Cell Metastatic Behavior Research Articles

Cationic Modulation of Voltage-Gated Sodium Channel (Nav1.5): Neonatal Versus Adult Splice Variants-2. Divalent (Cd2+) and Trivalent (Gd3+) Ions.

Background: A "neonatal" splice-form of the voltage-gated sodium channel Nav1.5 is functionally expressed in human cancers and potentiates metastatic cell behaviors. Splicing causes the replacement of 7 amino acids, including a negatively charged aspartate211 in the "adult" Nav1.5 (aNav1.5) to a positively charged lysine in the "neonatal" (nNav1.5). These changes occur in the region surrounding the DI:S3-S4 extracellular linker. The splice variants respond differently to changes in extracellular H+ and this could be of pathophysiological significance. However, how the two differentially charged splice variants would react to cations of higher valency is not known. Materials and Methods: We used patch-clamp recording to compare the electrophysiological effects of Cd2+ and Gd3+ on "adult" and "neonatal" Nav1.5 expressed stably in EBNA-293 cells. Several parameters were determined for the two channels and statistically compared. Results: Both cations inhibited peak I Na through reducing G max and induced a positive shift in the voltage range of activation. However, unlike Gd3+, Cd2+ had only a weak effect on voltage dependence of activation, and no effect on voltage dependence of inactivation, recovery from inactivation, or the kinetics of activation/inactivation. Conclusions: The electrophysiological effects of Cd2+ and Gd3+ studied were essentially the same for "neonatal" and "adult" Nav1.5, although these splice variants possess differences in their external charges. In contrast, the effects of H+ were shown earlier to be significantly differential. Taken together, these results suggest that limited adjustment of the charged structure of pharmacological agents could enable selective targeting of neonatal Nav1.5 associated with several cancers.

Abstract 1781: Transcriptional activation of CBFβ by CDK11p110 is necessary to promote osteosarcoma cell proliferation

Abstract Aberrant expression and activation of cyclin-dependent protein kinase (CDK) is a hallmark of cancer. CDK11 is a protein kinase in the CDK family and plays a crucial role in cancer cell growth and proliferation. However, the molecular mechanisms of CDK11 in osteosarcoma and CDK11 transcriptional regulated genes are largely unknown. In this study, we performed global transcriptional analysis using gene array technology to investigate the transcriptional role of CDK11 in osteosarcoma. The promoter luciferase assay, chromatin immunoprecipitation assay, and Gel Shift assay were used to identify direct transcriptional target of CDK11. Clinical significance relevance and function of CBFβ were further accessed in osteosarcoma tissue microarray and in osteosarcoma cell lines. We identified a transcriptional role of protein-DNA interaction for CDK11p110, but not CDK11p58, in the regulation of core-binding factor subunit beta (CBFβ) expression in osteosarcoma cells. The CBFβ promoter luciferase assay, chromatin immunoprecipitation assay, and Gel Shift assay confirmed that CBFβ is a direct transcriptional target of CDK11. High expression of CBFβ is associated with poor outcome in osteosarcoma patients. Expression of CBFβ contributes to the proliferation and metastatic behavior of osteosarcoma cells. These data establish CBFβas a mediator of CDK11p110dependent oncogenesis and suggest that targeting the CDK11-CBFβ pathway may be a promising therapeutic strategy for osteosarcoma treatment. Citation Format: Yong Feng, Francis Hornicek, Zhenfeng Duan. Transcriptional activation of CBFβ by CDK11p110 is necessary to promote osteosarcoma cell proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1781.

The protein secretion modulator TMED9 drives CNIH4/TGF\u03b1/GLI signaling opposing TMED3-WNT-TCF to promote colon cancer metastases

How cells in primary tumors initially become pro-metastatic is not understood. A previous genome-wide RNAi screen uncovered colon cancer metastatic suppressor and WNT promoting functions of TMED3, a member of the p24 ER-to-Golgi protein secretion family. Repression of canonical WNT signaling upon knockdown (kd) of TMED3 might thus be sufficient to drive metastases. However, searching for transcriptional influences on other family members here we find that TMED3 kd leads to enhanced TMED9, that TMED9 acts downstream of TMED3 and that TMED9 kd compromises metastasis. Importantly, TMED9 pro-metastatic function is linked to but distinct from the repression of TMED3-WNT-TCF signaling. Functional rescue of the migratory deficiency of TMED9 kd cells identifies TGFα as a mediator of TMED9 pro-metastatic activity. Moreover, TMED9 kd compromises the biogenesis, and thus function, of TGFα. Analyses in three colon cancer cell types highlight a TMED9-dependent gene set that includes CNIH4, a member of the CORNICHON family of TGFα exporters. Our data indicate that TGFA and CNIH4, which display predictive value for disease-free survival, promote colon cancer cell metastatic behavior, and suggest that TMED9 pro-metastatic function involves the modulation of the secretion of TGFα ligand. Finally, TMED9/TMED3 antagonism impacts WNT-TCF and GLI signaling, where TMED9 primacy over TMED3 leads to the establishment of a positive feedback loop together with CNIH4, TGFα, and GLI1 that enhances metastases. We propose that primary colon cancer cells can transition between two states characterized by secretion-transcription regulatory loops gated by TMED3 and TMED9 that modulate their metastatic proclivities.

Neuroblastoma Characteristics and Embryonic Origin of The Primary Lesion Site: A SEER Study

Background: Neuroblastoma, one of the most prevalent infant malignancies, is heterogeneous and easily spreads into other organs causing life-threatening consequences. Different characters of organs (such as the germ layers where the organs derived from) exert different growth microenvironments and potentially influence the behavior of metastatic neuroblastoma cells and the prognosis of patients. However, limited information is been known about this in neuroblastoma. Objectives: To compare characteristics of neuroblastoma, primarily originating from the same germ layer (the seeds), in different tissues (microenvironment) derived from different germ layers. Methods: We performed retrospective analysis of SEER (Surveillance, Epidemiology, and End Results) data (1973 - 2014), patients with malignant neuroblastoma were grouped based on the primary lesion site (mesoderm-, ectoderm- or endoderm-derived tissue). Baseline demographic and clinical characteristics were compared between groups. Due to difficulties of processing incomplete SEER data, therapeutic method and survival rates were analyzed using cases from another SEER database (2000 - 2014). Results: The analysis included 3701 patients: 1970 (53.2%) in the mesoderm group, 1017 (27.5%) in the ectoderm group and 714 (19.3%) in the endoderm group. Tumor histology differed between groups (P < 0.01): the ectoderm group had mostly neuroblastoma (79.2%), as did the mesoderm group (71.1%), whereas the endoderm group contained mainly olfactory neuroblastoma (94.7%). The tumors (mean size: 69.14 ± 58.37 mm) were most commonly poorly differentiated with local extension, although lymph node invasion and distant metastasis occurred in a minority of cases. Compared with the other groups, the endoderm group had smaller (43.89 ± 20.84 mm) and better-differentiated tumors and a lower prevalence of lymph node invasion and metastasis (P < 0.05). Despite this, overall survival was poorest for the endoderm group (P < 0.05). Radiotherapy improved overall survival in the endoderm and ectoderm groups but worsened overall survival in the mesoderm group (P < 0.05). Conclusions: Malignant neuroblastoma characteristics may be influenced by the tumor microenvironment. In the youngest patients, decision-making regarding the best choice of therapy should be delayed until accurate risk stratification is possible.

The role of microRNA-30a and downstream snail1 on the growth and metastasis of melanoma tumor.

Objective(s):Growing evidences have indicated microRNAs as modulators of tumor development and aggression. On the other hand, a phenomenon known as epithelial-mesenchymal transition (EMT) that indicates a transient phase from epithelial-like features to mesenchymal phenotype is a key player in tumor progression. In this study, we aimed to assess the potential impacts of miR-30a-5p as an inhibitor of melanoma progression and metastasis.Materials and Methods:MiR-30a-5p was transfected into B16-F10 melanoma cells. Then, the B16-F10 cells were injected subcutaneously or intravenously (IV) in to C57BL/6 mice. Then, the mice were euthanized and tumor size, tumor weight, snail1 protein expression and nodules in the lungs were evaluated.Results:The migration of cancerous cells was significantly suppressed in vitro following the ectopic presentation of miR-30a-5p into B16-F10 melanoma cells. Furthermore, the metastatic behavior of the neoplastic cells was further suppressed in a xenograft mouse model of melanoma as observed with limited lung infiltration. We also found that transfected miR-30a-5p into melanoma cells could decrease snail1 and N-cadherin expression. Conclusion:MiR-30a-5p may represent an effective therapeutic target for the management of melanoma and other snail-overexpressing neoplasms.

Contribution of voltage-gated sodium channel \u03b2-subunits to cervical cancer cells metastatic behavior

BackgroundVoltage-gated sodium (NaV) channels are heteromeric proteins consisting of a single pore forming α-subunit associated with one or two auxiliary β-subunits. These channels are classically known for being responsible of action potential generation and propagation in excitable cells; but lately they have been reported as widely expressed and regulated in several human cancer types. We have previously demonstrated the overexpression of NaV1.6 channel in cervical cancer (CeCa) biopsies and primary cultures, and its contribution to cell migration and invasiveness. Here, we investigated the expression of NaV channels β-subunits (NaVβs) in the CeCa cell lines HeLa, SiHa and CaSki, and determined their contribution to cell proliferation, migration and invasiveness.MethodsWe assessed the expression of NaVβs in CeCa cell lines by performing RT-PCR and western blotting experiments. We also evaluated CeCa cell lines proliferation, migration, and invasion by in vitro assays, both in basal conditions and after inducing changes in NaVβs levels by transfecting specific cDNAs or siRNAs. The potential role of NaVβs in modulating the expression of NaV α-subunits in the plasma membrane of CeCa cells was examined by the patch-clamp whole-cell technique. Furthermore, we investigated the role of NaVβ1 on cell cycle in SiHa cells by flow cytometry.ResultsWe found that the four NaVβs are expressed in the three CeCa cell lines, even in the absence of functional NaV α-subunit expression in the plasma membrane. Functional in vitro assays showed differential roles for NaVβ1 and NaVβ4, the latter as a cell invasiveness repressor and the former as a migration abolisher in CeCa cells. In silico analysis of NaVβ4 expression in cervical tissues corroborated the downregulation of this protein expression in CeCa vs normal cervix, supporting the evidence of NaVβ4’s role as a cell invasiveness repressor.ConclusionsOur results contribute to the recent conception about NaVβs as multifunctional proteins involved in cell processes like ion channel regulation, cell adhesion and motility, and even in metastatic cell behaviors. These non-canonical functions of NaVβs are independent of the presence of functional NaV α-subunits in the plasma membrane and might represent a new therapeutic target for the treatment of cervical cancer.

Exosomal miRNAs from highly metastatic cells can induce metastasis in non-metastatic cells

AimsBreast cancer is a high prevalence cancer among women worldwide. 15–20% of breast cancer cases are triple-negative with a poor prognosis. miRNA aberrant expression is one of the reasons of cancer development and metastasis. Exosomes are vesicles that carry cargos such as miRNAs to other cells. Therefore, we hypothesized that miRNAs transported by exosomes to other cells can induce malignant transformation. Materials and methodsWe extracted exosomes from highly metastatic MDA-MB-231 cells and characterized them using Dynamic light scattering, scanning and transmitting electron microscopy as well as western blot. Then, we treated non-metastatic MCF-7 cells with the exosomes. Afterwards, we evaluated exosome uptake by MCF-7 cells using PHK67 staining. Finally, we used soft agar colony formation, migration, and invasion assays to explore any increase in/induction of metastatic behavior of exosome-treated MCF-7 cells. Key findingsOur result indicated that the particles extracted from MDA-MB-231 cells' supernatant were actually exosomes. PKH67 staining and confocal microscopy showed that the exosomes were actively taken up by MCF-7 cells. Treatment of MCF-7 cells with the exosomes resulted in increased ability of MCF-7 cells to grow independent of anchorage. In addition, migration and invasion capacity of exosome-treated MCF-7 cells increased in a dose-dependent manner. SignificanceAlong with our previous study, we here indicate that highly metastatic MDA-MB-231 cells' exosomes and exosomal miRNAs may induce malignant transformation in non-metastatic MCF-7 cells, thus introducing a novel route of cancer development and metastasis.

Cirsiliol Suppressed Epithelial to Mesenchymal Transition in B16F10 Malignant Melanoma Cells through Alteration of the PI3K/Akt/NF-κB Signaling Pathway.

Malignant melanoma is a highly aggressive form of skin cancer which has a propensity for metastasis. Epithelial mesenchymal transition (EMT) plays a primordial role in the progression of metastatic disease. Metastatic melanoma is resistant to conventional therapies. Hence, researchers have been exploring alternative approaches, including the utility of bioactive phytochemicals to manage metastatic disease. In the present study, we investigated the potential of cirsiliol, a flavonoid isolated from Centaurea jacea L., in modulating the aggressive behavior of B16F10 metastatic melanoma cells, including EMT, and associated molecular mechanisms of action. Cirsiliol was found to be effective in restraining the colony formation and migration of fibronectin-induced B16F10 metastatic melanoma cells. Cirsiliol inhibited the activity and expression of matrix metalloproteinase-9 (MMP-9). Cirsiliol also suppressed the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (also known as Akt)/nuclear factor-κB (NF-κB) signaling pathway which, in turn, caused upregulation of E-cadherin and downregulation of N-cadherin, Snail and Twist. Based on these results, cirsiliol may be considered a promising compound against EMT in the therapeutic management of malignant melanoma.

Anticancer Activity of Dendriplexes against Advanced Prostate Cancer from Protumoral Peptides and Cationic Carbosilane Dendrimers.

The interaction of neuropeptides, vasoactive intestinal peptide (VIP), or growth hormone-releasing hormone (GHRH), with a cationic carbosilane dendrimer forms dendriplexes with antitumoral behavior in advanced prostate cancer cells PC3. At the concentrations used for dendriplexes formation, the free peptides were protumoral and prometastatic in advanced prostate cancer, while dendrimer only showed low cytotoxicity, but did not avoid the metastatic behavior of PC3 cells. However, these nanoplexes favored also cell adhesion and avoided cell migration. Also, the dendriplexes were not toxic for no tumoral prostate cells (RPWE-1) or fibroblasts. The use of labeled GHRH peptide (rhodamine labeled) and a dendrimer (fluorescein labeled) allowed us to observe that both systems reach the intracellular milieu after dendriplex formation. The treatment of PC3 cells with the nanoplexes reduced expression of vascular endothelial growth factor (VEGF) and cyclic adenosine monophosphate (cAMP). Molecular modeling analysis highlights the important contribution of the carbosilane framework in the stabilization of the dendriplex, since dendrimer interacts with a peptide region where hydrophobic amino acids are presented.

Pancreatic cancer-derived exosomes promoted pancreatic stellate cells recruitment by pancreatic cancer.

Cancer-associated fibroblasts (CAFs), which are an important component of the tumor microenvironment, have been identified in the blood circulation of patients with cancer metastasis, and metastatic cancer cells can recruit circulating CAFs. However, primary carcinoma sites usually regulate the behavior of metastatic cancer cells through exosomes. Here, we hypothesized that cancer-derived exosomes could enhance CAF recruitment. Exosomes secreted by pancreatic cancer cells (PANC-1 and MIA PaCa-2) were isolated and characterized. The ability of pancreatic cancer to recruit pancreatic stellate cells (PSCs) was assessed with Transwell assays in vitro and bioluminescent imaging in a mouse model in vivo, and the underlying molecular mechanism was also investigated. The results showed that pancreatic cancer cell-derived exosomes (Exo-Pan and Exo-Mia) promoted the pancreatic cancer recruitment of PSCs. This effect was mediated partially by the transfer of the exosomal protein Lin28B to the recipient cells to activate the Lin28B/let-7/HMGA2/PDGFB signaling pathway. These results suggested that exosomes derived from local cancer could promote the formation of distant metastases through transferring the exosomal protein Lin28B to the metastatic cancer cells.

Silencing growth hormone receptor inhibits estrogen receptor negative breast cancer through ATP-binding cassette sub-family G member 2

Growth hormone receptor (GHR) plays a vital role in breast cancer chemoresistance and metastasis but the mechanism is not fully understood. We determined if GHR could be a potential therapeutic target for estrogen receptor negative (ER−ve) breast cancer, which are highly chemoresistant and metastatic. GHR was stably knocked down in ER-ve breast cancer cells and its effect on cell proliferation, metastatic behavior, and chemosensitivity to docetaxel (DT) was assessed. Microarray analysis was performed to identify potential GHR downstream targets involved in chemoresistance. GHR and ATP-binding cassette sub-family G member 2 (ABCG2) overexpression and knockdown studies were performed to investigate the mechanism of GHR-induced chemoresistance. Patient-derived xenografts was used to study the effect of GHR and ABCG2. Immunohistochemical data was used to determine the correlation between GHR, pAKT, pmTOR, and ABCG2 expressions. GHR silencing drastically reduced the chemoresistant and metastatic behavior of ER-ve breast cancer cells and also inhibited AKT/mTOR pathway. In contrast, activation, or overexpression of GHR increased chemoresistance and metastasis by increasing the expression and promoter activity, of ABCG2. Inhibition of JAK2/STAT5 signaling repressed GHR-induced ABCG2 promoter activity and expression. Further, ABCG2 knockdown significantly increased the chemosensitivity. Finally, patient-derived xenograft studies revealed the role of GHR in chemoresistance. Overall, these findings demonstrate that targeting GHR could be a novel therapeutic approach to overcome chemoresistance and associated metastasis in aggressive ER-ve breast cancers.

Humanization of the Prostate Microenvironment Reduces Homing of PC3 Prostate Cancer Cells to Human Tissue-Engineered Bone

The primary tumor microenvironment is inherently important in prostate cancer (PCa) initiation, growth and metastasis. However, most current PCa animal models are based on the injection of cancer cells into the blood circulation and bypass the first steps of the metastatic cascade, hence failing to investigate the influence of the primary tumor microenvironment on PCa metastasis. Here, we investigated the spontaneous metastasis of PC3 human PCa cells from humanized prostate tissue, containing cancer-associated fibroblasts (CAFs) and prostate lymphatic and blood vessel endothelial cells (BVECs), to humanized tissue-engineered bone constructs (hTEBCs) in NOD-SCID IL2Rγnull (NSG) mice. The hTEBC formed a physiologically mature organ bone which allowed homing of metastatic PCa cells. Humanization of prostate tissue had no significant effect on the tumor burden at the primary site over the 4 weeks following intraprostatic injection, yet reduced the incidence and burden of metastases in the hTEBC. Spontaneous PCa metastases were detected in the lungs and spleen with no significant differences between the humanized and non-humanized prostate groups. A significantly greater metastatic tumor burden was observed in the liver when metastasis occurred from the humanized prostate. Together, our data suggests that the presence of human-derived CAFs and BVECs in the primary PCa microenvironment influences selectively the metastatic and homing behavior of PC3 cells in this model. Our orthotopic and humanized PCa model developed via convergence of cancer research and tissue engineering concepts provides a platform to dissect mechanisms of species-specific PCa bone metastasis and to develop precision medicine strategies.

Fluorometric Quantification of Single-Cell Velocities to Investigate Cancer Metastasis.

Hematogenous metastasis is a multistep, selectin-regulated process whose mechanisms remain poorly understood. To investigate this biological pathway of cancer dissemination and better understand circulating cancer cells, we developed a high-throughput methodology that integrates organ-on-chip-like microfluidic and photoconvertible protein technologies. Our approach can ascribe single-cell velocity as a traceable cell property for off-chip analysis of the direct relationships between cell molecular profiles and adhesive phenotypes in the context of physiologically relevant fluid flow. We interrogate how natively expressed selectin ligands relate to colon cancer cell rolling frequencies and velocities and provide context for previously reported disparities in invitro and invivo models of selectin-mediated adhesion and metastasis. This integrated methodology represents a versatile approach for the development of anti-metastatic therapeutics as well as to generate and test mechanistic hypotheses regarding spatiotemporal processes that occur over timescales of seconds to hours with single-cell resolution.

SSeCKS/Akap12 suppresses metastatic melanoma lung colonization by attenuating Src-mediated pre-metastatic niche crosstalk.

SSeCKS/Gravin/AKAP12 (SSeCKS) controls metastasis-associated PKC and Src signaling through direct scaffolding activity. SSeCKS is downregulated in the metastases of many human cancer types, and its forced re-expression suppresses the metastatic behavior of prostate cancer cells. SSeCKS is also downregulated in breast and prostate cancer stroma, and SSeCKS-null mice (KO) are metastasis-prone, suggesting a role in suppressing formation of the pre-metastatic niche. Here, we show that lung colonization and metastasis formation by B16F10 and SM1WT1[BrafV600E] mouse melanoma cells is 9-fold higher in syngeneic KO compared to WT hosts, although there is no difference in orthotopic tumor volumes. Although melanoma cells adhered equally to KO or WT lung fibroblasts (LF), co-injection of melanoma cells with KO (vs. WT) LF increased lung macrometastasis formation in WT hosts, marked by increased melanoma colonization at foci of leaky vasculature. Increased melanoma adhesion on KO lung endothelial cells (LEC) was facilitated by increased E-Selectin levels and by increased STAT3-regulated secretion of senescence-associated factors from KO-LF, such as Vegf. Finally, the ability of SSeCKS to attenuate IFNα-induced Stat3 activation in KO-LF required its Src-scaffolding domain. Taken together, these data suggest that SSeCKS normally suppresses metastatic colonization in the lung by attenuating the expression of Selectin adhesion proteins, which can be controlled autonomously by local endothelial cells or enhanced by senescence factors secreted by neighboring fibroblasts in a SSeCKS-regulated, Src/Stat3-dependent manner.

Aberrant expression of PDZ-binding kinase/T-LAK cell-originated protein kinase modulates the invasive ability of human pancreatic cancer cells via the stabilization of oncoprotein c-MYC.

High frequency of mortality in patients with pancreatic ductal adenocarcinoma (PDAC) is vastly associated with the invasive and metastatic nature of these cancer cells. Little is known about the factors involved in this invasive/metastatic process. The current challenge in the treatment of these patients is the lack of viable options besides gemcitabine. The aim of this study was to evaluate the role of PDZ-binding kinase (PBK)/T-LAK cell-originated protein kinase (TOPK) in invasive PDAC cells and to determine whether PBK/TOPK expression drives invasiveness in PDAC. Using gain-of-function and loss-of-function studies in established and patient-derived xenograft-PDAC cell lines, and examining patient-derived archival tissue samples, we demonstrate for the first time that PBK/TOPK is upregulated in pancreatic cancer and expression levels are closely associated with the invasive property of pancreatic cancer cells. Modulation of PBK/TOPK causally regulates the invasive ability of PDAC cells. We also demonstrate that two key players in metastatic invasion, matrix metalloproteinases-2 (MMP-2) and MMP-9 gelatinase activity and gene promoter activities, are regulated by PBK/TOPK. Moreover, we demonstrate for the first time that PBK/TOPK provides stability of an oncoprotein, c-MYC, which transcriptionally regulates MMP-2 and MMP-9 in these invasive PDAC cells. Our in vitro and in situ data corroborate that PBK/TOPK is closely associated with the invasive nature of PDAC and reveal a novel mechanism by which the metastatic behavior of human pancreatic cancer cells is regulated. These findings provide a rationale for targeting PBK/TOPK for the therapeutic intervention of invasive/metastatic pancreatic cancer in human.

Feedback control of the CXCR7/CXCL11 chemokine axis by estrogen receptor α in ovarian cancer.

Ovarian cancer (OC) is one of the most intractable diseases, exhibiting tremendous molecular heterogeneity and lacking reliable methods for screening, resulting in late diagnosis and widespread peritoneal dissemination. Menopausal estrogen replacement therapy is a well‐recognized risk factor for OC, but little is known about how estrogen might contribute to this disease at the cellular level. This study identifies chemokine receptor CXCR7/ACKR3 as an estrogen‐responsive gene, whose expression is markedly enhanced by estrogen through direct recruitment of ERα and transcriptional active histone modifications in OC cells. The gene encoding CXCR7 chemokine ligand I‐TAC/CXCL11 was also upregulated by estrogen, resulting in Ser‐118 phosphorylation, activation, and recruitment of estrogen receptor ERα at the CXCR7 promoter locus for positive feedback regulation. Both CXCR7 and CXCL11, but not CXCR3 (also recognized to interact with CXCL11), were found to be significantly increased in stromal sections of microdissected tumors and positively correlated in mesenchymal subtype of OC. Estrogenic induction of mesenchymal markers SNAI1, SNAI2, and CDH2 expression, with a consequent increase in cancer cell migration, was shown to depend on CXCR7, indicating a key role for CXCR7 in mediating estrogen upregulation of mesenchymal markers to induce invasion of OC cells. These findings identify a feed‐forward mechanism that sustains activation of the CXCR7/CXCL11 axis under ERα control to induce the epithelial–mesenchymal transition pathway and metastatic behavior of OC cells. Such interplay underlies the complex gene profile heterogeneity of OC that promotes changes in tumor microenvironment and metastatic acquisition.

Antimetastatic effect of the pharmacological inhibition of serine/arginine-rich protein kinases (SRPK) in murine melanoma

The Serine/arginine-rich protein kinases (SRPK) are involved in pre-mRNA splicing control through the phosphorylation of the SR protein family of splicing factors. Over the last years, several studies have shown the relevance of SRPK for human cancers and their potential as promising drug targets. In this context, we have previously selected three trifluoromethyl arylamides (named here as SRVIC24, SRVIC30 and SRVIC36) with improved in vitro antileukemia effect and ability of impairing the cellular activity of SRPK. Given the increasing amount of reports on the implication of these kinases in metastatic cancers, in this study, we have evaluated the antimetastatic effect of these compounds and the known SRPK inhibitor (SRPIN340) on a murine model of metastatic melanoma. The compounds were able to impact the melanoma cell metastatic behavior by decreasing migration, invasion, adhesion, and colony formation in in vitro assays. Also, they presented antimetastatic in vivo activity, without apparent signs of systemic toxicity after treatments, as revealed by the histology of organs and analysis of key serum biochemical markers. Moreover, the effect of the treatments on SRPK1 nuclear translocation and SR protein phosphorylation was observed. Finally, molecular docking studies were carried out to gain structural information on the SRPK-compound complexes. Together, these data suggest that SRPK pharmacological inhibition should be considered as an interesting therapeutic strategy against metastatic cancers.

Abstract B086: Monocytes-produced Chitinase-3-like 1 is a driver of metastatic behavior in advanced prostate cancer patients

Abstract Purpose: The identification, functional status, distribution, and interactions of immune cells in PCa patients have yet to be fully characterized. Understanding these details could provide key insights as to how promising current and developing immunotherapies might be directed toward PCa. Recruited myeloid cells promote cancer initiation, malignant progression, metastasis, and resistance to therapy in the tumor niche. In the present study, we tested the hypothesis that circulating blood monocytes from advanced prostate cancer (PCa) patients exhibit a tumor-promoting phenotype and directly influence the tumor microenvironment in response to tumor-derived signals. Experimental Design: Monocytes from metastatic (PCa-M) and nonmetastatic (PCa-N) patient blood samples were isolated, cultured, and conditioned media (CM) was obtained. To evaluate the role of monocytes in metastatic behavior of PCa cells, in vitro invasion (via Matrigel) and motility assays (Incucyte Live Cell Analysis System) were performed using monocyte-CM. To identify the candidate PCa-M monocyte-secreted protein(s) that might be implicated in tumor progression, we analyzed the CM using Proteome Profiler Analysis (R&amp;D). Data were validated using ELISA and qPCR. Results: Herein, we report that CM from circulating monocytes in patients with PCa/mCRPC (metastatic castration-resistant PCa) increased motility and invasion of epithelial PCa cells. Proteome Profiler Analysis revealed that monocyte-derived CM from metastatic PCa/mCRPC patients presented high levels of YKL-40 (Chitinase-3-like 1; CHI3L1) when compared with PCa-N patients and healthy controls (HC), classified as individuals with no known history of cancer. The only described receptor for YKL-40, interleukin-13 receptor α2 (IL-13Rα2), was significantly upregulated in ARCaPm cells; accordingly, we observed that the activation of IL-13Rα2 from PCa-M CM increased the invasiveness of ARCaPm. In addition, siRNA directed against this receptor in ARCaPm cells caused a significant reduction in the invasiveness of these cells in the presence of CM from PCa-M patients. Conclusions: We show that circulating monocytes from metastatic PCa/mCRPC patients exert a tumor-promoting role via the secretion of YKL-40, and YKL-40 demands further exploration as a possible therapeutic target in advanced PCa. Citation Format: Karen A. Cavassani, Rebecca Meza, David Habiel, Jie-Fu Chen, Alexander Montes, Manisha Tripathi, Gislaine Martins, Timothy R. Crother, Cory M. Hogaboam, Sungyong You, Neil Bhowmick, Edwin Posadas. Monocytes-produced Chitinase-3-like 1 is a driver of metastatic behavior in advanced prostate cancer patients [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B086.

Abstract A57: Antisense oligonucleotide therapeutics with receptor-targeted delivery in triple-negative breast cancer cells via microRNA blockade without passenger strand side effects

Abstract Triple-negative breast cancer (TNBC) attacks &amp;gt;40,000 young women annually in the US, and has no molecular targeted therapies. MicroRNAs drive many forms of cancer, and they modulate multiple gene pathways simultaneously. Highly expressed microRNA miR-17-5p is distinctive in TNBC and contributes to cancer cell survival. We hypothesized that reduction of miR-17-5p activity by delivering oligonucleotide-based antagomiRs specifically to breast cancer cells via receptor-mediated endocytosis would inhibit metastatic behavior of TNBC cells. Using a luciferase reporter system harboring a miR-17-5p binding site in the 3′ untranslated region of the luciferase gene, we assessed the efficacy of various antagomiRs. Contrary to conventional wisdom, that only one of the two strands in a pre-miRNA duplex is active, such as miR-17-5p, we previously observed that a full-length miR-17-5p DNA-LNA blocker created additional off-target effects by mimicking miR-17-3p, implying that therapeutic microRNA blockers should be designed to avoid resemblance to the opposing strands. We designed guide strand specific blockers of miR-17-5p composed of gapmers with anionic backbone derivatives 2′-fluoro-arabino nucleic acid (FANA), and 2′-aminomethyl-bridged nucleic acid (NC-BNA). Since most breast cancer cells overexpress insulin-like growth factor receptor (IGF1R), a peptide derivative of the IGF1 was conjugated to anti-miR-17-5p antagomiRs to achieve breast cancer cell-specific delivery. Our results showed that in multiple TNBC cell lines, the NC-BNA 15mers displayed high efficacy with sub-nM activity for miR-17-5p blockade and significant inhibition of cell growth. Anti-miR-17-5p-IGF1 peptide conjugate successfully inhibited miR-17-5p activity in the luciferase reporter system. We are further examining the effects of NC-BNA-peptides in multiple TNBC cell lines. Our study provides a good basis for the development of a new TNBC targeted therapy. Supported by Bound Therapeutics LLC. Conflict of interest: pending TJU PCT/US2015/015681 patent application licensed to Bound Therapeutics LLC. Citation Format: Yuan-Yuan Jin, Eric Wickstrom. Antisense oligonucleotide therapeutics with receptor-targeted delivery in triple-negative breast cancer cells via microRNA blockade without passenger strand side effects [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A57.

Abstract A66: The tissue transglutaminase 2–fibronectin protein complex: A new target in ovarian cancer

Abstract Here we describe a small-molecule inhibitor (SMI) that disrupts the tissue transglutaminase 2 (TG2)-fibronectin (FN) interaction. TG2 is a multifunctional protein, with enzymatic (transglutaminase, isopeptidase, protein disulfide isomerase), GTPase, and extracellular matrix (ECM) scaffold activities. TG2 directly interacts with FN through its N-terminus domain, playing an important role in stabilizing ternary TG2/FN/integrin complexes, which regulate cell adhesion to the matrix. Our group has previously shown that TG2 overexpressed in ovarian cancer (OC) promotes epithelial-to-mesenchymal transition (EMT) contributing to OC cell invasiveness and metastatic behavior. We showed that the interaction with FN is critical to these functions. Proximity ligation assay was used to measure TG2/integrin beta 1 interaction in OC tumors and identified TG2/integrin beta 1 complex in &amp;gt;50% of ovarian tumors, irrespective of histologic type. Based on those findings, we conducted the high-throughput screening and identified TG53 as the lead TG2/FN inhibitory compound. Structure-activity relationship-based approach was used to optimize the physicochemical properties of TG53, leading to design and chemical synthesis of 6 analogues (MT1-6). All analogues blocked TG2/FN interaction, as measured by an ELISA assay, and OC cell adhesion to FN, as measured by a solid phase assay, MT4 being the most potent inhibitor. MT4 downregulates integrin-initiated signaling in OC cells, leading to downstream inhibition of FAK and ERK. Immunofluorescence staining demonstrated that the actin cytoskeleton adopts a cortical localization pattern in MT4-treated cells. MT4 caused membrane ruffling delaying the formation of stable focal contacts and mature adhesions points. Pretreatment with MT4 sensitized OC cells to paclitaxel, as measured by colony-forming assay. In an intraperitoneal OC mouse model, MT4 caused decreased OC cell adhesion to the peritoneum, inhibiting peritoneal dissemination. Hence, MT4 is a potential candidate to be developed and used in combination with current chemotherapeutics to target OC tumor progression. Further studies will continue to optimize this new class of TG2/FN SMIs for potential clinical use in OC. Citation Format: Livia Elena Sima, Bakhtiyor Yakubov, Salvatore Condello, Horacio Cardenas, Zhong Yin Zhang, Lan Chen, Daniela Elena Matei. The tissue transglutaminase 2–fibronectin protein complex: A new target in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A66.