Potent Antimetastatic Effects Research Articles

Role of gonadotropin-releasing hormone 2 and its receptor in human reproductive cancers.

Gonadotropin-releasing hormone (GnRH1) and its receptor (GnRHR1) drive reproduction by regulating gonadotropins. Another form, GnRH2, and its receptor (GnRHR2), also exist in mammals. In humans, GnRH2 and GnRHR2 genes are present, but coding errors in the GnRHR2 gene are predicted to hinder full-length protein production. Nonetheless, mounting evidence supports the presence of a functional GnRHR2 in humans. GnRH2 and its receptor have been identified throughout the body, including peripheral reproductive tissues like the ovary, uterus, breast, and prostate. In addition, GnRH2 and its receptor have been detected in a wide number of reproductive cancer cells in humans. Notably, GnRH2 analogues have potent anti-proliferative, pro-apoptotic, and/or anti-metastatic effects on various reproductive cancers, including endometrial, breast, placental, ovarian, and prostate. Thus, GnRH2 is an emerging target to treat human reproductive cancers.

Discovery of unglycosylated indolocarbazoles as ROCK2 isoform-selective inhibitors for the treatment of breast cancer metastasis

Breast cancer metastasis is a major challenge in clinical therapy because of the absence of effective treatments. Rho-associated coiled-coil kinase (ROCK), which is essential for cell invasion and migration, has recently been suggested as a potential target for the treatment of cancer metastasis. Herein, we report the structure−activity relationships (SAR) of indolocarbazoles against ROCK2 and reveal the crucial role of the C-3 hydroxyl for ROCK2 inhibition. The most potent unglycosylated aglycone THK01 was demonstrated to bind to and stabilize ROCK2 with potent anti-metastatic effects in breast cancer in vitro and in vivo with no obvious toxicities. Further mechanistic studies revealed that the anti-metastatic effect of THK01 was closely related to the suppression of STAT3Y705 activation. Moreover, THK01 exhibited excellent selectivity over the isoform protein ROCK1 (>100-fold). Taken together, with low toxicity, the ROCK2 inhibitor THK01 potently inhibited breast cancer metastasis through the ROCK2-STAT3 signaling pathway, which offers a new opportunity for the treatment of metastatic breast cancer.

Nanocarrier of Pin1 inhibitor based on supercritical fluid technology inhibits cancer metastasis by blocking multiple signaling pathways.

Cancer metastasis is the primary cause of all cancer-related deaths due to the lack of effective targeted drugs that simultaneously block multiple signaling pathways that drive the dissemination and growth of cancer cells. The unique proline isomerase Pin1 activates numerous cancer pathways, but its role in cancer metastasis and the inhibitory efficacy of Pin1 inhibitors on cancer metastasis are unknown. Moreover, the applicability of Pin1 inhibitor-all-trans retinoic acid (ATRA) is limited due to its several drawbacks. Herein, uniform ATRA-loaded polylactic acid-polyethylene glycol block copolymer nanoparticles (ATRA-NPs) with high encapsulation efficiency, good cellular uptake, excellent controlled release performance and pharmacokinetics are developed using supercritical carbon dioxide processing combined with an optimized design. ATRA-NPs exhibited excellent biosafety and significant inhibition on the growth and metastasis of hepatocellular carcinoma. Pin1 played a key role in cancer metastasis and was the main target of ATRA-NPs. ATRA-NPs exerted their potent anti-metastatic effect by inhibiting Pin1 and then simultaneously blocking multiple signaling pathways and cancer epithelial-mesenchymal progression. Since ATRA-NPs could effectively couple the inhibition of cancer cell dissemination with cancer growth, it provided a novel therapeutic strategy for efficiently inhibiting cancer metastasis.

Heterodimeric IL-15 (hetIL-15) reduces circulating tumor cells and metastasis formation improving chemotherapy and surgery in 4T1 mouse model of TNBC.

Immunotherapy has emerged as a viable approach in cancer therapy, with cytokines being of great interest. Interleukin IL-15 (IL-15), a cytokine that supports cytotoxic immune cells, has been successfully tested as an anti-cancer and anti-metastatic agent, but combinations with conventional chemotherapy and surgery protocols have not been extensively studied. We have produced heterodimeric IL-15 (hetIL-15), which has shown anti-tumor efficacy in several murine cancer models and is being evaluated in clinical trials for metastatic cancers. In this study, we examined the therapeutic effects of hetIL-15 in combination with chemotherapy and surgery in the 4T1 mouse model of metastatic triple negative breast cancer (TNBC). hetIL-15 monotherapy exhibited potent anti-metastatic effects by diminishing the number of circulating tumor cells (CTCs) and by controlling tumor cells colonization of the lungs. hetIL-15 treatment in combination with doxorubicin resulted in enhanced anti-metastatic activity and extended animal survival. Systemic immune phenotype analysis showed that the chemoimmunotherapeutic regimen shifted the tumor-induced imbalance of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in favor of cytotoxic effector cells, by simultaneously decreasing PMN-MDSCs and increasing the frequency and activation of effector (CD8+T and NK) cells. Tumor resection supported by neoadjuvant and adjuvant administration of hetIL-15, either alone or in combination with doxorubicin, resulted in the cure of approximately half of the treated animals and the development of anti-4T1 tumor immunity. Our findings demonstrate a significant anti-metastatic potential of hetIL-15 in combination with chemotherapy and surgery and suggest exploring the use of this regimen for the treatment of TNBC.

Discovery of 1-Benzhydryl-Piperazine-Based HDAC Inhibitors with Anti-Breast Cancer Activity: Synthesis, Molecular Modeling, In Vitro and In Vivo Biological Evaluation.

Isoform-selective histone deacetylase (HDAC) inhibition is promoted as a rational strategy to develop safer anti-cancer drugs compared to non-selective HDAC inhibitors. Despite this presumed benefit, considerably more non-selective HDAC inhibitors have undergone clinical trials. In this report, we detail the design and discovery of potent HDAC inhibitors, with 1-benzhydryl piperazine as a surface recognition group, that differ in hydrocarbon linker. In vitro HDAC screening identified two selective HDAC6 inhibitors with nanomolar IC50 values, as well as two non-selective nanomolar HDAC inhibitors. Structure-based molecular modeling was employed to study the influence of linker chemistry of synthesized inhibitors on HDAC6 potency. The breast cancer cell lines (MDA-MB-231 and MCF-7) were used to evaluate compound-mediated in vitro anti-cancer, anti-migratory, and anti-invasive activities. Experiments on the zebrafish MDA-MB-231 xenograft model revealed that a novel non-selective HDAC inhibitor with a seven-carbon-atom linker exhibits potent anti-tumor, anti-metastatic, and anti-angiogenic effects when tested at low micromolar concentrations.

108 (PB098) - MYC inhibition by Omomyc unveils a prognostic gene signature in Melanoma

Background: Melanoma is one of the most frequent causes of cancer death and despite significant treatment improvements, non-responder patients and those who develop resistance constitute a significant proportion of patients still in dire need of alternative and improved therapeutic options. Omomyc is a MYC inhibitor designed by Dr. Soucek that is extensively preclinically validated and currently being tested in a Phase I/IIa Clinical Trial. We have seen potent antitumour and antimetastatic effects when Omomyc is transgenically expressed in melanoma cells. Material and methods: We performed microarray analysis on BRAF-mutated A375 and BRAF wild-type SkMel147 human melanoma cells in vivo after one week of Omomyc expression and analysed them in R with Bioconductor. We assessed the overlap on transcriptional reprogramming exerted by Omomyc and investigated how these genes are associated with patient prognosis, by retrieving clinical and transcriptional data from the Skin Cutaneous Melanoma TCGA, PanCancer Atlas study in cBioPortal. Results: We found 620 differentially expressed genes in common, 436 downregulated by Omomyc (DN) and 184 upregulated (UP). DN genes cluster into Cell Cycle, RNA processing, Metabolism and Chromatin modification, organisation, and remodelling, while UP genes are related to Immune Response and several subcellular processes. Expression lower than the median of 45% of the DN genes or higher than the median of 54% of the UP genes are associated with significantly increased survival. We then generated a 6-gene signature combining DN and UP genes, that clearly separates patients in good and bad prognosis cohorts. Those with simultaneous low expression of these DN genes and high expression of these UP genes have significantly increased OS (205 (162-NA) vs. 42 (28– 63) months, p = 7 × 10−7) Additionally, 63% of UP genes are enriched in patients with Good Prognosis whereas 56% of DN genes are enriched in Poor Prognosis patients. Thus, Omomyc in vivo-regulated genes overlap considerably with those genes enriched in patients pointing to a reasonable similarity among in vivo results with Omomyc and clinical and transcriptional data from patients. Finally, pre-ranked GSEA in these patients showed that those with Good Prognosis have lower expression of MYC target genes and genes involved in melanoma relapse and metastases, and increased expression of Adaptive Immune Response. Conclusion: We show that specific gene sets reflecting decreased MYC transcriptional activity, restricted metastatic capacity and increased immune response are identified in Omomyc-expressing melanoma xenografts and indicate patients with Good Prognosis. We can therefore define an Omomyc-derived gene signature that clearly discriminates patients with different prognoses, whose transcriptional signatures resemble those of vehicle or Omomyc-expressing tumours in vivo. Conflict of interest: Ownership: L. Soucek and M. Beaulieu are cofounders and shareholders of Peptomyc, a company focused on developing Myc inhibitors for cancer treatment. M.F. Zacarias Fluck, S. Casacuberta-Serra, and J.R. Whitfield are shareholders of Peptomyc S.L.

Regulation of Cell-Signaling Pathways by Berbamine in Different Cancers.

Natural product research is a cornerstone of the architectural framework of clinical medicine. Berbamine is a natural, potent, pharmacologically active biomolecule isolated from Berberis amurensis. Berbamine has been shown to modulate different oncogenic cell-signaling pathways in different cancers. In this review, we comprehensively analyze how berbamine modulates deregulated pathways (JAK/STAT, CAMKII/c-Myc) in various cancers. We systematically analyze how berbamine induces activation of the TGF/SMAD pathway for the effective inhibition of cancer progression. We also summarize different nanotechnological strategies currently being used for proficient delivery of berbamine to the target sites. Berbamine has also been reported to demonstrate potent anti-cancer and anti-metastatic effects in tumor-bearing mice. The regulation of non-coding RNAs by berbamine is insufficiently studied, and future studies must converge on the identification of target non-coding RNAs. A better understanding of the regulatory role of berbamine in the modulation of non-coding RNAs and cell-signaling pathways will be advantageous in the effective translation of laboratory findings to clinically effective therapeutics.

IL-1β in breast cancer bone metastasis.

Bone is the most common site for advanced breast cancer to metastasise. The proinflammatory cytokine, interleukin-1β (IL-1β) plays a complex and contradictory role in this process. Recent studies have demonstrated that breast cancer patients whose primary tumours express IL-1β are more likely to experience relapse in bone or other organs. Importantly, IL-1β affects different stages of the metastatic process including growth of the primary tumour, epithelial to mesenchymal transition (EMT), dissemination of tumour cells into the blood stream, tumour cell homing to the bone microenvironment and, once in bone, this cytokine participates in the interaction between cancer cells and bone cells, promoting metastatic outgrowth at this site. Interestingly, although inhibition of IL-1β signalling has been shown to have potent anti-metastatic effects, inhibition of the activity of this cytokine has contradictory effects on primary tumours, sometimes reducing but often promoting their growth. In this review, we focus on the complex roles of IL-1β on breast cancer bone metastasis: specifically, we discuss the distinct effects of IL-1β derived from tumour cells and/or microenvironment on inhibition/induction of primary breast tumour growth, induction of the metastatic process through the EMT, promotion of tumour cell dissemination into the bone metastatic niche and formation of overt metastases.

Self-healing microcapsules synergetically modulate immunization microenvironments for potent cancer vaccination.

Therapeutic cancer vaccines that harness the immune system to reject cancer cells have shown great promise for cancer treatment. Although a wave of efforts have spurred to improve the therapeutic effect, unfavorable immunization microenvironment along with a complicated preparation process and frequent vaccinations substantially compromise the performance. Here, we report a novel microcapsule-based formulation for high-performance cancer vaccinations. The special self-healing feature provides a mild and efficient paradigm for antigen microencapsulation. After vaccination, these microcapsules create a favorable immunization microenvironment in situ, wherein antigen release kinetics, recruited cell behavior, and acid surrounding work in a synergetic manner. In this case, we can effectively increase the antigen utilization, improve the antigen presentation, and activate antigen presenting cells. As a result, effective T cell response, potent tumor inhibition, antimetastatic effects, and prevention of postsurgical recurrence are achieved with various types of antigens, while neoantigen was encapsuled and evaluated in different tumor models.

Integrin and Heparan Sulfate Dual-Targeting Peptide Assembly Suppresses Cancer Metastasis.

Metastasis is one of the ongoing challenges in cancer therapy which most treatments failed to address. Inspired by the upregulated expression of both integrin β1 and heparan sulfate in metastatic tumors, we developed an integrin/HS dual-targeting peptide assembly that selectively inhibits cancer cell migration and invasion. Particularly, the dual-targeting peptide self-assembles into nanofibrous microdomains specifically on the cancer cell membrane, triggering spatial organization of integrins, which form clusters on the apical membrane. Via the actin cytoskeleton that physically connects to integrin clusters, the oncogene yes-associated protein, which regulates cancer metastasis, is deactivated. We showed that in multiple cancer cell lines, including the highly metastatic pancreatic cancer cells, the dual-targeting peptide exerts potent and dose-dependent antimetastatic effects. Our work illustrates how basic biochemical insights can be exploited as the basis for nano-biointerface fabrication, which is potentially a general design strategy for nanomedicine development.

Expression of Concern: Synthesized multiple antigenic polypeptide vaccine based on B-cell epitopes of human heparanase could elicit a potent antimetastatic effect on human hepatocellular carcinoma in vivo.

Expression of Concern: Synthesized multiple antigenic polypeptide vaccine based on B-cell epitopes of human heparanase could elicit a potent antimetastatic effect on human hepatocellular carcinoma in vivo.

Specific Inhibition of Heparanase by a Glycopolymer with Well-Defined Sulfation Pattern Prevents Breast Cancer Metastasis in Mice.

Heparanase, the heparan sulfate polysaccharide degrading endoglycosidase enzyme, has been correlated with tumor angiogenesis and metastasis and therefore has become a potential target for anticancer drug development. In this systematic study, the sulfation pattern of the pendant disaccharide moiety on synthetic glycopolymers was synthetically manipulated to achieve optimal heparanase inhibition. Upon evaluation, a glycopolymer with 12 repeating units was determined to be the most potent inhibitor of heparanase (IC50 = 0.10 ± 0.36 nM). This glycopolymer was further examined for cross-bioactivity using a solution-based competitive biolayer interferometry assay with other HS-binding proteins (growth factors, P-selectin, and platelet factor 4), which are responsible for mediating angiogenic activity, cell metastasis, and antibody-induced thrombocytopenia. The synthetic glycopolymer has low affinity for these HS-binding proteins in comparison to natural heparin. In addition, the glycopolymer possessed no proliferative properties toward human umbilical endothelial cells (HUVECs) and a potent antimetastatic effect against 4T1 mammary carcinoma cells. Thus, our study not only establishes a specific inhibitor of heparanase with high affinity but also illustrates the high effectiveness of this multivalent heparanase inhibitor in inhibiting experimental metastasis in vivo.

A molecular basis for the synergy between 17‑allylamino‑17‑demethoxy geldanamycin with Capecitabine and Irinotecan in human colorectal cancer cells through VEFG and MMP-9 gene expression

Anti-proliferative, anti-metastatic and anti-angiogenic effects of 17‑allylamino‑17‑demethoxy geldanamycin (17-AAG) were studied alone and in combination with Capecitabine (Cap) and/or Irinotecan (IR) on HT-29 human colorectal carcinoma cells. Expression of MMP-9 (matrix metalloproteinase‑9) and VEGF (vascular endothelial growth factor) mRNA was analyzed by real-time PCR method. The study was further followed by wound scratch assay for migration assessment. Nitric oxide content, Malondialdehyde generation and total anti-oxidant capacity were also assessed. Results showed significant differences between mono- and double therapy (p < 0.05). Combination of 17-AAG with IR or Cap resulted in synergistic effect (Combination Index < 1). Among double combination groups only Cap/17-AAG showed significant differences in MMP-9 and VEGF genes expression and wound healing assay. Moreover, a significant decrease of wound area in our triple combination group was obtained, indicating the antagonistic effect. IR/17-AAG and IR/Cap double combination groups resulted in down-regulation of MMP-9 and VEGF mRNA expression, respectively. Significant generation of MDA and decrease in TAC values have been observed in all our tested groups, however, the IR/17-AAG combination was the only group that could elevate NO concentration, significantly. Our findings demonstrated potent anti-angiogenesis and anti-metastatic effects for 17-AAG when it is provided in double combination especially with Cap, suggesting a new protocol in colorectal cancer combination therapy. These findings may indicate that down-regulation of VEGF and MMP-9 genes is directly related to angiogenesis and metastasis.

Discovery of novel cathepsin inhibitors with potent anti-metastatic effects in breast cancer cells

It is still challenging to determine the potential targets of natural products, which is essential for further drug research and development. Due to its novel mechanism of action of inducing autophagy effects in breast cancer cells, asperphenamate has received our considerable attention. However, its unknown target inevitably impedes further study. In our previous work, the target enzyme of asperphenamate was predicted as cathepsin by the natural product consensus pharmacophore strategy. Then, asperphenamate and its three derivatives were chosen to study in detail by molecular docking calculations with AutoDock 4 suite. The docking results showed the three derivatives interacted more tightly with either cathepsin L or cathepsin S than with asperphenamate. The ortho-benzyloxyl phenylacetyl derivative 1 andp-toluenesulfonyl derivative 3 showed similar interactions with cathepsin L and adopted a better geometric shape within the binding pocket than did the N-CBZ-piperidyl analog 2. On the other hand, compound 2 formed more hydrogen bonds than 1 and 3 to make it tightly bind within cathepsin S. The cathepsin inhibitory activity assay verified the molecular simulation results. Compound 2 was remarkably less active than 1 and 3 against cathepsin L. However, compound 2 showed the strongest potency against cathepsin S with IC50 of 13.12 ± 0.29 μM. Considering that cathepsin S plays a vital role in the process of metastasis in breast cancer cells, the inhibitory effect of 2 on migration and invasion was further studied in human breast cancer MDA-MB-231 cells by wound healing and transwell chamber assays. The results illustrated that 2 exhibited an apparent inhibitory ability to the metastasis of MDA-MB-231 cells. Next, 2 will be chosen as a lead compound to develop novel double functional chemotherapeutic agents with both novel mechanisms of action against apoptosis-resistant cancer cells, such as inducing autophagy and inhibiting cancer metastasis.

Selective depletion of metastatic stem cells as therapy for human colorectal cancer.

Selective elimination of metastatic stem cells (MetSCs) promises to block metastatic dissemination. Colorectal cancer (CRC) cells overexpressing CXCR4 display trafficking functions and metastasis‐initiating capacity. We assessed the antimetastatic activity of a nanoconjugate (T22‐GFP‐H6‐FdU) that selectively delivers Floxuridine to CXCR4+ cells. In contrast to free oligo‐FdU, intravenous T22‐GFP‐H6‐FdU selectively accumulates and internalizes in CXCR4+ cancer cells, triggering DNA damage and apoptosis, which leads to their selective elimination and to reduced tumor re‐initiation capacity. Repeated T22‐GFP‐H6‐FdU administration in cell line and patient‐derived CRC models blocks intravasation and completely prevents metastases development in 38–83% of mice, while showing CXCR4 expression‐dependent and site‐dependent reduction in foci number and size in liver, peritoneal, or lung metastases in the rest of mice, compared to free oligo‐FdU. T22‐GFP‐H6‐FdU induces also higher regression of established metastases than free oligo‐FdU, with negligible distribution or toxicity in normal tissues. This targeted drug delivery approach yields potent antimetastatic effect, through selective depletion of metastatic CXCR4+ cancer cells, and validates metastatic stem cells (MetSCs) as targets for clinical therapy.

Turmeric extract, with absorbable curcumin, has potent anti-metastatic effect in vitro and in vivo

BackgroundCurcumin, a well-studied component in turmeric, exhibits potent antitumor effects in colorectal cancer. Previous studies showed that turmerones raised the accumulation of curcumin inside colonic cells, and curcumin present in turmeric ethanolic extract had enhanced anti-tumor activities in mice. Metastasis accounts for more than 90% colorectal cancer deaths. However, the anti-metastatic effect of turmeric extract on colorectal cancer is still unknown. MethodsIn the present study, colony formation, scratch, transwell and Western blot were used to assess colony formation, motility, migration and underlying mechanisms in vitro, respectively. Anti-tumor and anti-metastatic effects in vivo were investigated using an orthotopic xenograft model. ResultsTurmeric extract exhibited cytotoxic effect, inhibited colony formation, decreased cell motility, migration and epithelial–mesenchymal transitions through regulating multiple pathways including cofilin, FAK/p-Src, AKT, Erk and STAT3 signaling pathways in murine colorectal cancer cells. Furthermore, turmeric extract at 200 mg/kg could decrease colon tumor burden and inhibit liver and lung metastasis in vivo. Treatment of turmeric extract enhanced immunity through T cell stimulation, changed tumor microenvironment, exerted anti-metastatic effects which were shown for the first time in pre-clinical colorectal cancer models. The decrease of immunity after FOLFOX treatment was also firstly demonstrated in mouse model. ConclusionsTurmeric extract was demonstrated for the first time for its anti-tumor and anti-metastatic effects in both colorectal cancer cells and orthotopic mouse model through regulation of multiple targets. These findings strongly suggested the promising use of turmeric extract as chemopreventive or chemotherapeutic agent for colorectal cancer patients with metastasis.

ANTI‐NEOPLASTIC EFFECTS OF METFORMIN AGAINST RENAL CLEAR CELL CARCINOMA

Background and ObjectivesRenal clear cell carcinoma (RCCC) is the most common and lethal form of urological cancer diagnosed globally. Mutation of Von‐Hippel Lindau (VHL) tumor suppresser gene and the resultant overexpression of hypoxia‐inducible factor‐1 alpha (HIF‐1α) protein promotes cell proliferation, angiogenesis and epithelial‐mesenchymal transition (EMT) and leads to RCCC progression and metastasis. Moreover, cancer drug resistance continues to be a major impediment to the treatment of RCCC. Metformin, an activator of AMP‐activated kinase (AMPK), is primarily prescribed for the management of type‐2 diabetes; nonetheless, emerging studies suggest that metformin exerts anti‐neoplastic effects in various types of cancers. Thus, the objectives of this study were to investigate metformin's antineoplastic effects and the signaling pathways modulated by metformin in RCCC using a human renal carcinoma cell line ‐ Caki‐2.MethodsCaki‐2 cells, which lack functional VHL, were treated with metformin (ranging from 1 to 50 mM) for 48 h and assessed for cell viability (using Alamar blue assay), cell cycle progression (using Tali cell cycle kit), cell migration (using in vitro scratch migration technique at 6 and 24 h). Western blotting analysis was performed to assess the effects of metformin on various signaling pathways such as AMPK, mammalian target of rapamycin (mTOR), AKT, HIF‐1α, autophagy (LC3II) pathway and epithelial‐mesenchymal transition or EMT marker (α‐SMA).ResultsFollowing 48 h metformin treatment, a significant and dose‐dependent decrease in cell viability were observed, i.e., 81% in 2 mM, 69% in 5 mM, 60% in 10 mM, 47% in 20 mM and 34% in 50 mM in metformin treated cells compared to control (set as 100%). Similarly, cell cycle analysis revealed a significant and dose‐dependent cell cycle arrest at G0/G1 phase in metformin treated cells compared to control. A prominent decrease in the migration of Caki‐2 cells was noted after 6 and 24 h of metformin treatment (5 mM and 10 mM) compared to control. Western blot analysis revealed a dose‐dependent increase in the levels of phosphorylated (active) AMPK i.e., 115% in 2 mM, 148% in 5 mM and 172% in 10 mM in metformin treated cells in comparison to control (set at 100%). In contrast, a significant decrease in the levels of phosphorylated (active) forms of mTOR (49% in 2 mM, 34% in 5 mM and 20% in 10 mM) and AKT (84% in 5 mM and 66 % in 10 mM) were noted in metformin treated Caki‐2 cells. Moreover, marked downregulation of HIF‐1α expression (62%) and reductions in the levels of EMT marker ‐ α‐SMA (30%) and autophagy marker ‐ LC3II protein (67%) were observed in Caki‐2 cells treated with metformin at 10 mM.ConclusionOur findings indicate that metformin exerts potent anti‐neoplastic and anti‐metastatic effects in RCCC through concerted activation of AMPK pathway and repression of AKT, mTOR, HIF‐1α, LC3II protein and EMT signaling pathways. The modulation of the aforementioned signaling pathways might contribute to the cell cycle arrest at the G0/G1 phase and reductions in cell migration. Together, our study unveils the therapeutic potential of metformin to inhibit the progression of RCCC in vitro.Support or Funding InformationThis study was supported by the intramural research grants from Drake University and Qatar University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 124: Imipridone ONC201 promotes intra-tumoral accumulation of CD3+/NK+ cells that contribute to its anti-tumor efficacy

Abstract ONC201, a first-in-class oral anti-tumor agent, upregulates the pro-apoptotic immune cytokine TRAIL and activates the integrated stress response leading to upregulation of death receptor 5 in bulk tumor and cancer stem cells. We previously demonstrated that ONC201 exerts a dose- and schedule-dependent effect on tumor progression in vivo while suppressing Akt/ERK signaling in tumors in a dose/frequency-dependent manner (Wagner et al., AACR, 2016). We also provided evidence that ONC201 exhibits a potent anti-metastatic effect (Wagner et al., AACR, 2016). We observe accumulation and activation of TRAIL-secreting NK+ cells within ONC201-treated tumors in C57/BL6, Balb/c, and athymic nude tumor-bearing mice. Importantly, ONC201 exerts in vivo anti-tumor efficacy on tumor cell lines that are ONC201-resistant in vitro, including acquired stable resistance. Using the NK-depleting antibody GM1, we demonstrate that the activation and TRAIL secretion of NK cells by ONC201 significantly contributes to in vivo anti-tumor efficacy, including TRAIL/ONC201-resistant tumors. We are currently investigating how ONC201 recruits NK cells to the tumor by examining NK-recruiting chemokine factors within the tumor site. We have also demonstrated upregulation of CD3+ T cells by ONC201 in syngeneic mice. Finally, we observed an increase in activated TRAIL-secreting NK cells in the peripheral blood of patients upon ONC201 administration in the clinic. Our results demonstrate novel and potentially significant increases in cytotoxic NK cell recruitment to tumors. The results offer a unique pathway of immune stimulation for cancer therapy that may be combined with immune checkpoint or targeted cancer therapy strategies. We are currently investigating the role of NK cells and CD3+ cells in ONC201’s ability to inhibit metastasis by using a metastatic model that involves surgically removing the primary tumor and allowing metastases to grow in vivo before treatment. These findings indicate that ONC201 possess immunomodulatory activity and provide a rationale for combining ONC201 with PD-1/PDL-1 inhibitors, a combination we are currently testing in syngeneic immunocompetent mouse models. Citation Format: Jessica Wagner, C. Leah Kline, Lanlan Zhou, Andrew Zloza, Charles Chesson, Jenna Newman, Howard Kaufman, Joseph Bertino, Mark Stein, Wafik El-Deiry. Imipridone ONC201 promotes intra-tumoral accumulation of CD3+/NK+ cells that contribute to its anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 124. doi:10.1158/1538-7445.AM2017-124

Cucurbitacin B inhibits cell proliferation and induces apoptosis in human osteosarcoma cells via modulation of the JAK2/STAT3 and MAPK pathways.

Osteosarcoma (OS) is the most commonly diagnosed tumor of the bones in children and young adults. Even with conventional therapies the 5-year survival rate is ~65% in patients with OS. Considering the side effects and aggressiveness of malignant bone tumors, research is focussing on multi-targeted strategies in treatment. Cucurbitacin B, a triterpenoid compound has been demonstrated to induce apoptosis in various cancer cell types. The Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signalling cascades and mitogen activated protein kinases (MAPK) signalling cascades are critical regulators of tumorigenesis. The present study assessed the influence of cucurbitacin B on the viability and expression of MAPKs and proteins of the JAK2/STAT3 cascades in human OS cells (U-2 OS). Cucurbitacin B (20-100 µM) significantly reduced cell viability (P<0.05) and induced apoptosis, as assessed by MTT and Annexin V/propidium iodide staining, along with inhibiting cell migration. Gelatin zymography revealed supressed activities of matrix metalloproteinase (MMP-)2 and 9. Furthermore, cucurbitacin B effectively upregulated the apoptotic pathway and caused the effective inhibition of MAPK signalling and JAK2/STAT3 cascades. Multifold suppression of vascular endothelial growth factor by cucurbitacin B was also observed, indicating inhibition of angiogenesis. Thus, by downregulating major pathways-MAPK and JAK2/STAT3 and MMPs, cucurbitacin B has potent anti-proliferative and anti-metastatic effects that require further investigation with regards to cancer treatment.

Effect of an anti-MARCKS peptide (BIO-11006) on metastasis of lung cancer in vivo.

e23017 Background: We have previously reported that administration of peptides that inhibit the function of MARCKS (myristoylated alanine-rich C Kinase Substrate) inhibit metastasis of human lung cancer cells orthotopically-injected into the left lung of SCID mice (Chen et al: Oncogene 33:3696–3706, 2014; Chen et al: Am J Resp Crit Care Med 190:1127-1138, 2015). Methods: Here, we administered an anti-MARCKS peptide, BIO-11006, as an inhaled aerosol in 2 models of lung cancer in SCID mice: PC-9 cells orthotopically injected into the left lung, and A549 cells injected into the tail vein. In the orthotopic model, initiation of treatment with BIO-11006 at day 4 post injection of cells and treatment every day thereafter for 4 weeks resulted in dramatic attenuation of tumor metastasis into the lung, heart and diaphragm, with similar attenuation whether the peptides were administered IP or via inhaled aerosol. In additional studies with this model, we waited until day 26 post orthotopic cell injection, a time when there was already extensive metastasis to the lung and distal organs, to begin daily treatment with BIO-11006 via inhalation. Results: There was complete inhibition of further metastasis and primary tumor growth when the animals were examined at day 38 post orthotopic injection of cells. Similar results with the tail vein model were observed: when the peptide was administered by aerosol starting at day 3 post tail vein inoculation, metastasis observed 8 weeks later in distal organs was inhibited by ~ 95% by treatment with inhaled BIO-11006. When PC-9 cells were examined via confocal microscopy, BIO-11006 was seen to cause a cytoskeletal rearrangement so that the cells took on a morphology favoring adherence to the substrate rather than movement. Conclusions: The results suggest that inhaled BIO-11006, already completed in phase 2 clinical trials in patients with COPD, appears to have potent antimetastatic effects in two different mouse models of lung cancer. Clinical trials of inhaled BIO-11006 in patients with advanced lung cancer were initiated in January of 2017.