiveness and tumorigenesis of breast cancer cells in vitro and in vivo: preliminary study of the underlying mechanism

ObjectivesCitron kinase (CIT-K), as a key Rho effector, functions to maintain proper structure of the midbody during cell mitosis. This study assessed CIT-K expression and its role in breast cancer cells.MethodsParaffin-embedded breast cancer and para-tumor tissues from 43 invasive breast cancer patients and 33 normal mammary glands were collected for immunohistochemistry. CIT-K expression knockdown was achieved using lentivirus carrying CIT-K shRNA in a wide range of breast cancer cell lines. Cells were then subjected to Western blot, qRT-PCR, cell proliferation, colony formation, transwell, and flow cytometric assays. The tumorigenicity of CIT-K knocked-down breast cancer cells was assessed using the nude mouse xenograft assay. Microarray analysis was performed to elucidate the underlying gene regulation after CIT-K silencing.ResultsCIT-K protein was overexpressed in breast cancer tissues, which is associated with advanced tumor stage, HER-2 expression and Ki-67 expression, whereas knockdown of CIT-K expression reduced breast cancer cell proliferation and colony formation, but promoted tumor cell apoptosis and cell-cycle arrest. Knockdown of CIT-K expression also inhibited breast cancer cell migration and invasion capacity. Moreover, CIT-K knockdown suppressed the tumorigenicity of breast cancer cells in nude mice. Molecularly, the expression of a variety of signaling genes, such as cyclin D1, EGFR, JAK1, TGF-α, PTK2, RAF1, RALB, SOS1, mTOR, and PTGS2, were altered after CIT-K knockdown.ConclusionsThis study demonstrated that CIT-K is associated with aggressive breast cancer behavior and targeting CIT-K may be a novel strategy for the future control of breast cancer.

FPR1 mediates the tumorigenicity of human cervical cancer cells.

PurposeThe present study aimed to investigate the role of FPR1 and the downstream effectors such as NF-κB and IL-6/8 in the development of cervical cancer.Patients and methodsFPR1 protein expression was detected via immunohistochemical staining in tissue microarrays containing cervical cancer tissues from 185 patients. Following FPR1 silencing in SiHa cells using lentiviral siRNA delivery, biological characteristics and tumor formation were evaluated in vitro and in vivo, respectively. Phosphorylated NF-κB levels were detected by Western blotting, while IL-6 and IL-8 secretion were detected by ELISA in both FPR1 knockdown and control SiHa cells. Human umbilical vein endothelial cell tube formation assays were performed to evaluate the angiogenesis-promoting ability of IL-6 and IL-8 secretion in FPR1 knockdown and control SiHa cells. Neovascularization, proliferation and apoptosis markers were detected by immunohistochemical staining to analyze the tumorigenic role of FPR1.ResultsImmunohistochemistry of cervical cancer tissues from 185 patients revealed high FPR1 expression levels in patients with advanced-stage disease and/or poor prognosis. Compared with control cells, cervical cancer cells in which FPR1 was silenced exhibited inhibition of cell invasion, migration and proliferation and higher levels of apoptosis. NF-κB was inhibited in FPR1 knockdown in SiHa cells. IL-6/8 upregulation by FPR1 activation stimulated angiogenesis. FPR1 deficiency inhibited the tumorigenicity of cervical cancer cells in nude mice. FPR1, IL-6, IL-8, CD31 and Ki67 levels were all reduced, whereas cleaved caspase-3 was upregulated, in the FPR1 knockdown group compared with the levels in the control group.ConclusionHigh FPR1 expression was associated with advanced stage and poor prognosis in cervical cancer patients. FPR1 activation induced NF-κB nuclear translocation to promote cervical cancer development through the upregulation of IL-6 and IL-8 expression. Inhibiting FPR1 activity may thus have potential therapeutic value in cervical cancer patients.

SALL4 activates TGF-β/SMAD signaling pathway to induce EMT and promote gastric cancer metastasis.

BackgroundIncreasing evidence suggests that SALL4 plays oncogenic roles in cancer development and progression. We have previously shown that SALL4 is highly expressed in gastric cancer, and its upregulation is associated with lymph node metastasis and poor prognosis. The role of SALL4 in gastric cancer metastasis and the underlying mechanism remain unclear.Materials and methodsThe biological roles of SALL4 in gastric cancer cell mobility, migration, and invasion were investigated by wound healing, transwell migration assay, and Matrigel invasion assay. The effects of SALL4 on epithelial–mesenchymal transition (EMT) in gastric cancer cells were examined by quantitative real-time PCR and Western blot. The downstream target genes of SALL4 were identified by microarray. The regulation of TGF-β1 by SALL4 in gastric cancer cells was analyzed by luciferase reporter assay and chromatin immunoprecipitation assay.ResultsSALL4 knockdown inhibited, while SALL4 overexpression promoted the motility, migration, and invasion abilities of gastric cancer cells in vitro. SALL4 knockdown also suppressed the peritoneal metastasis of gastric cancer cells in nude mice. SALL4 knockdown suppressed, while SALL4 overexpression induced the activation of TGF-β/SMAD signaling pathway and triggered EMT in gastric cancer cells. TGF-β1 was identified as a direct target gene of SALL4. The results of chromatin immunoprecipitation study and luciferase reporter assay further confirmed that SALL4 bound to the promoter of TGF-b1 gene and activated its expression. TGF-β1 knockdown reversed SALL4-mediated promotion of gastric cancer cell motility, migration, and invasion, indicating that TGF-β1 acts as a downstream effector of SALL4. Furthermore, the expression of TGF-β1 was found to be closely associated with that of SALL4 in gastric cancer tissues.ConclusionSALL4 promotes the metastasis of gastric cancer, at least partly, by directly activating TGF-β1, suggesting that SALL4 may serve as a new target for gastric cancer therapy.

LncRNA PVT1 regulates VEGFC through inhibiting miR-128 in bladder cancer cells.

Long noncoding RNA PVT1 is considered to be an oncogene in multiple cancers. Our previous studies indicated that PVT1 levels were higher in bladder cancer tissue and correlated with clinical progression and poor prognosis in bladder cancer patients. A bioinformatics analysis showed that PVT1 may regulate VEGFC expression through miR-128 as a competing endogenous RNA (ceRNA). In this study, we demonstrated that PVT1 expression levels affect the proliferation and migration ability of bladder cancer cells. Moreover, PVT1 knockdown significantly decreased the proliferation capacity of bladder cancer cells in nude mice. Luciferase assays and RNA-binding protein immunoprecipitation were performed to investigate the potential mechanism of ceRNAs in the regulation of PVT1 and VEGFC. The results showed that the increased number of PVT1 transcripts interacted directly with miR-128 to decrease miR-128 binding to the VEGFC 3'-untranslated region. This effect suppressed VEGFC mRNA degradation by miR-128. In conclusion, these results indicated that PVT1 might play a critical role in bladder cancer tumorigenesis via miR-218 and VEGFC. Therefore, PVT1 could be a new biomarker for bladder cancer diagnosis and therapy.

MicroRNA‑320a suppresses tumour cell proliferation and invasion of renal cancer cells by targeting FoxM1.

An increasing body of evidence has indicated that microRNAs (miRNAs/miRs) may play an important role in tumourigenesis and tumour progression. Recent studies have demonstrated that miR-320a is aberrantly expressed in a variety of different types of human cancer. The results of the present study confirmed that the expression of miR-320a was decreased in clinical specimens and cell lines. Expression of miR-320a inhibited the growth and invasive ability of ACHN and Caki-1 cells. Bioinformatics analysis and a luciferase reporter assay demonstrated that forkhead box protein M1 (FoxM1) was directly regulated by miR-320a. Rescue experiments in vitro revealed that the upregulation of FoxM1 antagonized the miR-320a-mediated malignant phenotype in renal cancer. Furthermore, experiments employing a xenograft mouse model revealed that the upregulation of miR-320a inhibited the proliferation of renal cancer cells in nude mice when FoxM1 protein expression was reduced. Collectively, the present study demonstrated a novel molecular interaction regulated by miR-320a, which may provide a novel insight into the treatments for renal cancer.

Abstract 1379: INCB059872, a novel FAD-directed LSD1 Inhibitor, is active in prostate cancer models and impacts prostate cancer stem-like cells

Abstract Cancer of the prostate is one of the most common malignancies and the second leading cause of cancer death in men in developed countries. There is increasing evidence that cancer stem-like cells (CSCs) are implicated in CRPC disease progression and treatment resistance. Transcriptional, epigenetic and metabolic reprogramming are key features for the acquisition and maintenance of stem-like properties. Understanding the factors regulating self-renewal and survival of prostate CSCs may offer novel targets for innovative therapeutic strategies. LSD1/KDM1A is a lysine demethylase for histone and non-histone proteins and functions as transcriptional corepressor or coactivator depending on the binding partners and substrates. LSD1 is a key epigenetic modifier controlling the fate of pluripotent stem cells in several tissues. Overexpression of LSD1 is found in many human cancers and has been linked to tumorigenic and CSC-like features. The involvement of LSD1 in stem cell pluripotency and differentiation suggests that LSD1 inhibitors, such as INCB059872, might be useful to target the prostate CSC subpopulation. Here, we examined the effects of INCB059872 on the growth properties, self-renewal and tumorigenic capability of prostate CSCs derived from human cell lines and the Pb-Cre4;Ptenflox/flox;Rosa26ERG/ERG (ERG/PTEN) mice, which develop highly invasive prostate adenocarcinomas. In ex vivo tumor-sphere assays, INCB059872 significantly suppressed the growth of tumor-initiating stem-like cells isolated from prostatic tumors generated in ERG/PTEN mice. Furthermore, treatment with INCB059872 inhibited colony and tumor-sphere formation by human prostate cancer cells. Conversely, the effects on proliferation and viability of bulk tumor cells were limited and required long-term exposure to the drug. These effects were observed in multiple prostate cancer cell lines, irrespective of the distinctive genetic features and AR status. Importantly, genetic knockdown of LSD1 by siRNAs and shRNAs recapitulated the effects of INCB059872. Both transient and stable knockdown of LSD1 had limited effects on proliferation and viability of bulk tumor cells, whereas they significantly reduced growth of colony and tumor-sphere forming stem-like cancer cells. Furthermore, LSD1 knockdown drastically reduced tumor growth and tumorigenic stem-like cells in subcutaneous xenografts of human prostate cancer cells in nude mice. These results support the hypothesis that LSD1 has a major role in sustaining the stem-like and tumorigenic subpopulation in prostate tumors and its inhibition by chemical or genetic approaches prevents survival and self-renewal capability of prostate CSCs. These data suggest that INCB059872 could be a valid addition to the current treatment strategies for prostate cancer. INCB059872 is currently in phase1/2 clinical studies Citation Format: Gianluca Civenni, Giada Zoppi, Ramiro Vazquez, Dhreeraj Shinde, Alyssa Paganoni, Aleksandra Kokanovic, Sang Hyun Lee, Bruce Ruggeri, Giuseppina M. Carbone, Carlo V. Catapano. INCB059872, a novel FAD-directed LSD1 Inhibitor, is active in prostate cancer models and impacts prostate cancer stem-like cells [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 1379.

FOXA2 promotes the proliferation, migration and invasion, and epithelial mesenchymal transition in colon cancer.

The present study determined the expression and biological functions of FOXA2 gene in colon cancer in tissues, cells and animals. A total of 66 patients with colon cancer were included in the present study. Using The Human Protein Atlas database, expression and distribution of FOXA2 in colon cancer tissues were analyzed. Using immunohistochemistry, the expression and distribution of FOXA2 in colon cancer cells were studied. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to determine the expression of FOXA2 mRNA in colon cancer tissues. Following in vitro transfection with FOXA2 interference sequence (siR-FOXA2), the proliferation, cell cycle, migration and invasion of colon cancer HCT116 and HT29 cells were investigated using Cell Counting Kit-8 assay, flow cytometry, and Transwell assay, respectively. Furthermore, flow cytometry was used to determine apoptosis of HCT116 and HT29 cells. Western blotting was used to determine the expression of epithelial mesenchymal transition (EMT) proteins, E-Cadherin and Vimentin. Laser scanning confocal microscopy was performed to observe the cytoskeleton in HCT116 and HT29 cells. Results indicated tumorigenesis of colon cancer cells in nude mice. In addition, the expression of FOXA2 in colon cancer tissues was elevated and associated with the metastasis and clinical staging of colon cancer. Notably, inhibition of FOXA2 reduced the proliferation of colon cancer cells in vitro and reduced expression of FOXA2 was able to decrease the migration and invasion abilities of colon cancer cells. Furthermore, FOXA2 promoted EMT, inhibited apoptosis and enhanced the invasion ability of colon cancer cells. Decreased expression of FOXA2 inhibited tumorigenesis of colon cancer cells in nude mice. To conclude, the present study demonstrated that the expression of FOXA2 in colon cancer tissues was elevated and associated with the metastasis and clinical staging of colon cancer. As an oncogene, FOXA2 may promote the proliferation, migration and invasion and EMT in colon cancer.

Discovery and cellular stress pathway analysis of 1,4-naphthoquinone derivatives with novel, highly potent broad-spectrum anticancer activity

BackgroundChemotherapy and targeted therapies have made important strides in cancer treatment yet they often fail and new therapies are still needed. Here, we employed a phenotypic screen to identify and analyze the mechanism of action of novel small molecules that interfere with critical pathways involved in tumor cell growth, using chemoresistant A375 melanoma cells as a model.MethodsCell culture studies were performed in ATCC-recommended media. Compounds, and compound libraries were obtained from Boston University or purchased commercially. Effects on A375 cell viability, proliferation and morphology were determined by Celigo Image Cytometer and viability staining. Anticancer activity of the lead compound was tested in a xenograft nude mouse model. Signaling and cell death pathways were analyzed by SDS-PAGE and immunoblotting, and/or fluorescence microscopy.ResultsAfter evaluating 4477 compounds, one hit compound CB533 was identified that caused significant reduction of A375 cell growth. CB533 is an unexplored 1,4-naphthoquinone (NQ) derivative which unlike 1,4-NQ, induced rapid cell death without generating reactive oxygen species (ROS). Structure-activity relationship analysis showed that a pyrrolidine in the 1,4-NQ nucleus in lead compound Pyr-1 yielded optimal activity. CB533 and Pyr-1 had growth-suppressing effects on a large variety of chemotherapy-resistant cancer cell lines in the nano to picomolar range. Pyr-1 also significantly reduced growth of MDA-MB-231 breast cancer cells in nude mice. Pyr-1 rapidly induced activation of major stress pathways and autophagy, which was efficiently blocked by ERK, and somewhat by PI3K inhibitors.ConclusionCB533 and lead Pyr-1 represent novel broad-spectrum, anticancer compounds that are up to 1000-fold more potent than plumbagin, a natural 1,4-NQ with known anticancer activity. Since the growth suppression activities of CB533 and Pyr-1 are unaffected by the chemotherapy resistance of cancer cells, these compounds have promising therapeutic potential. The pyrrolidine in the 3 position of the 1,4-NQ nucleus of Pyr-1 is a critical component of the pharmacophore. Pyr-1-induced cellular stress was mediated by an ERK, and to a lesser extent by an AKT-dependent pathway without involving apoptosis. Our data suggest that Pyr-1 derives its greatly enhanced antitumor activity via mimicking ROS-induced stress signaling without generating ROS, and likely committing cells to autophagy.

HAase-sensitive dual-targeting irinotecan liposomes enhance the therapeutic efficacy of lung cancer in animals.

Among all cancers, lung cancer is one of the most common and serious types of cancer. It is challenging for site-specific delivery of anticancer therapeutics to tumor cells. Herein, we developed a novel“smart” dual-targeting liposomal platform to respond to the highly expressed hyaluronidase (HAase) in the tumor microenvironment and improve tumor targeting and antitumor efficacy.Methods: In our design, the HA was used as a sensitive linker between a liposomal lipid and long chain PEG block to synthesize three functional conjugates in order to prepare“smart” liposomal platform modified with epidermal growth factor receptor (EGFR) antibody (GE11) and cell-penetrating peptide (TATp). Using irinotecan as a model therapeutic, evaluations were performed on the human lung adenocarcinoma A549 cells as well as the xenografted A549 cancer cells in nude mice.Results: The GE11/HA/TATp-irinotecan liposomes evidently increased the uptake of irinotecan and showed significant antitumor efficacy in the xenografted A549 cancer cells in nude mice by intravenous administration. The mechanisms were defined to be two aspects: GE11 exhibits high affinity for EGFR binding and the degradation of the HA by HAase results in the long-chain PEG removal and exposure of the previously hidden surface-attached TATp to enhance the target cell internalization.Conclusion: Our findings suggest that this functional liposomal platform may provide a novel strategy for treating lung cancers because of effective intracellular delivery.

SOX4 contributes to TGF-β-induced epithelial–mesenchymal transition and stem cell characteristics of gastric cancer cells

SOX4 is highly expressed in gastric cancer (GC) and is associated with tumor grade, metastasis and prognosis, however the mechanism is not clear. We report herein that SOX4 was upregulated and overexpression of SOX4 was associated with increased expression of the markers of Epithelial–mesenchymal transition (EMT) and stemness in clinic patient samples. In vitro, overexpression of SOX4 promoted the invasion as showed by Transwell assay and stemness of GC cells as assessed by sphere formation assay, which was suppressed by silencing SOX4 with shRNA. Further studies showed that SOX4 up-regulated the expression of EMT transcription factors Twist1, snail1 and zeb1 and stemness transcription factors SOX2 and OCT4, and promoted the nuclear translocation of β-catenin. Moreover, we revealed that TGF-β treatment significantly up-regulated the expression of SOX4 and silencing SOX4 reversed TGF-β induced invasion and sphere formation ability of GC cells. Finally, we showed that SOX4 promoted the lung metastasis and tumor formation ability of gastric cancer cells in nude mice. Our results suggest that SOX4 is a target TGF-β signaling and mediates TGF-β-induced EMT and stem cell characteristics of GC cells, revealing a novel role of TGF-β/SOX4 axis in the regulation of malignant behavior of GC.

Lewis y antigen promotes p27 degradation by regulating ubiquitin-proteasome activity

As a tumor-associated carbohydrate antigen, elevated expression of Lewis y promotes the malignant behaviors of tumor cells. Although our preliminary study showed that the increased expression of Lewis y antigen decreased the expression of cell cycle inhibitor protein p27, the relevant mechanism remains unclear. Autophagy and the ubiquitin-proteasome system are two main ways of intracellular protein degradation, whose abnormal activities are closely associated with progression of malignant tumors. In our present study, we constructed two stable transfected cell lines with high expression of Lewis y antigen, named CAOV3-FUT1 and SKOV3-FUT1. We showed that the proportion of cells at S phase was significantly increased after FUT1 transfection, whereas p27 protein was obviously decreased. The autophagy activity, the levels of ubiquitination, and chymotrypsin-like protease activity were increased remarkably in the transfected cells. Interestingly, Lewis y antigen promoted the degradation of p27 by increasing ubiquitin-proteasome activity. In the vivo studies, Lewis y antigen improved the tumorigenic ability of ovarian cancer cells in nude mice and reduced the expression of p27. These findings suggested that Lewis y antigen activated both the autophagy and ubiquitin-proteasome activity and promoted the degradation of p27 through the ubiquitin-proteasome pathway.

TIPE1 suppresses invasion and migration through down-regulating Wnt/β-catenin pathway in gastric cancer.

Epithelial–mesenchymal transition (EMT) plays an important role in the invasiveness and metastasis of gastric cancer. Therefore, identifying key molecules involved in EMT will provide new therapeutic strategy for treating patients with gastric cancer. TIPE1 is a newly identified member of the TIPE (TNFAIP8) family, and its contributions to progression and metastasis have not been evaluated. In this study, we found that the levels of TIPE1 were significantly reduced and inversely correlated with differentiation status and distant metastasis in primary gastric cancer tissues. We further observed overexpression of TIPE1 in aggressive gastric cancer cell lines decreased their metastatic properties both in vitro and in vivo as demonstrated by markedly inhibiting EMT and metastasis of gastric cancer cells in nude mice. Consistently, gene silencing of TIPE1 in well‐differentiated gastric cancer cell line (AGS) inhibited these processes. Mechanistically, we found that TIPE1‐medicated Wnt/β‐catenin signalling was one of the critical signal transduction pathways that link TIPE1 to EMT inhibition. Importantly, TIPE1 dramatically restrained the expression and activities of MMP2 and MMP9 which are demonstrated to promote tumour progression and are implicated in EMT. Collectively, these findings provide new evidence for a better understanding of the biological activities of TIPE1 in progression and metastasis of gastric cancer and suggest that TIPE1 may be an innovative diagnostic and therapeutic target of gastric cancer.

Herbal formula YYJD inhibits tumor growth by inducing cell cycle arrest and senescence in lung cancer

Lung cancer represents a major cause of cancer-related death worldwide. Although various tactics and anti-tumor drugs have been used to improve curative effects, five-year survival rate of lung cancer patients remains poor. In this study, we investigated the action and underlying mechanisms of our recently optimized Chinese herbal formula Yangyinjiedu (YYJD) against lung cancer. YYJD significantly inhibits the proliferation of lung cancer cell lines (95-D, A549, H460 and H1975) by inducing cell cycle arrest and senescence in a dose-dependent manner. In particular, YYJD induces significant G2/M phase arrest and inhibits the colony formation of lung cancer cells. Moreover, we found that administration of YYJD could inhibit the growth of xenografted lung cancer cells in nude mice without loss in body weight. Our findings suggest that the herbal formula YYJD is a potential anti-tumor agent against lung cancer.

Complement component 1, q subcomponent binding protein (C1QBP) in lipid rafts mediates hepatic metastasis of pancreatic cancer by regulating IGF‐1/IGF‐1R signaling

Pancreatic cancer shows a remarkable predilection for hepatic metastasis. Complement component 1, q subcomponent binding protein (C1QBP) can mediate growth factor-induced cancer cell chemotaxis and distant metastasis by activation of receptor tyrosine kinases. Coincidentally, insulin-like growth factor-1 (IGF-1) derived from the liver and cancer cells itself has been recognized as a critical inducer of hepatic metastasis. However, the mechanism underlying IGF-1-dependent hepatic metastasis of pancreatic cancer, in which C1QBP may be involved, remains unknown. In the study, we demonstrated a significant association between C1QBP expression and hepatic metastasis in patients with pancreatic cancer. IGF-1 induced the translocation of C1QBP from cytoplasm to lipid rafts and further drove the formation of CD44 variant 6 (CD44v6)/C1QBP complex in pancreatic cancer cells. C1QBP interacting with CD44v6 in lipid rafts promoted phosphorylation of IGF-1R and thus activated downstream PI3K and MAPK signaling pathways which mediated metastatic potential of pancreatic cancer cells including proliferation, apoptosis, invasion, adhesion and energy metabolism. Furthermore, C1QBP knockdown suppressed hepatic metastasis of pancreatic cancer cells in nude mice. We therefore conclude that C1QBP in lipid rafts serves a key regulator of IGF-1/IGF-1R-induced hepatic metastasis from pancreatic cancer. Our findings about C1QBP in lipid rafts provide a novel strategy to block IGF-1/IGF-1R signaling in pancreatic cancer and a reliable premise for more efficient combined modality therapies.

MiR-187 inhibits the growth of cervical cancer cells by targeting FGF9.

MicroRNAs (miRNAs) are a cluster of short non-coding RNAs playing critical roles in human cancers. miR-187 was recently found to be a novel cancer-related microRNA. However, the expression and function of miR-187 in cervical cancer have not been investigated. In this study, we found that miR-187 level was decreased in cervical cancer tissues and cell lines. Patients with low level of miR-187 had significantly decreased rate of overall survival (OS) and progression-free survival (DFS). miR-187 overexpression inhibited proliferation and promoted apoptosis of cervical cancer cells, whereas miR-187 knockdown promoted proliferation and inhibited apoptosis of cervical cancer cells. Forced expression of miR-187 inhibited the subcutaneous growth of cervical cancer cells in nude mice. Furthermore, FGF9 was found to be the downstream target of miR-187 in cervical cancer cells. Importantly, targeting FGF9 was required for miR-187 exerting its tumor suppressive roles in cervical cancer cells.

In Vivo Selection of Intermediately- and Highly-Malignant Variants of Triple-negative Breast Cancer in Orthotopic Nude Mouse Models.

Triple-negative breast cancer (TNBC), defined by the absence of receptors for estrogen, progesterone and human epithelial receptor 2 (HER2), is a recalcitrant disease in need of effective therapy. We previously isolated very-highly metastatic variants of the TNBC cell line MDA-MB-231 using serial orthotopic implantation of MDA-MB-231 human breast cancer cells in nude mice. MDA-MB-231 cells expressing red fluorescent protein (MDA-MB-231-RFP) (1×107 cells/site) were initially injected subcutaneously in the flank of nude mice. After the subcutaneous tumors grew, they were harvested and cut into small pieces for orthotopic implantation into the right lower mammary gland. After the orthotopic tumors grew, they were resected and cut into small pieces and orthotopically re-implanted into the mammary gland of nude mice. The tumors grew and metastasized to lymph nodes. The lymph node metastases were harvested and cut into small pieces and orthotopically re-implanted into the mammary gland of nude mice. After the orthotopic tumors grew, the tumor was removed leaving residual cancer cells, which grew and metastasized to lymph nodes. The lymph node metastases were harvested, cut into pieces and orthotopically re-implanted into the mammary gland of nude mice for three or seven cycles in order to isolate intermediately, or highly-metastatic variants, respectively. The degree of malignancy of isolated variants of MDA-MB-231 depends on the extent of orthotopic transplantation. Serial transplantation resulted in MDA-MB-231-RFP which significantly produced larger tumors compared with the parental MDA-MB-231-RFP. Serial orthotopic implantation for three cycles resulted in intermediately-metastatic variants of MDA-MB-231-RFP. MDA-MB-231-RFP serially orthotopically transplanted seven times significantly metastasized more to lymph nodes compared with parental MDA-MB-231-RFP cells and the intermediately-metastatic variant. The highly-metastatic variant MDA-MB-231-RFP cells significantly metastasized to the lung to a greater extent compared with parental MDA-MB-231-RFP and intermediate variants of MDA-MB-231-RFP. These results suggest that the number of serial orthotopic transplantations of MDA-MB-231-RFP correlates positively with tumor aggressiveness, and the intermediately- and highly-metastatic variants can serve as models of metastatic progression of TNBC.

Polyphyllin I inhibits the growth of ovarian cancer cells in nude mice.

Polyphyllin I (PPI) is an active component in Rhizoma Paridis, which displays extensive pharmacological antitumor activities. In a previous study, we found that polyphyllin I exhibited inhibitory effects on cell growth in the human ovarian cancer HO-8910PM cell line, as well as promoting apoptosis and the inhibition of cell migration. Furthermore, gene expression was also profiled by microarray, which showed that numerous genes were altered by PPI; three genes were of particular note that were associated with tumor progression, namely, Caspase-9, C-jun and Wnt5a. In the present study, the effect of PPI on subcutaneous tumor growth (HO-8910PM cells) in nude mice was further evaluated, and immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) were used to examine the expression of Caspase-9, C-jun and Wnt5a in subcutaneous and lung metastatic tumor tissues, in order to investigate the possible mechanisms involved. The results showed that PPI significantly inhibited the tumor growth in vivo without a marked impact on body weight, and through use of immunohistochemical staining and RT-PCR, it was found that the expression of Caspase-9 and Wnt5a was decreased, while the expression of C-jun was increased, in subcutaneous and lung metastatic tumor tissue; this was consistent with the in vitro results. In conclusion, the present study showed that PPI exerted antitumor activity on ovarian cancer cells in vivo, and indicated that the modulation of Caspase-9, C-jun and Wnt5a may be involved in the antitumor effect of PPI.

Enhanced Metastatic Recurrence Via Lymphatic Trafficking of a High-Metastatic Variant of Human Triple-Negative Breast Cancer After Surgical Resection in Orthotopic Nude Mouse Models.

We previously developed and characterized a highly invasive and metastatic triple-negative breast cancer (TNBC) variant by serial orthotopic implantation of MDA-MB-231 human breast cancer cells in nude mice. Eventually, a highly invasive and metastatic variant of human TNBC was isolated after lymph node metastases was harvested and orthotopically re-implanted into the mammary gland of nude mice for two cycles. The variant thereby isolated is highly invasive in the mammary gland and metastasized to lymph nodes in 10 of 12 mice compared to 2 of 12 of the parental cell line. In the present report, we observed that high-metastatic MDA-MB-231H-RFP cells produced significantly larger subcutaneous tumors compared with parental MDA-MB-231 cells in nude mice. Extensive lymphatic trafficking by high-metastatic MDA-MB-231 cells was also observed. High-metastatic MDA-MB-231 developed larger recurrent tumors 2 weeks after tumor resection compared with tumors that were not resected in orthotopic models. Surgical resection of the MDA-MB-231 high-metastatic variant primary tumor in orthotopic models also resulted in rapid and enhanced lymphatic trafficking of residual cancer cells and extensive lymph node and lung metastasis that did not occur in the non-surgical mice. These results suggest that surgical resection of high metastatic TNBC can greatly increase the malignancy of residual cancer. J. Cell. Biochem. 118: 559-569, 2017. © 2016 Wiley Periodicals, Inc.

Phosphorylation of LSD1 at Ser112 is crucial for its function in induction of EMT and metastasis in breast cancer.

LSD1 is overexpressed in various cancers including breast cancer, but its functional roles in tumourigenesis are not fully understood. This study aims at revealing the role of LSD1 in breast cancer development. In addition, it has been reported that phosphorylation of the Serine 112 residue of LSD1 by PKCα is crucial for its function in gene regulation. We also explored whether this phosphorylation affects LSD1's role in breast cancer development. This study includes LSD1 IHC data generated with tissue microarrays of 163 cases of breast cancer samples and 72 normal tissues. In vitro, role of LSD1, LSD1 S112D mutant (a phosphorylation simulation) and LSD1 S112A mutant (an unphosphorylation simulation) in induction of EMT is evaluated. Mechanismly, we checked the role of LSD1 and its mutant on E-cadherin promoter histone modifications. We also investigated the role of LSD1 and its mutants in metastasis with a nude mice model. We found LSD1 is expressed at a higher level in breast cancer tissues compared with that in normal tissues, and LSD1 expression is closely linked to breast cancer metastasis. LSD1 potentiates EMT in breast epithelia cells by repressing E-cadherin expression through demethylating H3K4me at gene's promoter, during which phosphorylation of LSD1 Ser112 is crucial for its binding and demethylation activity. In vivo, knockdown of LSD1 impairs the metastatic ability of MDA-MB-231 breast cancer cells in nude mice. Ectopic overexpression of either LSD1 or LSD1 S112D mutant (a phosphorylation simulation) facilitates metastasis, whereas the LSD1 S112A mutant (an unphosphorylation simulation) fails to affect the metastasis. Data presented in this report indicate that LSD1 is able to induce EMT and to promote metastasis in breast cancer, and phosphorylation at LSD1 Ser112 is crucial for these functions.

Abstract 1015: Repurposing the FDA-approved drug carbidopa to treat human cancers

Abstract Carbidopa is used in combination with L-DOPA to treat Parkinson's disease; it does not have any therapeutic use by itself in Parkinson's disease, but when used along with L-DOPA prevents the conversion of the latter into dopamine in the periphery by inhibiting aromatic amino acid decarboxylase. Carbidopa however does not cross the blood-brain barrier; thus does not impact on the conversion of L-DOPA into dopamine in the brain. We hypothesized that carbidopa might have potential as an anticancer drug with the following rationale: (a) carbidopa is an amino acid derivative and therefore might block the entry of amino acids into cancer cells via certain amino acid transporters; (b) the stress hormones epinephrine and norepinephrine are known to promote cancer progression, and carbidopa as an inhibitor of aromatic amino acid decarboxylase might interfere with the generation of these tumor-promoting hormones; (c) carbidopa is also an analog of phenylhydrazine, which is an inhibitor of the immunosuppressive enzyme indoleamine-2,3-dioxygenase, a drug target for cancer treatment; carbidopa might inhibit this enzyme and thus enhance the ability of the immune system to recognize cancer cells as foreign and fight against them. Based on this rationale, we examined the efficacy of carbidopa to treat pancreatic and breast cancer. We found carbidopa to be effective in blocking the proliferation of pancreatic cancer cell in vitro. We then examined its efficacy in vivo using xenografts of pancreatic cancer cells in nude mice; again, the drug was effective in decreasing the growth of the xenografted tumor cells into tumors. We also tested its efficacy on proliferation of breast cancer cell lines; we used ZR-75.1, MB-231, and HCC-1937 breast cancer cell lines as models for ER-positive, ER-negative and BRCA-1 mutant breast cancers, respectively. Carbidopa decreased the proliferation of all three cell lines. We then examined its in vivo efficacy against breast cancer using the MMTV-PyMT-transgenic mouse as a model of spontaneous breast cancer. In this model, breast cancer develops initially as an ER-positive subtype but then turns into an ER-negative subtype. Carbidopa markedly decreased the growth of breast cancer in this mouse model. Based on these in vitro and in vivo data, we conclude that carbidopa has promise for use as an anticancer drug. As the drug potentially elicits its anticancer effects by targeting multiple pathways, the anticancer efficacy of the drug is likely to be broad against different types of human cancers. For in vivo studies, we used the drug intraperitoneally at a dose of 1 mg/mouse that approximately translates to a human dose of 300-400 mg/day. As the drug has been shown to have no detectable side effects in humans at doses as high as 400 mg/day, it can be taken to clinical trials readily to test its efficacy in humans as an anticancer drug. Citation Format: Vadivel Ganapathy, Ellappan Babu, Sabarish Ramachandran, Yangzom D. Bhutia. Repurposing the FDA-approved drug carbidopa to treat human cancers. [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 1015.