Growth Of Subcutaneous Xenografts Research Articles

Abstract P157: Targeting Trop2 for treatment of prostate and breast cancer

Abstract Background: Prostate cancer and breast cancer are the most commonly diagnosed non-skin cancers and the second leading cause of cancer related mortalities in men and women respectively in the United States. Fifteen percent of breast cancer is TNBC characterized with lack of receptors for estrogen, progesterone, and HER-2/neu making it resistant to hormone therapies and therapies targeting estrogen, progesterone, and HER-2/neu signaling. The first line of treatment for men with advanced prostate cancer is androgen depravation therapy. While initial responses are observed, unfortunately, the disease frequently recurs as a lethal metastatic form referred to as castration-resistant prostate cancer (CRPC). We recently identified that the cell surface receptor, Trop2, is a new regulator of TNBC and CRPC. We demonstrated that Trop2 is highly expressed in CRPC and TNBC. In this study, we set out to test the therapeutic potential of new fully humanized human anti-Trop2 antibody fragments in CRPC and NEPC. Methods: We have generated new human anti-Trop2 antibody fragments. Lentiviral infection was used to generate prostate and breast cancer cell lines with over-expression of Trop2 and knock-down of Trop2. In vivo functional assays were performed including subcutaneous xenograft growth and metastasis. Results: High protein levels of Trop2 are observed in metastatic CRPC and TNBC. Furthermore, loss of Trop2 significantly delays growth, migration and invasion of prostate and breast cancer cells in vitro and delays tumor growth and metastasis of prostate and breast cancer in vivo. Overexpression of Trop2 enhances tumor growth and metastasis of prostate cancer cells and drives CRPC. We generated new fully humanized human anti-Trop2 antibody fragments that have therapeutic efficacy in preclinical models of prostate and breast cancers. Conclusions: In a previous study, we identified cell surface receptor, Trop2, as a novel driver of metastatic CRPC. Herein, our new findings reveal that Trop2 represents a promising therapeutic target for prostate, breast, and potentially other epithelial cancers. We identified lead antibody candidates that bind to human Trop2 protein with high affinity and stability. Furthermore, our lead anti-Trop2 Abs significantly delay the tumors growth in CRPC and TNBC without measurable toxicity and provide new therapeutic strategy for metastatic CRPC and TNBC. Citation Format: Tanya Stoyanova. Targeting Trop2 for treatment of prostate and breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P157.

Multiple Antitumor Molecular Mechanisms Are Activated by a Fully Synthetic and Stabilized Pharmaceutical Product Delivering the Active Compound Sulforaphane (SFX-01) in Preclinical Model of Human Glioblastoma

Frequent relapses and therapeutic resistance make the management of glioblastoma (GBM, grade IV glioma), extremely difficult. Therefore, it is necessary to develop new pharmacological compounds to be used as a single treatment or in combination with current therapies in order to improve their effectiveness and reduce cytotoxicity for non-tumor cells. SFX-01 is a fully synthetic and stabilized pharmaceutical product containing the α-cyclodextrin that delivers the active compound 1-isothiocyanato-4-methyl-sulfinylbutane (SFN) and maintains biological activities of SFN. In this study, we verified whether SFX-01 was active in GBM preclinical models. Our data demonstrate that SFX-01 reduced cell proliferation and increased cell death in GBM cell lines and patient-derived glioma initiating cells (GICs) with a stem cell phenotype. The antiproliferative effects of SFX-01 were associated with a reduction in the stemness of GICs and reversion of neural-to-mesenchymal trans-differentiation (PMT) closely related to epithelial-to-mesenchymal trans-differentiation (EMT) of epithelial tumors. Commonly, PMT reversion decreases the invasive capacity of tumor cells and increases the sensitivity to pharmacological and instrumental therapies. SFX-01 induced caspase-dependent apoptosis, through both mitochondrion-mediated intrinsic and death-receptor-associated extrinsic pathways. Here, we demonstrate the involvement of reactive oxygen species (ROS) through mediating the reduction in the activity of essential molecular pathways, such as PI3K/Akt/mTOR, ERK, and STAT-3. SFX-01 also reduced the in vivo tumor growth of subcutaneous xenografts and increased the disease-free survival (DFS) and overall survival (OS), when tested in orthotopic intracranial GBM models. These effects were associated with reduced expression of HIF1α which, in turn, down-regulates neo-angiogenesis. So, SFX-01 may have potent anti-glioma effects, regulating important aspects of the biology of this neoplasia, such as hypoxia, stemness, and EMT reversion, which are commonly activated in this neoplasia and are responsible for therapeutic resistance and glioma recurrence. SFX-01 deserves to be considered as an emerging anticancer agent for the treatment of GBM. The possible radio- and chemo sensitization potential of SFX-01 should also be evaluated in further preclinical and clinical studies.

TRIM22 inhibits the proliferation of gastric cancer cells through the Smad2 protein

TRIM22 is involved in tumorigenesis and development, but its mechanism is not clear. In this study, we investigated the expression and biological role of TRIM22 in gastric cancer. We found that TRIM22 mRNA and protein expression was abnormally low in gastric cancer tissues and cells and correlated with tumor size and depth of invasion. Overexpression of TRIM22 significantly inhibited the proliferation, colony formation, and migration of gastric cancer cells and downregulated the expression of HSPA6. However, the HSPA6-siRNA complementation test showed that TRIM22 did not regulate cell proliferation through HSPA6. Furthermore, overexpression of TRIM22 downregulated the phosphorylation of Smad2 and Smad3. In addition, TRIM22 directly binds to Smad2, and overexpression of Smad2 can reverse the inhibition of cell proliferation and migration induced by TRIM22. In vivo, overexpression of TRIM22 significantly inhibited the growth of subcutaneous xenografts in nude mice. Our study indicates that TRIM22 has an important role in the development of gastric cancer and may inhibit the proliferation of gastric cancer cells through Smad2.

Abstract 1122: Novel targets for therapy resistant pancreatic neuroendocrine tumors

Abstract Pancreatic Neuroendocrine Tumors (PNETs) remains an unmet clinical problem and epidemiologic studies indicate that their incidence has significantly increased over the years. Surgery remains the only curative option in patients with localized tumors. However, there is no effective therapy in patients with advanced or metastatic disease. Thus, unfortunately, 65% of PNET patients with advanced/unresectable disease die within 5 years after diagnosis. Current therapeutic approaches for advanced PNET patients include chemotherapy (Capecitabine, Temolozomide, 5FU etc), targeted therapies (everolimus, and sunitinib), hormonal therapies [somatostatin analogs (octreotide or lanreotide)] and the novel peptide receptor radionuclide therapy. Nevertheless, all these therapeutic modalities show only minimal response in patients with PNETs in the clinic. Therefore, novel targets need to be identified that could improve the dismal outcome of advanced PNETs. An analysis of PNET tissue identified p21-activated Kinase 4 (PAK4) and nicotinamide Phosphoribosyltransferase (NAMPT) as two new therapeutic targets. PAK4 is the downstream effector of Rac1 (members of the Rho family of GTPases) and is involved in critical cellular processes such as cell motility, proliferation, and survival. More importantly, PAK4 protein has been implicated in the activation of Ras/Raf/Mek/Erk and PI3K/Akt/mTOR signaling in cancers. Similarly, NAMPT is an enzyme that catalyzes the rate-limiting step in the principal salvage pathway of NAD biosynthesis in mammals. Tumor cells have highly active glycolytic, pentose and fatty acid synthesis pathways that require persistent high levels of NAD. Consequently, most cancers rely more heavily on NAMPT for rapid NAD biosynthesis. Earlier we demonstrated that biological or chemical inhibition of PAK4-NAMPT by RNAi or by a dual inhibitor (KPT-9274 a Phase I drug and its analogs) can suppress PNET proliferation and reduce the growth of subcutaneous xenografts. Our new studies show that PAK4-NAMPT dual inhibition can suppress PNET anchorage independent growth and metabolomic analysis of KPT-9274 treated PNET cells revealed significant alterations in a series of metabolites related to NAD signaling. KPT-9274 synergistically enhanced the anti-tumor activity of everolimus (combination index <1). Molecular analysis of combination treatment showed down-regulation of known everolimus resistance drivers including mTORC1, mTORC2, PI3K, ERK, FAK, RICTOR, ß-catenin. Importantly, combination treatment of KPT-9274 (150mg/kg) and everolimus (2.5 mg/kg used at sub-optimal dose) caused reduction of PNET xenografts. Our investigations demonstrate that PAK4 and NAMPT are two viable therapeutic targets in the difficult to treat PNETs that warrant further clinical investigations. Citation Format: Gabriel B. Mpilla, Amro Aboukameel, Md Hafiz Uddin, Mohammed N. Al-Hallak, Bayan Al-Share, Yosef Landesman, Yiwei Li, Steve Kim, Rafic Beydoun, Ramzi M. Mohammad, Philip A. Philip, Bassel El-Rayes, Asfar S. Azmi. Novel targets for therapy resistant pancreatic neuroendocrine tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1122.

Silence of circBANP increases radiosensitivity of colorectal cancer cells and inhibits growth of subcutaneous xenografts by up-regulating miR-338-3p expression

Objective: To investigate the effect of circBANP on radiosensitivity of colorectal cancer cells and subcutaneous transplanted tumor in nude mice and its potential molecular mechanism. Methods: The carcinoma and adjacent normal mucosal tissues of 20 patients with colorectal cancer who were surgically resected in Henan People's Hospital from January 2018 to January 2019 were selected. The radio-resistant colorectal cancer cell LoVo/R was established. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expressions of circBANP and miR-338-3p. The radiation sensitivity was determined by cell clone formation experiment. Cell vitality was detected by using methyl thiazolyl tetrazolium (MTT). The expressions of autophagy-related protein microtubule-associated protein light chain 3 (LC3) and p62 were detected by western blot. The fluorescence intensity of LC3 in cells was detected by immunofluorescence assay. The downstream microRNAs (miRNAs) of circBANP were predicted by Circular RNA Interactome website and further verified by dual luciferase reporter gene assay. The transplanted tumor model of LoVo/R cells in nude mice was established, and the effect of circBANP on the growth of transplanted tumor after radiation was observed. Results: The expression levels of circBANP and miR-338-3p in colorectal cancer tissues were 3.21+ 0.29 and 0.47+ 0.04, respectively, which were significantly higher than 1.00+ 0.07 and 1.00+ 0.05 in adjacent tissues (P<0.05). The circBANP expression level of LoVo/R cells was 3.21±0.34, higher than 1.00±0.07 of LoVo cells (P<0.05), and the expression level of miR-338-3p of LoVo/R cells was 0.33±0.04, lower than 1.00±0.08 of LoVo cells (P<0.05). After 4 Gy irradiation, compared with the control group, the viability of LoVo/R cells in the circBANP silencing group [(34±4)% vs (62±6)%, P<0.05], the cell survival fraction (0.07±0.02 vs 0.27±0.04, P<0.05) were decreased, and the radiation sensitization ratio was 1.843, the expression of LC3Ⅱ/Ⅰin LoVo/R cells increased while p62 expression decreased, the cell autophagy was observed. Autophagy inhibitor chloroquine reversed the increased expression of LC3Ⅱ/Ⅰ and inhibited expression of p62 in LoVo/R cells induced by radiation, and promoted the suppression of cell viability and survival induced by radiation, the radiotherapy sensitization ratio was 1.780. Compared with control group after 4 Gy irradiation, the relative fluorescence intensity of LC3 in circBANP silencing LoVo/R cells decreased (0.11±0.01 vs 1.00±0.12, P<0.05), the expression of LC3-Ⅱ/Ⅰdecreased (1.25±0.13 vs 3.84±0.39, P<0.05) while p62 expression increased (2.76±0.29 vs 1.00±0.08, P<0.05). As predicted by Circular RNA Interactome website and confirmed by double luciferase reporter gene assay, miR-338-3p was the target gene of circBANP. The relative fluorescence intensity of LC3 in circBANP silencing + anti-miR-338-3p + 4 Gy group increased (7.32±0.72 vs 1.00±0.09, P<0.05), the expression level of LC3-Ⅱ/Ⅰ increased (4.13±0.43 vs 2.31±0.23, P<0.05) while p62 expression decreased (0.34±0.03 and 1.00±0.11, P<0.05), the radiotherapy sensitization ratio was 0.596. Nude mice subcutaneously transplanted tumor experiment showed that the tumor volume and weight of circBANP silencing group on 13, 16, 19, 22, 25, 28, and 31 days were lower than those of control group (P<0.05), while the tumor volume and weight of circBANP silencing + anti-miR-338-3p group on days of 13, 16, 19, 22, 25, 28 and 31 after inoculated were higher than those of circBANP+ anti-miR-NC group (P<0.05). Conclusions: CircBANP can regulate the radiosensitivity of colorectal cancer cells by regulating the expression of miR-338-3p, and affect the growth of transplanted tumor in nude mice. CircBANP may be a potential target for enhancing radiosensitivity of colorectal cancer cells.

Antitumor efficacy of 2-(6,8-dimethyl-5-nitro-4-chloroquinoline-2-yl)-5,6,7-trichloro-1,3-tropolone in lung cancer xenografts.

e15008 Background: Lung cancer is the most common cancer worldwide. The efficacy of chemotherapy for this tumor does not exceed 40%. Moreover, all cytotoxic agents cause many side effects. The search for new substances with an antitumor effect seems to be relevant. Tropolone alkaloids, which are seven-membered non-benzenoid aromatic compounds, are promising inhibitors of tumor growth. The purpose of the study was to evaluate the antitumor efficacy of 2-(6,8-dimethyl-5-nitro-4-chloroquinoline-2-yl)-5,6,7-trichloro-1,3-tropolone in subcutaneous xenografts of lung cancer A549 cells in immunodeficient Balb/c Nude mice. Methods: The study was performed on a PDX model of lung cancer created by subcutaneous injection of A549 cell suspension, 5x106 cells in 0.2 ml of a solution of serum-free nutrient medium 199 and Matrigel (1:1). Animals were equally divided into 5 groups (each n = 5). Experimental groups 1, 2, 3 and 4 received 2-(6,8-dimethyl-5-nitro-4-chloroquinoline-2-yl)-5,6,7-trichloro-1,3-tropolone at doses of 0.0055, 0.055, 0.55 and 2.75 mg/g, respectively; the control group received 1% starch gel. The dynamics of the growth of a subcutaneous xenograft was evaluated by measuring tumor nodes. The volumes of tumor nodes were calculated using the Shrek’s formula for the ellipsoid: V = a×b×c×p/6, where V is the tumor volume (mm3), and a, b, c are the maximum dimensions of the ellipsoid in three planes (mm). The data were statistically analyzed using the Exel and Medstatistic progams. Results: The dynamics of subcutaneous xenograft growth in groups 1, 2, 3 and 4 significantly differed from the control group (p = 0.023). The average tumor volumes on the 39th day after the implantation in the control group and in experimental groups 1, 2, 3 and 4 were 1159.2, 895.3, 565.8, 80.7 and 76.7 mm3, respectively. The inhibition of tumor growth (ITG) was in direct proportion to the dose of the administered substance. Lower ITG(%) (32.4, 23.2 and 10.3% in experimental groups 2, 3 and 4, respectively) was associated with lower concentrations of О154 (0.55 mg/g, 0.055 mg/g and 0.0055 mg/g, respectively). Conclusions: The study demonstrated statistically significant differences in xenograft volume indices in all experimental groups compared to the control group.

Effects of silencing circRNA ABCB10 expression on biological properties of colorectal cancer cells

Objective: To investigate the expression of circular ribonucleic acid ABCB10 (circABCB10) in colorectal cancer tissues and cells and its effects on cell biological behavior, radiosensitivity and growth of subcutaneous xenografts. Methods: The tumor tissue and adjacent tissue from colorectal cancer patients treated in Henan People's Hospital were collected from January 2018 to December 2018. Quantitative polymerase chain reaction (qPCR) was used to detect the expressions of circABCB10 and miR-217, cell viability was detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT), cell apoptosis rate was detected by flow cytometry, cell migration and invasion were detected by Transwell method, cell radiosensitivity was detected by colony formation assay. The downstream miRNAs of circABCB10 were predicted by Circular RNA Interactome and verified by the dual luciferase reporter gene experiment. The effect of circABCB10 on the growth of transplanted tumor was examined in nude mice. Results: The expression level of circABCB10 mRNA in colorectal cancer tissues was (3.97±2.12), higher than (1.13±0.64) in adjacent tissues (P<0.05). The expression level of circABCB10 mRNA in FHC cells was (1.00±0.09), lower than that (4.53±0.44) in SW480, (3.12±0.32) in HCT116 and (3.51±0.36) in HT29 cells, respectively (all P<0.05). The MTT results showed that the absorbance values of SW480 cells in si-circABCB10-1 group at 48 and 72 hours after transfection were (0.36±0.04) and (0.43±0.04), lower than (0.48±0.05) and (0.82±0.08) in circ-negative control (NC) group, respectively (all P<0.05). The number of migrating cells and invasive cells in si-circABCB10-1 group were (45±8) and (34±7), lower than (106±21) and (84±15) in circ-NC group, respectively (all P<0.01). The radiosensitization ratio was 1.632. The results of subcutaneous transplantation assay showed that the tumor volume and tumor weight of the si-circABCB10-1 group were significantly lower than circ-NC group after 8 days of inoculation ( all P<0.05). MiR-217 is a target gene of circABCB10. Inhibition of miR-217 reversed the inhibitory effect of circABCB10 silencing on cell proliferation, migration, invasion and subcutaneous xenograft growth in nude mice and the radiosensitization activity. Conclusion: Silence of circABCB10 can up-regulate the expression of miR-217 to inhibit the proliferation, migration, invasion and growth of subcutaneous xenografts and increase the radiosensitivity of SW480 cells, which reveals the underlying molecular mechanism of colorectal cancer progression and provides a new sensitizing target for clinical radiotherapy of colorectal cancer.

Abstract PS17-09: Development of a potent mutant-ESR1 targeted agent, ERX-245, for treating metastatic therapy-resistant breast cancer

Abstract Background: ESR1 mutations are acquired following ERα targeted therapies and are a major determinant of therapy-resistance. These ESR1 mutations maintain ESR1 signaling, albeit in a ligand-independent manner. Effective drugs targeting these mutant (MT) ERα proteins represent a significant unmet clinical need. We had previously shown that ERX-11, an ESR1-coregulator binding inhibitor, could block the function of these MT ERα proteins. In this study, we sought to leverage recently published structures of MT ERα to develop more potent analogues of ERX-11. Methods: Virtual screening of &amp;gt;250,000 derivatives of ERX-11 was performed with simulated docking on the MT ERα to identify and design analogues of ERX-11. Several hundred analogues were synthesized and tested in vitro using multiple BC model cells that express wild type (WT) ESR1 or mutant (MT) ESR1 (Y537S or D538G). Mechanistic studies were performed using RNA-Seq, Western blotting, qRT-PCR and reporter gene assays. The in vivo efficacy of the most potent ERX-11 analogue ERX-245 was examined using xenograft, PDX and metastatic models of MT-ER driven BC. Results: From our virtual and functional screen, we identified an ERX-11 analogue, ERX-245 as the most potent hit to target MT-ERα. Docking studies modeled a better fit of ERX-245 into the ligand binding domain of both the Y537S and D538G MT-ERα. ERX-245 potently reduced (IC50 ~250 nM) the cell viability of both WT-ERα and MT-ERα driven BC cells but not ERα negative BC cells. ERX-245 significantly reduced the growth (colony formation, clonogenic and mammosphere assays) of MT-ERα BC cells. ERX-245 exhibited synergistic activity in combination with CDK4/6 inhibitors. In distinction to classic SERDs like fulvestrant (which degrade ERα with in 4h), ERX-245 treatment decreased MT-ERα protein levels over 24 hours. PK studies indicated that ERX-245 is more polar and has better solubility and pharmacokinetic properties than ERX-11. ERX-245 reduced tumor growth of subcutaneous xenograft and PDX models driven by MT-ERα as well as the proliferation of xenograft derived MT-ERα explant models. ERX-245 significantly reduced the invasive capability of MT-ERα BC cells in vitro and inhibited both the metastatic capability and growth of metastatic tumors derived from MT-ERα BC cells injected by intracardiac or intratibial routes. Conclusions: Taken together, these results indicate that ERX-245 is a potent and pharmacologically translatable analog of ERX-11, with activity against both primary and metastatic tumors driven by MT-ERα. Citation Format: Ganesh V Raj, Suryavathi Viswanadhapalli, Karla Parra, Shihong Ma, Tae-Kyung Lee, Xihui Liu, Kara Kassees, Weiwei Tang, Junhao Liu, Zexuan Liu, Uday P Pratap, Behnam Ebrahimi, Rajeshwar Rao Tekmal, Jung-Mo Ann, Ratna K Vadlamudi. Development of a potent mutant-ESR1 targeted agent, ERX-245, for treating metastatic therapy-resistant breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-09.

Effects of different combined regimens of cisplatin, metformin, and quercetin on nasopharyngeal carcinoma cells and subcutaneous xenografts

Cisplatin, metformin, and quercetin are all reliable anticancer drugs. However, it is unclear how effective their different combination regimens are on the growth of nasopharyngeal carcinoma cell line Sune-1 and subcutaneous xenograft in nude mice. This study evaluated the effects of single-drug, two-drug, and three-drug simultaneous or sequential combined application of these drugs on the growth of Sune-1 cells and subcutaneous xenograft tumors in nude mice. The results showed that the different combination regimens of cisplatin, metformin and quercetin all had significant inhibitory effects on the proliferation of Sune-1 cells and the growth of subcutaneous xenografts in nude mice (P < 0.01), and the inhibition rate of the three drugs simultaneous combined application was significant Higher than the two-drug combination or single-drug application (P < 0.05), the contribution level of each drug in the three-drug combination application from high to low were cisplatin > metformin > quercetin. In summary, our results indicate that the simultaneous combination of cisplatin, metformin, and quercetin may synergistically inhibit the growth of Sune-1 cells and subcutaneous xenografts in nude mice through their different anticancer mechanisms, which may be clinically refractory and provide reference for chemotherapy of patients with recurrent nasopharyngeal carcinoma.

Dual inhibition of DNA-PKcs and mTOR by CC-115 potently inhibits human renal cell carcinoma cell growth.

CC-115 is a dual inhibitor of DNA-PKcs and mTOR, both are valuable therapeutic targets for renal cell carcinoma (RCC). Our results showed that CC-115 inhibited survival and proliferation of established RCC cell lines (786-O and A489) and primary human RCC cells. The dual inhibitor induced selective apoptosis activation in RCC cells, as compared to no cytotoxicity nor apoptotic effects toward normal renal epithelial cells. CC-115 inhibited DNA-PKcs and mTORC1/2 activation in RCC cells. It was however ineffective in DNA-PKcs-mTOR double knockout (DKO) 786-O cells. CC-115 induced feedback autophagy activation in RCC cells. Autophagy inhibitors or Beclin-1/Light chain 3 (LC3) silencing potentiated CC-115-induced anti-RCC cell activity. Conversely, ectopic overexpression of Beclin-1 inhibited CC-115-induced cytotoxicity. At last CC-115 oral administration inhibited 786-O subcutaneous xenograft growth in nude mice. Taken together, dual inhibition of DNA-PKcs and mTOR by CC-115 potently inhibited RCC cell growth.

Interaction of BACH2 with FUS promotes malignant progression of glioma cells via the TSLNC8-miR-10b-5p-WWC3 pathway.

Glioma, a common malignant tumour of the human central nervous system, has poor prognosis and limited treatment options. Dissecting the biological mechanisms underlying glioma pathogenesis can facilitate the development of better therapies. Here, we investigated the endogenous expression of BTB and CNC homolog 2 (BACH2), fused in sarcoma (FUS), TSLNC8 and microRNA (miR)-10b-5p in glioma cells and tissues. We studied the interaction between BACH2 and FUS and its contribution to glioma progression. We demonstrated that the interaction between BACH2 and FUS promoted glioma progression via transcriptional inhibition of TSLNC8. Overexpression of TSLNC8 restrained glioma progression by suppressing miR-10b-5p. Binding of TSLNC8 to miR-10b-5p attenuated the suppression of WWC family member 3 (WWC3) by miR-10b-5p and activated the Hippo signalling pathway. Growth of subcutaneous xenografts could be inhibited by knockdown of BACH2 or FUS, by overexpressing TSLNC8 or a combination of the three, also leading to a prolonged survival in nude mice. Our results indicate that the BACH2 and FUS/TSLNC8/miR-10b-5p/WWC3 axis is responsible for glioma development and could serve as a potential target for the development of new glioma therapies.

MicroRNA-134 inhibits tumor stem cell migration and invasion in oral squamous cell carcinomas via downregulation of PI3K-Akt signaling pathway by inhibiting LAMC2 expression.

Oral squamous cell carcinoma (OSCC) is the most common malignant neoplasm of the mouth. Some studies have found that multiple microRNAs (miRs) participate in OSCC physiological and pathological processes. We explored the mechanism of action of miR-134 in OSCC involving the PI3K-Akt signaling pathway. Different bioinformatics methods were used to analyze the potential genes and their related miRs in OSCC. Tumor stem cells were separated from OSCCs through magnetic cell sorting. Regulatory pattern between miR-134 and LAMC2 in OSCC was evaluated by ectopic expression, knockdown and reporter assay experiments. The expression of miR-134, LAMC2, genes in PI3K-Akt signaling pathway, and apoptosis-related genes was detected. Cell proliferation was assessed by MTT assay, cell invasion by scratch test, cell migration by Transwell assay, cell cycle and apoptosis by flow cytometry, and cell growth and migration by xenograft tumor in nude mice. LAMC2 was predicted as the crucial factor related to OSCC using different chip data, and miR-134 was predicted to specifically bind LAMC2 in all five databases. Overexpressed miR-134 or silenced LAMC2 was observed to inhibit cell proliferation, migration, invasion of OSCC cells, growth of subcutaneous xenograft in nude mice, as well as promote OSCC cell apoptosis. LAMC2, a target gene of miR-134, decreased following miR-134 promotion, while the PI3K-Akt signaling pathway was inactivated following LAMC2 knockdown. Furthermore, we also observed that the effect of overexpressed miR-134 was enhanced when LAMC2 was knocked down. Taken together, these findings suggest that miR-134-mediated direct downregulation of LAMC2 inhibits migration and invasion of tumor stem cells in OSCC by suppressing the PI3K-Akt signaling pathway.

Isolation and characterization of castration-resistant prostate cancer LNCaP95 clones.

The androgen receptor (AR) is a validated therapeutic target for prostate cancer and has been a focus for drug development for more than six decades. Currently approved therapies that inhibit AR signaling, such as enzalutamide, rely solely on targeting the AR ligand-binding domain and, therefore, have limited efficacy on prostate cancer cells that express truncated, constitutively active AR splice variants (AR-Vs). The LNCaP95 cell line is a human prostate cancer cell line that expresses both functional full-length AR and AR-V7. LNCaP95 is a heterogeneous cell population that is resistant to enzalutamide, with its proliferation dependent on transcriptionally active AR-V7. The purpose of this study was to identify a LNCaP95 clone that would be useful for evaluating therapies for their effectiveness against enzalutamide-resistant prostate cancer cells. Seven clones from the LNCaP95 cell line were isolated and characterized using morphology, in vitro growth rate, and response to ralaniten (AR N-terminal domain inhibitor) and enzalutamide (antiandrogen). In vivo growth of the clones as subcutaneous xenografts was evaluated in castrated immunodeficient mice. All of the clones maintained the expression of full-length AR and AR-V7. Cell proliferation of the clones was insensitive to androgen and enzalutamide but importantly was inhibited by ralaniten, which is consistent with AR-Vs driving the proliferation of parental LNCaP95 cells. In castrated immunodeficient animals, the growth of subcutaneous xenografts of the D3 clone was the most reproducible compared to the parental cell line and other clones. These data support that the enzalutamide-resistant LNCaP95-D3 subline may be suitable as a xenograft tumor model for preclinical drug development with improved reproducibility.

Inhibition of scutellarin on differentiation of colonic cancer stem cells via hedgehog signaling pathway

The objective of this study was to investigate the inhibitory effect of scutellarin on the differentiation of colonic cancer stem cells in vitro and in vivo and to explore its underlying hedgehog signaling-based mechanism. The effect of scutellarin on the growth in vitro of HT-29 cells-derived cancer stem-like cells(HT-29 CSC) was observed with 3 D cell culture. The effect of scutellarin on the transformation of HT-29 CSC cells was assessed by soft agar colony formation assay. Fetal calf serum was used to induce differentiation of stem cells and observe the effect of scutellarin on HT-29 CSC cells differentiation in vitro. The effects of scutellarin on mRNA expressions of Lgr5, c-Myc, CK20 and Nanog in HT-29 CSC cells were determined by quantitative Real-time polymerase chain reaction(qRT-PCR). The effects of scutellarin on protein expressions of c-Myc, Gli1 and Lgr5 in HT-29 CSC cells were examined by Western blot. After subcutaneous implantation of HT-29 CSC cells in nude mice, the effect of scutellarin on the mouse body weight and the growth of HT-29 CSC-derived tumor were explored. qRT-PCR was used for evaluating the effect of scutellarin on mRNA levels of CD133, Lgr5, Gli1, Ptch1, c-Myc, Ki-67, CK20 and Nanog in tumor. Western blot and immunohistochemistry analysis were used to detect the effect of scutellarin on protein expressions of c-Myc, Gli1, Lgr5, CD133 and Ki-67 in tumor. The in vitro experiments showed that scutellarin inhibited the growth, transformation and differentiation of HT-29 CSC cells, significantly down-regulated the mRNA levels of Lgr5, c-Myc, CK20 and Nanog in HT-29 CSC cells as well as the protein expression levels of c-Myc, Gli1 and Lgr5 in HT-29 CSC cells. Additionally, animal experiments showed that scutellarin significantly inhibited the growth of subcutaneous xenografts in nude mice, and down-regulated the mRNA expressions of CD133, Lgr5, Gli1, Ptch1, c-Myc, Ki-67, CK20 and Nanog as well as the protein levels of c-Myc, Gli1, Lgr5, CD133 and Ki-67 of xenografts in nude mice. Taken together, scutellarin could inhibit the differentiation of colo-nic cancer stem cells in vitro and in vivo, potentially by down regulation of hedgehog signaling pathway activity.

Upregulated NTF4 in colorectal cancer promotes tumor development via regulating autophagy.

Autophagy plays a key role in colorectal cancer (CRC) development and reduces the sensitivity of CRC cells to treatment. The present study reported a novel tumor-suppressive role for autophagy, which was demonstrated to be regulated through the novel oncogene neurotrophin-4 (NTF4). NTF4 was significantly overexpressed in tumor tissue compared with non-tumor mucosa, and the upregulation of NTF4 in CRC was associated with poor overall survival and advanced TNM stage. The genetic knockdown of NTF4 using short hairpin RNA in CRC cells prevented epithelial-to-mesenchymal transition and activated autophagy; this was regulated through the interaction between autophagy-associated gene 5 (Atg5) and the mitogen-activated protein kinase pathway. In addition, the knockdown of NTF4 inhibited cell invasion, migration, proliferation and colony formation, and promoted cell cycle arrest. Treatment of the cells with the autophagy inhibitor chloroquine (CQ) rescued these functions and promoted cell invasion, migration, proliferation and colony formation. Finally, the knockdown of NTF4 inhibited the growth of subcutaneous xenografts in Balb/c-nu mice. In conclusion, these findings suggested that NTF4 may be a diagnostic marker associated with the overall survival and progression of patients with CRC. NTF4 was found to promote tumorigenesis and CRC development through autophagy regulation.

Interleukin 1β/1RA axis in colorectal cancer regulates tumor invasion, proliferation and apoptosis via autophagy.

Interleukin (IL)-1β is a member of the IL-1 family of proteins. IL-1 receptor antagonist (IL-1RA) is an agent that binds to the IL-1 receptor, preventing IL-1 from transmitting signals to cells. The present study aimed to identify the role of the IL-1β/1RA axis in epithelial-mesenchymal transition (EMT), cell invasion, migration, proliferation and clone formation in colorectal cancer (CRC) and to determine its underlying mechanisms of action. Significantly increased expression of both IL-1β and IL-1RA was identified in CRC patient data uploaded in The Cancer Genome Atlas database, and in tumor tissues when compared with matched control tissue. High expression of IL-1β was associated with an increased rate of overall survival and recurrence-free survival. Further research revealed that the IL-1β gene was co-expressed with the IL-1RA gene in tumors of CRC patients. It was additionally determined that recombinant human (rh)IL-1β suppressed autophagy as well as EMT in HCT-116 cells compared with control-treated cells, whereas rhIL-1RA exhibited the opposite effect. In addition, autophagy activator rapamycin (RAPA) rescued the inhibition of EMT in rhIL-1β-treated HCT-116 cells. Moreover, rhIL-1β inhibited cell invasion, migration, proliferation and colony-formation ability, when compared with a control treatment. Compared with a control treatment rhIL-1RA promoted cell invasion, migration, proliferation, but had no effect on clone formation ability. Furthermore, both rhIL-1RA and RAPA rescued inhibition of cell invasion, migration and clone formation ability in rhIL-1β-treated HCT-116 cells. RAPA, but not rhIL-1RA, rescue inhibited proliferation in rhIL-1β-treated HCT-116 cells compared with controls. In addition, it was confirmed that rhIL-1β inhibited the growth of subcutaneous xenografts in nude mice, when compared with control treatments. These results indicated that upregulation of the IL-1β/1RA axis in CRC regulated EMT, cell invasion and migration, proliferation and clone formation via autophagy.

New advances on the inhibition of Siwei Xiaoliuyin combined with Temozolomide in glioma based on the regulatory mechanism of miRNA21/221.

To provide evidence for the mechanism of Chinese medicine to treat glioma. We observe the effects of Si wei xiao xiu yin combined with chemotherapy on the growth of subcutaneous xenografts in nude mice and the expression of miRNA-21 and miRNA-221 in tumor tissues. The subcutaneous transplantation model of nude mice was established by subcutaneous inoculation of glioma U87 cell suspension. They were randomly divided into saline group, traditional Chinese medicine group, temozolomide group and traditional Chinese medicine combined with temozolomide group to observe the changes in body weight, and the tumor weight, length, short diameter, volume of mice. The relative expression levels of miRNA-21 and miRNA-221 in tumor tissues were detected by qRT-PCR, and the differences between groups were compared. After 28 days of gavage, the tumor growth of the other three groups was slower than that of saline group, and the difference was most significant in the combination group (P=0.008<0.05), besides, the relative expression of the three groups of miRNA-21 and miRNA-221 was significantly inhibited compared with saline group, and the difference was significant in the combination group (F=8.918, P=0.010<0.05). To some extent, Si wei xiao xiu yin combined with temozolomide can inhibit the growth of subcutaneous xenografts in glioma nude mice. The mechanism may be related to the inhibition of miRNA-21 and miRNA-221 expression.

Abstract C052: FGF trapping impairs multiple myeloma growth through c-Myc degradation-induced mitochondrial oxidative stress

Abstract Multiple myeloma (MM) remains incurable because of chemotherapeutic resistance. Fibroblast growth factors (FGF) play a pivotal role in MM by acting as an autocrine/paracrine mitogen on plasma cells, bone marrow-derived endothelial cells and fibroblasts. Also, a recurrent chromosomal translocation t(4;14) is associated with FGFR3 upregulation in MM patients. Thus, agents able to hamper FGF signaling may represent a novel approach for MM treatment. Recently we have identified the PTX3-derived small molecule NSC12 as the first orally active antitumor pan-FGF trap. Here, the role of FGF/FGFR system in MM cell survival and the therapeutic potential of NSC12 were investigated. In vitro effects of NSC12 were tested on four human MM cell lines harboring (KMS-11 and OPM-2 cells) or not (U-266 and RPMI8226 cells) the t(4:14) translocation and on patient-derived MM cells (N = 26). In vivo effects of FGF blockade were investigated using subcutaneous (KMS-11 and RPMI8226 cells) and systemic (MM-1S cells) xenografts grown in NOD/SCID mice. In addition, gene expression profiling (GEP) and gene set enrichment analyses (GSEA) were performed in NSC12-treated MM cells. NSC12 blocked the proliferation of all human MM cell lines tested, causing inhibition of FGFR signaling, downregulation of the anti-apoptotic protein mcl-1 and caspase 3 activation. Cytofluorimetric analysis revealed a significant increase of apoptotic cells as early as 6 hrs after NSC12 treatment, with 100% of cell death after 24 hrs even when MM cells were co-cultured with patient-derived bone marrow stromal cells. In vivo, NSC12 blocked FGFR activation and reduced the growth of subcutaneous xenografts and MM cell dissemination in the BM. GEP and GSEA analyses of NSC12-treated cells revealed the upregulation of oxidative stress-induced genes and the downmodulation of c-Myc targets. Accordingly, NSC12 caused the rapid proteasomal degradation of c-Myc paralleled by GSH depletion, mitochondrial ROS production and depolarization, DNA damage and finally apoptosis in KMS-11 cells but not in KMS-11 cells expressing the undegradable mutated (T58A) form of c-Myc. Interestingly, these findings were confirmed on Bortezomib-resistant MM cells as well as on bone marrow-derived primary MM cells from newly diagnosed and relapsed/refractory patients, including plasma cells bearing the t(4;14) translocation obtained from high-risk MM patients. Our data reveal that FGF blockade triggers MM mitochondrial oxidative stress, DNA damage and apoptotic cell death via the proteasomal degradation of the c-Myc oncoprotein. Altogether, our findings dissect for the first time the mechanism by which the FGF/FGFR system plays a non-redundant role in MM cell survival and disease progression, and indicate that FGF targeting may represent a therapeutic approach for MM patients with poor prognosis and advanced disease stage. Citation Format: Roberto Ronca, Gaia Cristina Ghedini, Federica Maccarinelli, Antonio Sacco, Silvia Laura Locatelli, Eleonora Foglio, Sara Taranto, Elisabetta Grillo, Sara Matarazzo, Riccardo Castelli, Vanessa Desantis, Nadia Cattane, Annamaria Cattaneo, Marco Mor, Carmelo Carlo-Stella, Aldo Maria Roccaro, Marco Presta, Arianna Giacomini. FGF trapping impairs multiple myeloma growth through c-Myc degradation-induced mitochondrial oxidative stress [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C052. doi:10.1158/1535-7163.TARG-19-C052

Upregulated NTF4 in Colorectal Cancer Promotes Tumor Development via Regulating Autophagy

Background: Previous studies of colorectal cancer (CRC) mainly confirmed that autophagy promotes the cancer development and reduces the sensitivity to treatment. But our study explained the new role of autophagy in tumor suppression based on a new oncogene: NTF4. Methods: NTF4 expression in CRC patients was confirmed and analyzed with clinical data. Then, NTF4-knockdown CRC cells was generated to research its role in vitro. Epithelial mesenchymal transition (EMT) and autophagy associated markers were detected by Western Blotting and RT-qPCR. Autophagy flow and Autophagosome were investigated by Immunofluorescence (IF) transmission electron microscopy (TEM) respectively. Cell proliferation was detected by CCK8 and clone formation assay, cell invasion and migration were detected by transwell and wound healing assays, cell cycle and apoptosis were detected by flow cytometry. Finally, effect of NTF in CRC was confirmed in vivo via mice model. Findings: Upregulation of NTF4 is associated with poor OS and advanced TNM stage. Knockdown of NTF4 in CRC cells repressed EMT, whereas activated autophagy. The NTF4-konckdown induced autophagy may come from interaction with Atg5 and regulation of MAPK pathway. In addition, Knockdown of NTF4 inhibited cell invasion, migration, proliferation, clone formation and promoted cell cycle arrest. After treated with autophagy inhibitor, inhibited cell invasion, migration, proliferation, clone formation and increased cell cycle arrest were all rescued. Finally, we confirmed knockdown of NTF4 inhibited growth of subcutaneous xenografts in nude mice. Interpretation: NTF4 is a meaningful diagnostic marker associated with OS and progression of CRC patients. NTF4 promotes tumorigenesis and development via autophagy. Funding Statement: This study was funded by Academic Leaders Training Program Supported by Pudong Health Bureau of Shanghai (Grant No. PWRd2016-05) and Natural Science Foundation of Shanghai (Grant No.16411972800). Declaration of Interests: The authors declare no conflict of interests. Ethics Approval Statement: All procedures involving human participants were performed in accordance with the Ethics Committee of Shanghai Pudong Hospital and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. All patients provided their written informed consent. The study protocol was approved by the Pudong Hospital Committee on human research. All detailed experimental procedures were approved by the Institutional Animal Care and Utilization Committee of Fudan University Pudong Animal Experimental Center.

LncRNA TPT1-AS1 promotes tumorigenesis and metastasis in epithelial ovarian cancer by inducing TPT1 expression.

Increasing numbers of studies have confirmed that long noncoding RNA (lncRNA) play a critical role in epithelial ovarian cancer (EOC) progression. However, the potential function of the lncRNA tumor protein translationally controlled 1 (TPT1) antisense RNA 1 (TPT1‐AS1) in EOC is unclear. In this study, we aimed to uncover the biological roles and regulatory mechanisms of TPT1‐AS1 in EOC progression and metastasis. First, TPT1‐AS1 expression was significantly higher in EOC metastatic tissue and cell lines than in their respective control counterparts. In addition, ectopic TPT1‐AS1 expression was strongly associated with unfavorable EOC clinicopathological features, including FIGO stage, tumor size and tumor differentiation. TPT1‐AS1 overexpression remarkably induced cell proliferation, migration and invasion, and significantly attenuated cell adhesion ability in vitro and facilitated nude mouse subcutaneous xenograft growth and intraperitoneal metastasis in vivo, while the downregulation of TPT1‐AS1 expression produced the opposite effect in vitro. Mechanistically, TPT1‐AS1 was proven to be primarily distributed in EOC cell nuclei and positively modulated TPT1 promoter activity and transcription. Moreover, the oncogenic effects of TPT1‐AS1 could be reversed by TPT1 depletion, and the PI3K/AKT signaling pathway downstream of TPT1 was also altered. These results suggested that TPT1‐AS1 induced EOC tumor growth and metastasis through TPT1 and downstream PI3K/AKT signaling and that TPT1‐AS1 may be a promising therapeutic target for EOC.