Reduced Cell Motility Research Articles

Mitotic Centromere-Associated Kinesin (MCAK/KIF2C) Regulates Cell Migration and Invasion by Modulating Microtubule Dynamics and Focal Adhesion Turnover.

Simple SummaryThe microtubule (MT) cytoskeleton is a crucial factor for organized cell motility and migration of cancer as well as benign cells. Mitotic centromere-associated kinesin (MCAK/KIF2C) is a member of the kinesin-13 family, which is important for the regulation of MT dynamics. Its overexpression has been reported to be related to increased metastasis in various tumor entities. Our study further elucidate how MCAK’s is able to modulate cell migration and invasion. Interfering with the precise regulated expression of MCAK led to impaired FA protein composition and altered their phosphorylation status, disturbed the assembly and disassembly rate of FA, delayed cell adhesion, and compromised the plus-tip dynamics of MTs. MCAK regulates these processes by affecting the actin-MT cytoskeleton dynamics, providing molecular mechanisms by which a deregulation of MCAK could promote tumor metastasis.The microtubule (MT) cytoskeleton is crucial for cell motility and migration by regulating multiple cellular activities such as transport and endocytosis of key components of focal adhesions (FA). The kinesin-13 family is important in the regulation of MT dynamics and the best characterized member of this family is the mitotic centromere-associated kinesin (MCAK/KIF2C). Interestingly, its overexpression has been reported to be related to increased metastasis in various tumor entities. Moreover, MCAK is involved in the migration and invasion behavior of various cell types. However, the precise molecular mechanisms were not completely clarified. To address these issues, we generated CRISPR/dCas9 HeLa and retinal pigment epithelium (RPE) cell lines overexpressing or downregulating MCAK. Both up- or downregulation of MCAK led to reduced cell motility and poor migration in malignant as well as benign cells. Specifically, it’s up- or downregulation impaired FA protein composition and phosphorylation status, interfered with a proper spindle and chromosome segregation, disturbed the assembly and disassembly rate of FA, delayed cell adhesion, and compromised the plus-tip dynamics of MTs. In conclusion, our data suggest MCAK act as an important regulator for cell motility and migration by affecting the actin-MT cytoskeleton dynamics and the FA turnover, providing molecular mechanisms by which deregulated MCAK could promote malignant progression and metastasis of tumor cells.

Lactate Dehydrogenase A or B Knockdown Reduces Lactate Production and Inhibits Breast Cancer Cell Motility in vitro.

Background: Lactate dehydrogenase (LDH) plays an important role in cancer pathogenesis and enhanced expression/activity of this enzyme has been correlated with poor prognosis. In this study we determined the expression profile of LDH-A and B in normal as well as in endocrine-resistant and -responsive breast cancer cells and the effect of their knockdown on LDH activity, lactate production, proliferation and cell motility. Methods: Knockdown experiments were performed using siRNA and shRNA. The expression profile of LDH and signaling molecules was determined using PCR and western blotting. Intracellular LDH activity and extracellular lactate levels were measured by a biochemical assay. Cell motility was determined using wound healing, while proliferation was determined using MTT assay. Results: LDH-A was expressed in all of the tested cell lines, while LDH-B was specifically expressed only in normal and endocrine-resistant breast cancer cells. This was correlated with significantly enhanced LDH activity and lactate production in endocrine resistant breast cancer cells when compared to normal or endocrine responsive cancer cells. LDH-A or -B knockdown significantly reduced LDH activity and lactate production, which led to reduced cell motility. Exogenous lactate supplementation enhanced cell motility co-incident with enhanced phosphorylation of ERK1/2 and reduced E-cadherin expression. Also, LDH-A or -B knockdown reduced ERK 1/2 phosphorylation. Conclusion: Enhanced cell motility in endocrine resistant breast cancer cells is at least in part mediated by enhanced extracellular lactate levels, and LDH inhibition might be a promising therapeutic target to inhibit cancer cell motility.

Aged Breast Extracellular Matrix Drives Mammary Epithelial Cells to an Invasive and Cancer-Like Phenotype.

Age is a major risk factor for cancer. While the importance of age related genetic alterations in cells on cancer progression is well documented, the effect of aging extracellular matrix (ECM) has been overlooked. This study shows that the aging breast ECM alone is sufficient to drive normal human mammary epithelial cells (KTB21) to a more invasive and cancer‐like phenotype, while promoting motility and invasiveness in MDA‐MB‐231 cells. Decellularized breast matrix from aged mice leads to loss of E‐cadherin membrane localization in KTB21 cells, increased cell motility and invasion, and increased production of inflammatory cytokines and cancer‐related proteins. The aged matrix upregulates cancer‐related genes in KTB21 cells and enriches a cell subpopulation highly expressing epithelial‐mesenchymal transition‐related genes. Lysyl oxidase knockdown reverts the aged matrix‐induced changes to the young levels; it relocalizes E‐cadherin to cell membrane, and reduces cell motility, invasion, and cytokine production. These results show for the first time that the aging ECM harbors key biochemical, physical, and mechanical cues contributing to invasive and cancer‐like behavior in healthy and cancer mammary cells. Differential response of cells to young and aged ECMs can lead to identification of new targets for cancer treatment and prevention.

CdGAP promotes prostate cancer metastasis by regulating epithelial-to-mesenchymal transition, cell cycle progression, and apoptosis

High mortality of prostate cancer patients is primarily due to metastasis. Understanding the mechanisms controlling metastatic processes remains essential to develop novel therapies designed to prevent the progression from localized disease to metastasis. CdGAP plays important roles in the control of cell adhesion, migration, and proliferation, which are central to cancer progression. Here we show that elevated CdGAP expression is associated with early biochemical recurrence and bone metastasis in prostate cancer patients. Knockdown of CdGAP in metastatic castration-resistant prostate cancer (CRPC) PC-3 and 22Rv1 cells reduces cell motility, invasion, and proliferation while inducing apoptosis in CdGAP-depleted PC-3 cells. Conversely, overexpression of CdGAP in DU-145, 22Rv1, and LNCaP cells increases cell migration and invasion. Using global gene expression approaches, we found that CdGAP regulates the expression of genes involved in epithelial-to-mesenchymal transition, apoptosis and cell cycle progression. Subcutaneous injection of CdGAP-depleted PC-3 cells into mice shows a delayed tumor initiation and attenuated tumor growth. Orthotopic injection of CdGAP-depleted PC-3 cells reduces distant metastasic burden. Collectively, these findings support a pro-oncogenic role of CdGAP in prostate tumorigenesis and unveil CdGAP as a potential biomarker and target for prostate cancer treatments.

The effect of temozolomide on Hsp60 and Hsp70 expression in extracellular vesicles derived from U87MG glioma cells

Abstract Objectives Temozolomide (TMZ) is an effective drug for glioblastoma multiforme (GBM), but the mechanism underlying TMZ resistance is poorly understood. New evidence has revealed that the release of heat shock proteins (Hsps) derived from extracellular vesicles (EVs) play an important role in cancer progression by modulating tumor microenvironment and cellular cross-talk. This study aims to evaluate the effects of TMZ on the expression of EV-derived and cellular Hsps and cell motility in U87MG human glioblastoma cell line. Methods Glial-EVs were isolated from the culture medium and characterized by SEM and immunoblotting. The effect of TMZ treatments (25, 200 and 750 µM) on cell proliferation (MTT assay), migration (scratch assay), and Hsp60 and Hsp70 levels (immunoblotting) were evaluated. Results TMZ treatments led to an increase in intracellular Hsp70 while decreasing EV-derived Hsp70. Cellular Hsp60 level was elevated at the low dose of TMZ, but it reduced at higher TMZ concentrations. Hsp60 was also decreased in EVs secreted from TMZ-treated cells. Besides, TMZ treatment reduced the proliferation and migration of glioma cells in a dose-dependent manner. Conclusions Our results suggest that TMZ has the potential to target both EV-derived and cellular Hsps for GBM treatment, thus it may reduce cell motility.

Allium willeanum Holmboe exerts anticancer activities on metastatic breast cancer cells MCF-7 and MDA-MB-231

Allium species are medically important plants, rich in bioactive molecules with antitumoral properties. In this study, we aimed to investigate the molecular composition and in vitro anticancer activities of Allium willeanum Holmboe, an endemic Allium species of the island of Cyprus. GC-MS analysis of ethanolic extracts of A. willeanum H. bulb (AWB) showed bioactive molecules, octadecanoic acid 2-hydroxy-1-(hydroxymethyl)ethyl ester (21.99 %), hexadecanoic acid (20.42 %), pentadecanoic acid (9.19 %), 1,2-benzenedicarboxylic acid, diethyl ester (8.79 %), with known anticancer activities. AWB exerted significant reduction in mitochondria dependent metabolic activity as a measure of cell growth on MCF-7 (weakly metastatic) and MDA-MB-231 (strongly metastatic) human breast cancer (BCa) cell lines with a more prominent effect on highly metastatic BCa cells. Both trypan blue and lactate dehydrogenase (LDH) cytotoxicity assays quantitatively revealed that exposure to AWB extract significantly reduced cancer cell viability in both tested cell types. Differential activation of caspases in the tested cell lines indicated faster apoptotic activity on MDA-MB-231 cells compared to MCF-7. A significant reduction in cell motility was demonstrated upon AWB exposure of MCF-7 and MDA-MB-231 cells. AWB bioactive molecules collectively act on cancer cells to exert significant cytotoxic, apoptotic activities on both MCF-7 and MDA-MB-231 cells and significantly reduce their lateral motility, elucidating AWB as a promising agent to further be utilized in anticancer studies.

Abstract 1332: Antiretroviral agent inhibits proliferation and motility of BT-20 cells through inhibition of human endogenous retrovirus type K

Abstract The purpose of the study was to establish the role of human endogenous retrovirus K (HERV-K) in cancer development and to explore the possibility of repurposing antiretroviral agents for cancer treatment. We screened different categories of antiretroviral agents and several breast cancer cell lines to study the ability of antiretroviral agents to inhibit cancer cell proliferation and to inhibit the natural expression of HERV-K in cancer cells. Cells were transduced with exogenous HERV-K genes or with HERV-K knockdown plasmids to investigate the relationship between cytotoxic effect of antiretroviral agents and HERV expression. We found that an integrase strand-transfer inhibitor, dolutegravir, inhibited HERV-K expression on both the RNA and protein levels in BT-20 cells. Transduction with both HERV-K env and pol genes rendered the cell line more resistant to dolutegravir. Knockdown of HERV-K with shRNA also increased resistance to dolutegravir compared to cells transduced with random double-stranded RNA. The cytotoxic effect of dolutegravir correlated with enhanced expression of E-cadherin and a reduction in cell motility. Interestingly, an HERV-K knockdown cell line also exhibited enhanced expression of E-cadherin and reduced motility. As a potential carcinogenic role of HERVs has raised interest in using antiretroviral agents as a new approach to treat cancer, we provide the first evidence that one antiretroviral agent actually inhibits the proliferation and motility of a cancer cell line through inhibiting an HERV. Citation Format: John Lin, Mason Farris, Lauren Robbins, Matthew Lee, Serratt Nong, Yingguang Liu. Antiretroviral agent inhibits proliferation and motility of BT-20 cells through inhibition of human endogenous retrovirus type K [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 1332.

Abstract 3049: Tumor treating fields induce DNA damage and apoptosis in medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant brain tumor in children and accounts for approximately 20% of all pediatric central nervous system tumors. Current multimodal treatment has led to a 70-90% five-year overall survival rate, however, the prognosis for patients with tumor dissemination and recurrent MB remains poor. Additionally, the majority of survivors exhibit long-term neurocognitive and neuroendocrine complications because of the cytotoxic drugs and high dose radiation. Consequently, more effective and less toxic treatments are necessary in order to raise the quality of life for these young patients. Tumor Treating Fields (TTFields) are an exciting new therapeutic opportunity for MB. TTFields are low intensity, alternating electric fields that disrupt cell division through physical interactions with key molecules during mitosis. The side effects from TTFields treatments are incredibly low, making it an ideal candidate for MB therapy. Currently TTFields were found to increase the survival of adult brain tumor patients by dysregulating the cytoskeletal network leading to mitotic arrest and as well as other varied multi-modal mechanisms of action. To determine if TTFields can be an effective treatment for MB, we conducted an in vitro study treating multiple MB cell lines with TTFields. We discovered that TTFields treatment reduced MB cell growth (Daoy, Med1-MB, D425, and D283) with an optimal frequency of 200-250 kHz. We also observed that TTFields treatment dysregulated actin polymerization that corresponded with a reduction in cell motility and invasion. TTFields treatment also caused DNA damage (γH2AX, 53BP1) that correlated with an increase in apoptotic cells. To expand on these initial findings, we conducted a combinatorial study with the small molecule inhibitor, CX-4945. CX-4945 is an inhibitor to casein kinase 2 and is currently being used in a phase I clinical trial to treat recurrent MB (NCT03904862). We discovered that CX-4945 enhanced the growth reduction of TTFields treatment. In addition, combining CX-4945 and TTFields increased the number of cells with dysregulated actin which correlated with a decrease in MB migration and invasion. Our findings demonstrate that TTFields may be a novel and less toxic method to treat MB patients. Citation Format: Ryan T. Nitta, Gordon Li. Tumor treating fields induce DNA damage and apoptosis in medulloblastoma [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 3049.

Thrombospondin-1 mediates Rho-kinase inhibitor-induced increase in outflow-facility.

Rho‐kinase (ROCK) inhibitors, a novel class of anti‐glaucoma agents, act by increasing the aqueous humor outflow through the conventional trabecular meshwork pathway. However, the downstream signaling consequences of the ROCK inhibitor are not completely understood. Our data show that Y39983, a selective ROCK inhibitor, could induce filamentous actin remodeling, reduced cell motility (as measured by cell migration), and transepithelial resistance in primary human TM (hTM) cells. After 2 days Y39983 treatment of hTM cells, a proteomic study identified 20 proteins whose expression was significantly altered. Pathway analysis of those proteins revealed the involvement of the p53 pathway, integrin signaling pathway, and cytoskeletal pathway regulation by Rho GTPase. Thrombospondin‐1 (TSP1), a matricellular protein that is increased in glaucoma patients, was downregulated fivefold following Y39983 treatment. More importantly, both TSP1 antagonist leucine–serine–lysine–leucine (LSKL) and small interfering RNA (siRNA) reduced TSP1 gene and protein expressions as well as hTM cell migration. In the presence of Y39983, no further inhibition of cell migration resulted after LSKL and TSP1 siRNA knockdown. Likewise, LSKL triggered a dose‐dependent increase in outflow facility in ex vivo mouse eyes, to a similar extent as Y39983 (83.8% increase by Y39983 vs. 71.2% increase by LSKL at 50 µM). There were no additive effects with simultaneous treatment with LSKL and Y39983, supporting the notion that the effects of ROCK inhibition were mediated by TSP1.

Neuropilin-2 Is Associated With Increased Hepatoblastoma Cell Viability and Motility.

The neuropilins NRP1 and NRP2 are multifunctional glycoproteins that have been implicated in several cancer-related processes including cell survival, migration, and invasion in various tumor types. Here, we examine the role of neuropilins in hepatoblastoma (HB), the most common pediatric liver malignancy. Using a combination of immunohistochemistry, RNA analysis and western blotting, we observed high level expression of NRP1 and NRP2 in 19 of 20 HB specimens and in a majority of human HB cell lines (HUH6 and five cell lines established from patient-derived xenografts) studied but not in normal hepatocytes. Silencing of NRP2 expression in HUH6 and HB-282 HB cells resulted in decreased cell viability, impaired cytoskeleton remodeling, and reduced cell motility, suggesting that NRP2 contributes to the malignant phenotype. We propose that neuropilins warrant further investigation as biomarkers of HB and potential therapeutic targets.

Antitumor Effect of Metformin in Combination with Binimetinib on Melanoma Cells.

Cutaneous melanoma is a fatal disease for patients with distant metastasis. Metformin is the most widely used anti-diabetic drug, and proved to suppress cell proliferation and metastasis in diverse cancers including melanoma. We previously reported that MEK inhibitor trametinib increases the expression of epithelial-mesenchymal transition (EMT) regulators and melanoma cell motility, which are suppressed by addition of metformin in A375 melanoma cells. To confirm our findings further, we first evaluated the effect of metformin in combination with another MEK inhibitor binimetinib on cell viability in G361 melanoma cells. We then investigated whether binimetinib affects the expression of EMT regulators and cell motility. We finally monitored the effect of metformin on binimetinib-induced cell migration. Cell viability assay showed that combination index (CI) value at ED50 is 0.80, suggesting synergy for the combination of metformin with binimetinib. Our results also revealed that binimetinib increased the expression of EMT regulators such as integrin αV, fibronectin and slug, which correlate well with the enhanced cell migration in wound healing assay. Metformin, on the contrary, suppressed the expression of sparc, integrin αV, fibronectin and N-cadherin with the reduced cell motility. The combination treatment showed that metformin counteracts the binimetinib-induced increase of cell motility. Overall, these results suggest that metformin with binimetinib might be useful as a potential therapeutic adjuvant against cell survival and metastatic activity in melanoma patients.

Organoruthenium Complexes with Benzo-Fused Pyrithiones Overcome Platinum Resistance in Ovarian Cancer Cells.

Simple SummaryOvarian cancer is the fifth most common cancer in the developing world, with many front-line treatments combining paclitaxel with platinum-based anticancer agents cisplatin/carboplatin. However, increased incidence of platinum resistance demands the development of new chemotherapeutic agents. The aim of our study was to explore a new family of organoruthenium(II) pyrithione complexes for their efficacy towards platinum-resistant ovarian cancer cells. We confirmed that this new class of compounds remain highly potent towards platinum-insensitive cells and appear to work by a mechanism that is not common to platinum agents.Drug resistance to existing anticancer agents is a growing clinical concern, with many first line treatments showing poor efficacy in treatment plans of some cancers. Resistance to platinum agents, such as cisplatin, is particularly prevalent in the treatment of ovarian cancer, one of the most common cancers amongst women in the developing world. Therefore, there is an urgent need to develop next generation of anticancer agents which can overcome resistance to existing therapies. We report a new series of organoruthenium(II) complexes bearing structurally modified pyrithione ligands with extended aromatic scaffold, which overcome platinum and adriamycin resistance in human ovarian cancer cells. The mechanism of action of such complexes appears to be unique from that of cisplatin, involving G1 cell cycle arrest without generation of cellular ROS, as is typically associated with similar ruthenium complexes. The complexes inhibit the enzyme thioredoxin reductase (TrxR) in a model system and reduce cell motility towards wound healing. Importantly, this work highlights further development in our understanding of the multi-targeting mechanism of action exhibited by transition metal complexes.

Stomatin‑like protein2 induces metastasis by regulating the expression of a rate‑limiting enzyme of the hexosamine biosynthetic pathway in pancreatic cancer.

Stomatin-like protein 2 (SLP-2) is associated with poor prognosis in several types of cancer, including pancreatic cancer (PC); however, the molecular mechanism of its involvement remains elusive. The present study aimed to elucidate the role of this protein in the development of PC. Human PC cell lines AsPC-1 and PANC-1 were transfected by a vector expressing SLP-2 shRNA. Analyses of cell proliferation, migration, invasion, chemosensitivity, and glucose uptake were conducted, while a mouse xenograft model was used to evaluate the functional role of SLP-2 in PC. Immunohistochemical analysis was retrospectively performed on human tissue samples to compare expression between the primary site (n=279) and the liver metastatic site (n=22). Furthermore, microarray analysis was conducted to identify the genes correlated with SLP-2. In vitro analysis demonstrated that cells in which SLP-2 was suppressed exhibited reduced cell motility and glucose uptake, while in vivo analysis revealed a marked decrease in the number of liver metastases. Immunohistochemistry revealed that SLP-2 was increased in liver metastatic sites. Microarray analysis indicated that this protein regulated the expression of glutamine-fructose-6-phosphate transaminase 2 (GFPT2), a rate-limiting enzyme of the hexosamine biosynthesis pathway. SLP-2 contributed to the malignant character of PC by inducing liver metastasis. Cell motility and glucose uptake may be induced via the hexosamine biosynthesis pathway through the expression of GFPT2. The present study revealed a new mechanism of liver metastasis and indicated that SLP-2 and its downstream pathway could provide novel therapeutic targets for PC.

Plasma cell dynamics in the bone marrow niche

SUMMARYUsing intravital imaging, we report that bone marrow (BM) plasma cells (PCs) are motile. BM PCs exhibit a unique migration pattern, characterized by intermittent periods of high motility and longer stretches of confined migration or arrest. BM PCs accumulate into clusters, which have reduced cell motility. APRIL promotes cluster formation and overall PC motility in the BM. Although CXCL12 and its receptor, CXCR4, promote PC motility in the BM, VLA4 activity promotes arrest. However, blocking either pathway promotes PC egress from the BM. Under steady-state conditions, BM PCs recirculate to other bones and spleen. In older mice, overall PC motility and recirculation increase, and this is correlated with increased CXCR4 expression, which depends on PC age or maturation rather than mouse age. Altogether, these results suggest that changes in PC motility and CXCR4 expression are linked with survival of long-lived PCs in the BM.

Synthesis of novel potential ROCK inhibitors and their antimigratory effects

Rho kinase (ROCK), an enzyme belonging to the serine-threonine kinase family, is involved in the regulation of basic cellular processes such as morphology, movement, division, differentiation and apoptosis. On the other hand, excessively activated ROCK can cause to cardiovascular and neurological disorders or cancer. In recent years, overactivation of Rho kinases has been associated with increased metastasis in various tumor types and has been explored as target for the development of new anticancer drugs. We report here the design and synthesis of five urea derivatives in search of novel inhibitors of cancer cell migration. Compounds evaluated for their cytotoxic activities against breast (MCF-7) cancer cell line. After determination of the ineffective concentrations of compounds on the proliferation of MCF-7 cells, wound healing experiments were conducted to investigate the antimigratory effects of compounds. While compounds 4 and 10 had no effect on cell migration, treatment of MCF-7 cells with compounds 5, 8 and 9 resulted in significant reduction in cell motility. Taken together our results suggest that the newly synthesized compounds 5, 8 and 9 had the potential antimigratory activity through possible ROCK inhibition in cancer cells.

The secondary bacterial infection caused by WSSV outbreaks impacts shrimp Marsupenaeus japonicus’ growth as well as its intestinal microbiota’s composition and function

Intestinal microbiota homeostasis is a complex ecosystem and is essential for promoting aquatic animals’ growth. Invading pathogens can cause dynamical composition and structure change in the hosts’ microbiota due to this affecting their functions. WSSV is one of the most common and dangerous shrimp pathogens, which leads to white spot disease with too high mortality. A few studies on shrimp intestinal microbiota mainly focused on the changes during acute infection stage. Marsupenaeus japonicus is one of the most cultured shrimp with a decent ability to cope with environmental changes. In the current research, using M. japonicus as a model, we compared the differences of intestinal microbiota between healthy shrimp and the shrimp that survived through the WSSV outbreak. Our study showed that compared to the healthy M. japonicus, the WSSV outbreak disrupted intestinal microbiota structure in the survived shrimp: There was less potential beneficial bacteria and more harmful bacteria. Furthermore, the diversity and total abundance of intestinal microbiota in WSSV-survived shrimp increased significantly. On top of that, a metagenomic analysis by PICRUSt suggested that the changed intestinal microbiota could help the host to combat the secondary bacterial infections caused by WSSV outbreaks by regulating cell growth and death, reducing cell motility, improving energy metabolism, and increasing intestinal enzyme activities regardless the smaller sizes of WSSV-survived shrimp. This could be a result of excessive energy consumption and reduced nutrient-absorbing intestinal microbiota. Our study indicated that shrimp intestinal microbiota plays a fundamental role in combating against secondary infections and regulating the hosts’ growth post WSSV outbreaks. The eventual goal is to development of more efficient diagnostics and therapeutic strategies.

Activation of β2-adrenergic receptor signals suppresses mesenchymal phenotypes of oral squamous cell carcinoma cells.

Metastasis is a primary reason related to the mortality of oral squamous cell carcinoma (OSCC) patients. A program called epithelial‐mesenchymal transition (EMT) has been shown to play a critical role in promoting metastasis in epithelium‐derived carcinoma. During EMT, epithelial cancer cells acquire motile mesenchymal phenotypes and detach from primary tumors. Recent lines of evidence have suggested that EMT confers cancer cells with tumor‐initiating ability. Therefore, selective targeting of EMT would lead to the development of effective therapeutic agents. In this study, using a chemical biology approach, we identified isoxsuprine, a β2‐adrenergic receptor (β2‐AR) agonist as a low‐molecular‐weight compound that interferes with the acquisition of mesenchymal phenotypes of oral cancer cells. Treatment of multiple types of oral cancer cells with isoxsuprine led to the downregulation of mesenchymal cell markers that was accompanied by reduced cell motility. Similar inhibitory effects were also observed for isoprenaline, a non‐selective β‐adrenergic receptor (β‐AR) agonist. In addition, inhibition of cell migration upon treatment with isoxsuprine was reverted by a non‐selective β‐AR antagonist, propranolol, and the CRISPR/Cas9 system‐mediated deletion of the β2‐AR gene, suggesting that the effects exerted by isoxsuprine involved signals mediated by β2‐AR. In addition, in a subcutaneous xenograft model of oral cancer cells, the administration of isoxsuprine effectively suppressed primary tumor growth, suggesting β2‐AR signals to be a promising cancer therapeutic target for treatment of OSCC.

Akt isoform-specific effects on thyroid cancer development and progression in a murine thyroid cancer model

The Akt family is comprised of three unique homologous proteins with isoform-specific effects, but isoform-specific in vivo data are limited in follicular thyroid cancer (FTC), a PI3 kinase-driven tumor. Prior studies demonstrated that PI3K/Akt signaling is important in thyroid hormone receptor βPV/PV knock-in (PV) mice that develop metastatic thyroid cancer that most closely resembles FTC. To determine the roles of Akt isoforms in this model we crossed Akt1−/−, Akt2−/−, and Akt3−/− mice with PV mice. Over 12 months, thyroid size was reduced for the Akt null crosses (p < 0.001). Thyroid cancer development and local invasion were delayed in only the PVPV-Akt1 knock out (KO) mice in association with increased apoptosis with no change in proliferation. Primary-cultured PVPV-Akt1KO thyrocytes uniquely displayed a reduced cell motility. In contrast, loss of any Akt isoform reduced lung metastasis while vascular invasion was reduced with Akt1 or 3 loss. Microarray of thyroid RNA displayed incomplete overlap between the Akt KO models. The most upregulated gene was the dendritic cell (DC) marker CD209a only in PVPV-Akt1KO thyroids. Immunohistochemistry demonstrated an increase in CD209a-expressing cells in the PVPV-Akt1KO thyroids. In summary, Akt isoforms exhibit common and differential functions that regulate local and metastatic progression in this model of thyroid cancer.

Estrogens Determine Adherens Junction Organization and E-Cadherin Clustering in Breast Cancer Cells via Amphiregulin.

SummaryEstrogens play an important role in the development and progression of human cancers, particularly in breast cancer. Breast cancer progression depends on the malignant destabilization of adherens junctions (AJs) and disruption of tissue integrity. We found that estrogen receptor alpha (ERα) inhibition led to a striking spatial reorganization of AJs and microclustering of E-Cadherin (E-Cad) in the cell membrane of breast cancer cells. This resulted in increased stability of AJs and cell stiffness and a reduction of cell motility. These effects were actomyosin-dependent and reversible by estrogens. Detailed investigations showed that the ERα target gene and epidermal growth factor receptor (EGFR) ligand Amphiregulin (AREG) essentially regulates AJ reorganization and E-Cad microclustering. Our results not only describe a biological mechanism for the organization of AJs and the modulation of mechanical properties of cells but also provide a new perspective on how estrogens and anti-estrogens might influence the formation of breast tumors.

Suppression of pancreatic cancer liver metastasis by secretion-deficient ITIH5

BackgroundPreviously, we identified ITIH5 as a suppressor of pancreatic ductal adenocarcinoma (PDAC) metastasis in experimental models. Expression of ITIH5 correlated with decreased cell motility, invasion and metastasis without significant inhibition of primary tumour growth. Here, we tested whether secretion of ITIH5 is required to suppress liver metastasis and sought to understand the role of ITIH5 in human PDAC.MethodsWe expressed mutant ITIH5 with deletion of the N-terminal secretion sequence (ITIH5Δs) in highly metastatic human PDAC cell lines. We used a human tissue microarray (TMA) to compare ITIH5 levels in uninvolved pancreas, primary and metastatic PDAC.ResultsSecretion-deficient ITIH5Δs was sufficient to suppress liver metastasis. Similar to secreted ITIH5, expression of ITIH5Δs was associated with rounded cell morphology, reduced cell motility and reduction of liver metastasis. Expression of ITIH5 is low in both human primary PDAC and matched metastases.ConclusionsMetastasis suppression by ITIH5 may be mediated by an intracellular mechanism. In human PDAC, loss of ITIH5 may be an early event and ITIH5-low PDAC cells in primary tumours may be selected for liver metastasis. Further defining the ITIH5-mediated pathway in PDAC could establish future therapeutic exploitation of this biology and reduce morbidity and mortality associated with PDAC metastasis.