Downregulation Of CD44 Research Articles

Auraptene Attenuates Malignant Properties of Esophageal Stem-Like Cancer Cells.

The high incidence of esophageal squamous cell carcinoma has been reported in selected ethnic populations including North of Iran. Low survival rate of esophageal carcinoma is partially due to the presence of stem-like cancer cells with chemotherapy resistance. In the current study, we aimed to determine the effects of auraptene, an interesting dietary coumarin with various biological activities, on malignant properties of stem-like esophageal squamous cell carcinoma, in terms of sensitivity to anticancer drugs and expression of specific markers. To do so, the half maximal inhibitory concentration values of auraptene, cisplatin, paclitaxel, and 5-fluorouracil were determined on esophageal carcinoma cells (KYSE30 cell line). After administrating combinatorial treatments, including nontoxic concentrations of auraptene + cisplatin, paclitaxel, or 5-fluorouracil, sensitivity of cells to chemical drugs and also induced apoptosis were assessed. In addition, quantitative real-time polymerase chain reaction was used to study changes in the expression of tumor suppressor proteins 53 and 21 ( P53 and P21), cluster of differentiation 44 ( CD44), and B cell-specific Moloney murine leukemia virus integration site 1 ( BMI-1) upon treatments. Results of thiazolyl blue assay revealed that auraptene significantly ( P < .05) increased toxicity of cisplatin, paclitaxel, and 5-fluorouracil in KYSE30 cells, specifically 72 hours after treatment. Conducting an apoptosis assay using flow cytometry also confirmed the synergic effects of auraptene. Results of quantitative real-time polymerase chain reaction revealed significant ( P < .05) upregulation of P53 and P21 upon combinatorial treatments and also downregulation of CD44 and BMI-1 after auraptene administration. Current study provided evidence, for the first time, that auraptene attenuates the properties of esophageal stem-like cancer cells through enhancing sensitivity to chemical agents and reducing the expression of CD44 and BMI-1 markers.

Fibroblastic reticular cells of the secondary lymphoid organ suppress T cell activation via COX2 mediated PGE2 secretion.

Abstract Accumulating evidence demonstrates that fibroblastic reticular cells (FRCs) in the T cell zone of the secondary lymphoid organs (SLO), characterized as CD45− gp38+CD31− cells, play an important role in maintaining T cell tolerance via presenting peripheral tissue–restricted antigens (PTAs) and/or interferon-induced nitric oxide during productive immunity. Here, using FRCs harvested from naïve mice, we revealed that FRCs also inhibit T cell activation via a soluble factor, independent of the aforementioned mechanisms. Specifically, FRCs suppressed CD3/CD28-ligation mediated or antigen induced antigen-specific T cell activation. This FRC-imposed suppression was demonstrated as (1) reduced Zap-70 phosphorylation and Calcium-flux within 30 minutes of TCR-ligation, (2) inhibited upregulation of surface CD69 and CD44 expression and downregulation of CD62L 6–14 hours post-activation, and (3) suppressed T cell effector cytokine production and proliferation. Further studies demonstrated that our observed inhibition of T cell activation by FRC is at least partially contributed by cyclooxygenase-2 (COX-2) mediated production of prostaglandin E2 (PGE2) because COX-2 inhibitor partially reversed FRC mediated inhibition. ELISA results confirmed high production level of PGE2 by FRC (~ 10 – 50ng/ml), at which level T cell activation is greatly suppressed. Taken together, our study illustrated another previously unrecognized mechanism of FRCs in maintaining T cell tolerance, which underscores the crucial role of FRCs in peripheral tolerance and immune regulation. The potential molecular mechanism by which the COX2-PGE2 pathway is activated in FRCs under physiological condition will be discussed.

Clinical, Pathological, and Molecular Features of Lung Adenocarcinomas with AXL Expression.

The receptor tyrosine kinase AXL is a member of the Tyro3-Axl-Mer receptor tyrosine kinase subfamily. AXL affects several cellular functions, including growth and migration. AXL aberration is reportedly a marker for poor prognosis and treatment resistance in various cancers. In this study, we analyzed clinical, pathological, and molecular features of AXL expression in lung adenocarcinomas (LADs). We examined 161 LAD specimens from patients who underwent pulmonary resections. When AXL protein expression was quantified (0, 1+, 2+, 3+) according to immunohistochemical staining intensity, results were 0: 35%; 1+: 20%; 2+: 37%; and 3+: 7% for the 161 samples. AXL expression status did not correlate with clinical features, including smoking status and pathological stage. However, patients whose specimens showed strong AXL expression (3+) had markedly poorer prognoses than other groups (P = 0.0033). Strong AXL expression was also significantly associated with downregulation of E-cadherin (P = 0.025) and CD44 (P = 0.0010). In addition, 9 of 12 specimens with strong AXL expression had driver gene mutations (6 with EGFR, 2 with KRAS, 1 with ALK). In conclusion, we found that strong AXL expression in surgically resected LADs was a predictor of poor prognosis. LADs with strong AXL expression were characterized by mesenchymal status, higher expression of stem-cell-like markers, and frequent driver gene mutations.

Regulation of a TGF-β1-CD147 self-sustaining network in the differentiation plasticity of hepatocellular carcinoma cells.

Cellular plasticity has an important role in the progression of hepatocellular carcinoma (HCC). In this study, the involvement of a TGF-β1-CD147 self-sustaining network in the regulation of the dedifferentiation progress was fully explored in HCC cell lines, hepatocyte-specific basigin/CD147-knockout mice and human HCC tissues. We demonstrated that TGF-β1 stimulation upregulated CD147 expression and mediated the dedifferentiation of HCC cells, whereas all-trans-retinoic acid induced the downregulation of CD147 and promoted differentiation in HCC cells. Overexpression of CD147 induced the dedifferentiation and enhanced the malignancy of HCC cells, and increased the transcriptional expression of TGF-β1 by activating β-catenin. CD147-induced matrix metalloproteinase (MMP) production activated pro-TGF-β1. The activated TGF-β1 signaling subsequently repressed the HNF4α expression via Smad-Snail1 signaling and enhanced the dedifferentiation progress. Hepatocyte-specific basigin/CD147-knockout mice decreased the susceptibility to N-nitrosodiethylamine-induced tumorigenesis by suppressing TGF-β1-CD147 signaling and inhibiting dedifferentiation in hepatocytes during tumor progression. CD147 was positively correlated with TGF-β1 and negatively correlated with HNF4α in human HCC tissues. Positive CD147 staining and lower HNF4α levels in tumor tissues were significantly associated with poor survival of patients with HCC. The overexpression of HNF4α and Smad7 and the deletion of CD147 by lentiviral vectors jointly reprogrammed the expression profile of hepatocyte markers and attenuated malignant properties including proliferation, cell survival and tumor growth of HCC cells. Our results highlight the important role of the TGF-β1-CD147 self-sustaining network in driving HCC development by regulating differentiation plasticity, which provides a strong basis for further investigations of the differentiation therapy of HCC targeting TGF-β1 and CD147.

Decreased FOXF1 promotes hepatocellular carcinoma tumorigenesis, invasion, and stemness and is associated with poor clinical outcome.

Forkhead box F1 (FOXF1), a member of the forkhead transcription factor superfamily, plays critical roles in the progression of certain types of cancers. However, the expression and function of FOXF1 in human hepatocellular carcinoma (HCC) are still unclear. Quantitative real-time reverse transcription polymerase chain reaction, Western blotting, and immunohistochemistry detected the relatively lower expression status of FOXF1 in HCC cases. Soft agar and transwell assays clearly demonstrated that FOXF1-knockdown cells showed significantly increased in vitro cell tumorigenesis and invasion, and FOXF1-overexpressing cells had significantly reduced growth and invasion potential. Our study also examined the role of FOXF1 in HCC cell stemness by sphere formation, aldehyde dehydrogenase (ALDH1) activity, and CD44/133-positive cell analysis. Enforced FOXF1 expression decreased HCC cell stemness, and the downregulation of FOXF1 promoted cancer cell stemness. The in vivo study showed that overexpressed FOXF1 inhibits nude mouse tumorigenicity with downregulation of CD44 and proliferating cell nuclear antigen. More importantly, loss of FOXF1 expression was linked to poor overall survival time by Kaplan–Meier analysis.

CD133 promotes gallbladder carcinoma cell migration through activating Akt phosphorylation.

Gallbladder carcinoma (GBC) is the fifth most common malignancy of gastrointestinal tract. The prognosis of gallbladder carcinoma is extremely terrible partially due to metastasis. However, the mechanisms underlying gallbladder carcinoma metastasis remain largely unknown. CD133 is a widely used cancer stem cell marker including in gallbladder carcinoma. Here, we found that CD133 was highly expressed in gallbladder carcinoma as compared to normal tissues. CD133 was located in the invasive areas in gallbladder carcinoma. Down-regulation expression of CD133 inhibited migration and invasion of gallbladder carcinoma cell without obviously reducing cell proliferation. Mechanism analysis revealed that down-regulation expression of CD133 inhibited Akt phosphorylation and increased PTEN protein level. The inhibitory effect of CD133 down-regulation on gallbladder carcinoma cell migration could be rescued by Akt activation. Consistent with this, addition of Akt inhibitor Wortmannin markedly inhibited the migration ability of CD133-overexpressing cells. Thus, down-regulation of CD133 inhibits migration of gallbladder carcinoma cells through reducing Akt phosphorylation. These findings explore the fundamental biological aspect of CD133 in gallbladder carcinoma progression, providing insights into gallbladder carcinoma cell migration.

Abstract P3-06-05: Importance of TGFβ-SMAD3 axis in resistance to anti-HER2 drugs

Abstract Aim: The aim of this study was to elucidate the role of transforming growth factor β (TGFβ) in the resistance of HER2-positive breast cancer cells to anti-HER2 drugs including trastuzumab and lapatinib. Methods: A HER2-positive breast cancer cell line, SKBR3, was cultured in the presence or absence of TGFβ for 14 days. Subsequently, TGFβ-treated cells were cultured for seven days with or without the anti-HER2 drugs. Sensitivity to trastuzumab and lapatinib was estimated by the WST-8 cell viability assay or absolute cell counts using In Cell Analyzer (GE Healthcare). Proportion of CD44+ CD24– breast cancer stem cells was estimated by flow cytometry of cells immunostained with anti-CD44 and anti-CD24 antibodies. For clinical study, 33 patients with HER2-positive breast cancer receiving neoadjuvant paclitaxel plus trastuzumab in our institution were analyzed. Among the cases, 27 biopsy samples obtained before any treatment from 27 patients who completed 12 cycles of weekly paclitaxel and trastuzumab were subjected to CD24 immunohistochemistry. Results: SKBR3 cells cultured with TGFβ for 14 days exhibited decreased sensitivity to both trastuzumab and lapatinib. Time course study revealed that continuous stimulation for 14 days with TGFβ was required for the resistance to anti-HER2 drugs. Activation of SMAD3, a downstream target molecule of TGFβ, was enhanced over time, judged by the increase in phosphorylation and in nuclear translocation. During 14 day culture with TGFβ, proportion of CD44+ CD24– cells were dramatically increased, and mammosphere formation, another marker of breast cancer stem cells, was significantly enhanced compared to cells treated without TGFβ. Among four HER2-positive breast cancer cell lines, only SKBR3 cells showed increased proportion of CD44+ CD24– cells and resistance to the anti-HER2 drugs, while other two cell lines exhibited epithelial-mesenchymal transition (EMT) in response to TGFβ. To explore the possibility of targeting TGFβ-SMAD3 axis to overcome resistance to anti-HER2 therapy, we used SIS3, a specific inhibitor of SMAD3. Importantly, SIS3 completely restored the sensitivity to both trastuzumab and lapatinib of TGFβ-treated SKBR3 cells, with the decrease in the proportion of CD44+ CD24– cells. These in vitro results suggest that CD24 downregulation can be a surrogate marker of resistance to anti-HER2 therapy. To establish this, we evaluated the CD24 expression in tumor samples of breast cancer patients who received paclitaxel plus trastuzumab in the neoadjuvant setting. Weak CD24 expression in tumor cells in biopsy samples obtained before any treatment was significantly correlated with poorer response to the drugs. Conclusion: These data clearly indicate the importance of TGFβ-SMAD3 axis in the acquired resistance to anti-HER2 drugs. Moreover, resistance to anti-HER2 therapy is associated with the property of breast cancer stem cells rather than EMT. Targeting TGFβ-SMAD3 axis warrants further investigation for overcoming resistance to anti-HER2 therapeutics. Citation Format: Shimoda M, Chihara Y, Kagara N, Naoi Y, Shimomura A, Shimazu K, Kim SJ, Noguchi S. Importance of TGFβ-SMAD3 axis in resistance to anti-HER2 drugs. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-06-05.

MiR-373 Inhibits Glioma Cell U251 Migration and Invasion by Down-Regulating CD44 and TGFBR2.

Glioblastoma multiforme (GBM) is the most malignant glioma, unveiling the underlying mechanisms of its aggressiveness could promote the discovery of potential targets for effective treatment. MicroRNAs (miRNAs) are important participants in both development and disease, its involvement in cancers has long been recognized. In this study, we investigated the role of miRNA-373 (miR-373) in GBM cell line U251, demonstrated that although miR-373 does not affect cell growth of U251, it inhibits migration and invasion of U251. Forced expression of miR-373 down-regulates the expressions CD44 and TGFBR2, while knockdown of CD44 and TGFBR2 presents the similar phenotype as miR-373 overexpression, suggesting that CD44 and TGFBR2 are functional targets of miR-373, down-regulation of CD44 and TGFBR2 by miR-373 are partly responsible for the migration, and invasion suppressive role of miR-373 in U251.

CD24 Induces the Activation of β-Catenin in Intestinal Tumorigenesis

Background: CD24 is a glycosylphosphatidylinositol-linked protein that functions as an adhesion molecule and is overexpressed at an early stage of CRC. The Wnt/β-catenin signaling pathway plays an important role in the CRC carcinogenesis process. We have previously shown that CD24 could affect the tumorigenesis process in ApcMin/+ mice. Methods: CD24-inducible 293T-RExTM cells previously developed in our lab, HT29 and SW480 CRC cells were used to study this interaction in vitro. ApcMin/+ and CD24 knockout (KO) mice, both on a C57BL/6J genetic background, were crossed to generate double KO transgenic mice. Large bowel polyps were counted macroscopically and small bowel polyps were verified microscopically. Results: In vitro Western blotting analyses showed that induced expression of CD24 led to the activation of β-catenin. Co-immunoprecipitation studies of CD24 and β-catenin indicated that these two proteins might be interacting. Cytoplasmic/nuclear fractionation showed that more active β-catenin enters the nucleus in cells that express CD24 compared to control cells. In addition, TOP/FOP luciferase reporter assay showed a significant increase in luciferase activity upon CD24 expression induction.Conversely, down-regulation of CD24 by mAbs and siRNA caused a decrease in the levels of active β-catenin. Depletion of CD24 alleles in ApcMin/+ mice led to a significant reduction in the number of polyps in the small and large intestine. The ApcMin/+mice displayed severe splenomegaly compared to ApcMin/+/CD24+/- mice and double KO mice were similar to WT mice with normal spleen size. HGB and RBC levels were significantly lower than in the double KO mice. Conclusions: CD24 plays a major role in intestinal tumorigenesis. It interacts with the Wnt pathway by activating β-catenin. Down regulation of CD24 reduces the polyp burden in mice and therefore it might serve as an important target in the therapy of CRC.

Baicalein inhibits hepatocellular carcinoma cells through suppressing the expression of CD24

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death and is the most common type of liver cancer. Current therapies for hepatocellular carcinoma are still rather limited and novel therapeutic strategies are required. Baicalein, extracted from Scutellaria baicalensis, has anticancer effects on HCC in vitro and vivo. However, the detailed mechanisms are not well studied yet. In the present study, we evaluated anticancer effects of purified botanical extracts on HCC cells using high-throughput screening and investigated the effects of baicalein on HCC cells using proliferation and apoptosis assays, RT-PCR, and Western blot. Transfection was used to explore the underlying mechanisms of these effects. Our results showed that baicalein is the most efficient botanical extract in a HCC cell line as compared with the other 13 extracts. Baicalein significantly decreased the expression of c-Myc, a crucial regulator of cell proliferation, apoptosis and cellular transformation, in dose- and time-dependent manners in HCC cells. Moreover, baicalein inhibited HCC cell proliferation and induced apoptosis. The mRNA and protein expressions of CD24 were downregulated by baicalein in HCC cells and ectopic overexpression of CD24 reversed baicalein-induced inhibition of cell proliferation and survival. Taken together, our results demonstrate efficient anticancer effects of baicalein on HCC cells and indicate that baicalein suppresses cell growth and cell survival through downregulation of CD24.

EtpE Binding to DNase X Induces Ehrlichial Entry via CD147 and hnRNP-K Recruitment, Followed by Mobilization of N-WASP and Actin

ABSTRACTObligate intracellular bacteria, such as Ehrlichia chaffeensis, perish unless they can enter eukaryotic cells. E. chaffeensis is the etiological agent of human monocytic ehrlichiosis, an emerging infectious disease. To infect cells, Ehrlichia uses the C terminus of the outer membrane invasin entry-triggering protein (EtpE) of Ehrlichia (EtpE-C), which directly binds the mammalian cell surface glycosylphosphatidyl inositol-anchored protein, DNase X. How this binding drives Ehrlichia entry is unknown. Here, using affinity pulldown of host cell lysates with recombinant EtpE-C (rEtpE-C), we identified two new human proteins that interact with EtpE-C: CD147 and heterogeneous nuclear ribonucleoprotein K (hnRNP-K). The interaction of CD147 with rEtpE-C was validated by far-Western blotting and coimmunoprecipitation of native EtpE with endogenous CD147. CD147 was ubiquitous on the cell surface and also present around foci of rEtpE-C-coated-bead entry. Functional neutralization of surface-exposed CD147 with a specific antibody inhibited Ehrlichia internalization and infection but not binding. Downregulation of CD147 by short hairpin RNA (shRNA) impaired E. chaffeensis infection. Functional ablation of cytoplasmic hnRNP-K by a nanoscale intracellular antibody markedly attenuated bacterial entry and infection but not binding. EtpE-C also interacted with neuronal Wiskott-Aldrich syndrome protein (N-WASP), which is activated by hnRNP-K. Wiskostatin, which inhibits N-WASP activation, and cytochalasin D, which inhibits actin polymerization, inhibited Ehrlichia entry. Upon incubation with host cell lysate, EtpE-C but not an EtpE N-terminal fragment stimulated in vitro actin polymerization in an N-WASP- and DNase X-dependent manner. Time-lapse video images revealed N-WASP recruitment at EtpE-C-coated bead entry foci. Thus, EtpE-C binding to DNase X drives Ehrlichia entry by engaging CD147 and hnRNP-K and activating N-WASP-dependent actin polymerization.

Immunohistochemical expression of cancer stem cell related markers CD44 and CD133 in endometrial cancer

The goal of this study was to detect the presence of cancer stem cell markers CD44 and CD133 in immunohistochemically stained samples of endometrial cancer and correlate their expression with clinicopathological variables to identify the impact of CD44 or CD133 expression on tumor behavior and endometrial carcinogenesis. Marker expression was analyzed in 62 endometrial cancer samples (57 endometrioid carcinoma and 5 carcinosarcoma) and 15 proliferative endometrium samples. We detected CD133 and CD44 expression in 87.09% and 79.03% respectively of the studied endometrial cancers, and the expression was significantly different from the normal group. CD44 expression decreased with myometrial invasive depth and lymph-vascular space invasion (LVSI), and these inverse relationships were significant (p=0.034, p=0.019, respectively). CD133 was more expressed by early stage tumor (FIGO I-II) compared with those having FIGO III to IV stage disease (p=0.021). The most notable conclusion of the present study is that CD44 and CD133 might participate in early-stage endometrial cancer carcinogenesis, and their overexpression may facilitate the early diagnosis of endometrial cancers. Analysis of our results supports the hypothesis that CD44 expression tends to decrease as the disease becomes invasive and progressive. So, we concluded that CD44 down-regulation might warn of a more aggressive course and may have a link with poorly prognosis carcinosarcomas. Further examination of the expression and function of CD44 and CD133 with a greater number of carcinosarcomas is warranted.

CD147 is increased in HCC cells under starvation and reduces cell death through upregulating p-mTOR in vitro.

Transarterial chemoembolization (TACE) is the standard of care for treatment of intermediate hepatocellular carcinoma (HCC), however, key molecules involved in HCC cell survival and tumor metastasis post-TACE remain unclear. CD147 is a member of the immunoglobulin superfamily that is overexpressed on the surface of HCC cells and is associated with malignant potential and poor prognosis in HCC patients. In this study, using an Earle's Balanced Salt Solution medium culture model that mimics nutrient deprivation induced by TACE, we investigated the regulation of CD147 expression on HCC cells under starvation conditions and its functional effects on HCC cell death. During early stages of starvation, the expression of CD147 was considerably upregulated in SMMC7721, HepG2 and HCC9204 hepatoma cell lines at the protein levels. Downregulation of CD147 by specific small interfering RNA (siRNA) significantly promoted starvation-induced cell death. In addition, CD147 siRNA-transfected SMMC7721 cells demonstrated significantly increased levels of both apoptosis and autophagy as compared to cells transfected with control siRNA under starvation conditions, whereas no difference was observed between the two treatment groups under normal culture conditions. Furthermore, silencing of CD147 resulted in a remarkable downregulation of phosphorylated mammalian target of rapamycin (p-mTOR) in starved SMMC7721 cells. Finally, the combined treatment of starvation and anti-CD147 monoclonal antibody exhibited a synergistic HCC cell killing effect. Our study suggests that upregulation of CD147 under starvation may reduce hepatoma cell death by modulating both apoptosis and autophagy through mTOR signaling, and that CD147 may be a novel potential molecular target to improve the efficacy of TACE.

Proteoglycan expression correlates with the phenotype of malignant and non-malignant EBV-positive B-cell lines.

The involvement of proteoglycans (PGs) in EBV-host interactions and lymphomagenesis remains poorly investigated. In this study, expression of major proteoglycans (syndecan-1, glypican-1, perlecan, versican, brevican, aggrecan, NG2, serglycin, decorin, biglycan, lumican, CD44), heparan sulphate (HS) metabolic system (EXT1/2, NDST1/2, GLCE, HS2ST1, HS3ST1/2, HS6ST1/2, SULF1/2, HPSE) and extracellular matrix (ECM) components (collagen 1A1, fibronectin, elastin) in primary B cells and EBV carrying cell lines with different phenotypes, patterns of EBV-host cell interaction and viral latency stages (type I-III) was investigated. Primary B cells expressed a wide repertoire of PGs (dominated by serglycin and CD44) and ECM components. Lymphoblastoid EBV+ B cell lines (LCLs) showed specific PG expression with down-regulation of CD44 and ECM components and up-regulation of serglycin and perlecan/HSPG2. For Burkitt's lymphoma cells (BL), serglycin was down-regulated in BL type III cells and perlecan in type I BL cells. The biosynthetic machinery for HS was active in all cell lines, with some tendency to be down-regulated in BL cells. 5'-aza-dC and/or Trichostatin A resulted in transcriptional upregulation of the genes, suggesting that low expression of ECM components, proteoglycan core proteins and HS biosynthetic system is due to epigenetic suppression in type I cells. Taken together, our data show that proteoglycans are expressed in primary B lymphocytes whereas they are not or only partly expressed in EBV-carrying cell lines, depending on their latency type program.

Impact of siRNA-mediated down-regulation of CD147 on human breast cancer cells

To investigate the influence of siRNA-mediated down-regulation of CD147 on growth, proliferation and movement of human breast cancer cell line MDA-MB-231. The protein expression of CD147, MMP-2 and TIMP-2 of the MDA-MB-231 cells were analyzed by ABC. Lentiviral expression vector of CD147 gene was constructed and transfected into MDA-MB-231 cells. RT-PCR and Western blot were used to detect the mRNA and protein level changes of CD147 genes to identify the optimal time point, followed by detection of changes of mRNA and protein expression of MMP-2 and TIMP-2 genes. CCK-8 reagent method and cell scratch test were used to detect the proliferation and migration change of MDA-MB-231 cells. The nude mouse model of breast cancer by hypodermic injection with MDA-MB-231 cells was established to document the effect of CD147 siRNA on the tumor transplants. After transfection of lentiviral expression vector of CD147 gene, protein of CD147, MMP-2 and TIMP-2 were weakly or negative expressed, significantly weaker than those of control group (P < 0.01). After 72 hours of transfection, average down-regulation rate of CD147 and MMP-2 were 96.03% ± 0.84% and 96.03% ± 0.84%, respectively. Both CD147 mRNA and MMP-2 mRNA expression were down-regulated (P < 0.05), while TIMP-2 mRNA expression showed no significant deference (P > 0.05). No less than 2 days after transfection, cell growth of MDA-MB-231 cell line was found significantly inhibited (P < 0.05). After 24 hours of transection, average migration distance of MDA-MB-231 cell line and control group were (0.64 ± 0.12) mm and (4.69 ± 0.85) mm, respectively, which indicated a lower migrate speed. Down regulation of CD147 led to reduction of volume and mass of nude mouses. The growth of the carcinoma transplant was inhibited upon siRNA-mediated down-regulation of CD147 (P < 0.05), with an average tumor mass of (1.85 ± 0.98) g and both reduction of tumor size and tumor mass. CD147 may alter the MMP-2/TIMP-2 balance in MDA-MB-231 cells. CD147 gene silencing inhibits the proliferation and migration of MDA-MB-231 cells and the growth of carcinoma transplants in nude mice.

HDAC inhibitor AR-42 decreases CD44 expression and sensitizes myeloma cells to lenalidomide.

Multiple myeloma (MM) is a hematological malignancy of plasma cells in the bone marrow. Despite multiple treatment options, MM is inevitably associated with drug resistance and poor outcomes. Histone deacetylase inhibitors (HDACi's) are promising novel chemotherapeutics undergoing evaluation in clinical trials for the potential treatment of patients with MM. Although in preclinical studies HDACi's have proven anti-myeloma activity, but in the clinic single-agent HDACi treatments have been limited due to low tolerability. Improved clinical outcomes were reported only when HDACi's were combined with other drugs. Here, we show that a novel pan-HDACi AR-42 downregulates CD44, a glycoprotein that has been associated with lenalidomide and dexamethasone resistance in myeloma both in vitro and in vivo. We also show that this CD44 downregulation is in part mediated by miR-9-5p, targeting insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), which directly binds to CD44 mRNA and increases its stability. Importantly, we also demonstrate that AR-42 enhances anti-myeloma activity of lenalidomide in primary MM cells isolated from lenalidomide resistant patients and in in vivo MM mouse model. Thus, our findings shed light on potential novel combinatorial therapeutic approaches modulating CD44 expression, which may help overcome lenalidomide resistance in myeloma patients.

The metastasis suppressor, NDRG1, inhibits "stemness" of colorectal cancer via down-regulation of nuclear β-catenin and CD44.

N-myc downstream-regulated gene 1 (NDRG1), has been identified as an important metastasis suppressor for colorectal cancer (CRC). In this study, we investigated: (1) the effects of NDRG1 on CRC stemness and tumorigenesis; (2) the molecular mechanisms involved; and (3) the relationship between NDRG1 expression and colorectal cancer prognosis. Our investigation demonstrated that CRC cells with silenced NDRG1 showed more tumorigenic ability and stem cell-like properties, such as: colony and sphere formation, chemoresistance, cell invasion, high expression of CD44, and tumorigenicity in vivo. Moreover, NDRG1 silencing reduced β-catenin expression on the cell membrane, while increasing its nuclear expression. The anti-tumor activity of NDRG1 was demonstrated to be mediated by preventing β-catenin nuclear translocation, as silencing of this latter molecule could reverse the effects of silencing NDRG1 expression. NDRG1 expression was also demonstrated to be negatively correlated to CRC prognosis. In addition, there was a negative correlation between NDRG1 and nuclear β-catenin and also NDRG1 and CD44 expression in clinical CRC specimens. Taken together, our investigation demonstrates that the anti-metastatic activity of NDRG1 in CRC occurs through the down-regulation of nuclear β-catenin and suggests that NDRG1 is a significant therapeutic target.

Ultrasound-targeted microbubble destruction-mediated downregulation of CD133 inhibits epithelial-mesenchymal transition, stemness and migratory ability of liver cancer stem cells.

Hepatocellular carcinoma (HCC) is an aggressive disease with a poor outcome due to the high incidence of metastasis. Cancer stem cells (CSCs) have been identified to be responsible for tumor progression and may be generated by epithelial-mesenchymal transition (EMT) characteristics. CD133 is a specific surface marker for liver cancer stem cells (LCSCs), which is also considered as an important functional factor for tumorigenesis and overall survival in HCC. Ultrasound-targeted microbubble destruction (UTMD) has recently been used as a novel, safe and effective gene transfection technology. The aim of the present study was to elucidate the regulatory mechanism of CD133 and EMT in LCSCs and whether the UTMD-based shRNA delivery system facilitated gene delivery in LCSCs. In the present study, CD133+ cells were isolated from the SMMC-7721 HCC cell line and then transfected with shCD133 mediated by UTMD and liposomes, respectively. Compared to the liposomes group, the UTMD group resulted in significantly improved transfection efficiency. The downregulation of CD133 reversed the EMT program, attenuated self-renewal, proliferation and migration of CD133+ LCSCs and suppressed the growth of CSC tumor xenografts. Additionally, the downregulation of CD133 led to downregulation of the nuclear factor-κB (NF-κB) pathway. The present study demonstrated that CD133 plays a critical role in the regulation of the EMT process, tumor-initiating properties and migratory ability of LCSCs. The UTMD technique targeted for CD133 downregulation may be examined as a potential therapeutic strategy for HCC.

Bidirectional crosstalk between PD-L1 expression and epithelial to mesenchymal transition: significance in claudin-low breast cancer cells.

BackgroundThe T-cell inhibitory molecule PD-L1 (B7-H1, CD274) is expressed on tumor cells of a subset of breast cancer patients. However, the mechanism that regulates PD-L1 expression in this group of patients is still not well-identified.MethodsWe have used loss and gain of function gene manipulation approach, multi-parametric flow cytometry, large scale gene expression dataset analysis and immunohistochemistry of breast cancer tissue sections.ResultsInduction of epithelial to mesenchymal transition (EMT) in human mammary epithelial cells upregulated PD-L1 expression, which was dependent mainly on the activation of the PI3K/AKT pathway. Interestingly, gene expression signatures available from large cohort of breast tumors showed a significant correlation between EMT score and the PD-L1 mRNA level (p < 0.001). Strikingly, very strong association (p < 0.0001) was found between PD-L1 expression and claudin-low subset of breast cancer, which is known to have high EMT score. On the protein level, significant correlation was found between PD-L1 expression and standard markers of EMT (p = 0.005) in 67 breast cancer patients. Importantly, specific downregulation of PD-L1 in claudin-low breast cancer cells showed signs of EMT reversal as manifested by CD44 and Vimentin downregulation and CD24 upregulation.ConclusionsWe have demonstrated a bidirectional effect between EMT status and PD-L1 expression especially in claudin-low subtype of breast cancer cells. Our findings highlights the potential dual benefit of anti-PD-L1 particularly in this subset of breast cancer patients that will likely benefit more from anti-PD-L1 targeted therapy as well as in monitoring biological changes upon treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0421-2) contains supplementary material, which is available to authorized users.

Abstract 2647: Anti-tumor activity of sHA8k, a HYAL1 hyaluronidase inhibitor, in bladder cancer cells

Abstract INTRODUCTION AND OBJECTIVE: Tumor cell-derived hyaluronidase HYAL1, which degrades hyaluronic acid (HA) into angiogenic fragments, promotes tumor growth and metastasis. HYAL-1 is an independent prognostic marker for predicting metastasis. Small molecular mass sulfated hyaluronic acid (sHA) derivatives (sHA8k) inhibit HYAL1 activity through a mixed inhibition mechanism. Antitumor activity of sHA8k and mechanism of action was evaluated in BCa models.. METHODS: Effect of sHA8k (0-40 ug/ml) on cell proliferation and apoptosis was examined in BCa cells (253J-Lung, HT1376, UMUC-3, T24, RT4) by cell counting and Cell Death ELISA kit. Matrigel invasion and Boyden chamber assays were used to test the effect of sHA8k on invasive activity. Effect of sHA on signaling, apoptosis cascade, HA receptor (CD44, RHAMM), EMT markers (β-catenin, E-cadherin, Snail, Twist) levels, was evaluated by Q-pCR and immunoblotting assays. Angiogenic fragment addition and mAkt transfection were performed to elucidate mechanism of action. Athymic mice bearing 253J-Lung xenografts were treated with sHA (25 and 50 mg/kg) by i.p. injection. RESULTS: sHA8k inhibited proliferation, motility and invasion in BCa cells that expressed HYAL-1. At IC50 for HAase activity inhibition (∼ 20-μg/ml), sHA induced &amp;gt; 3-fold apoptosis and inhibited invasive activity of BCa cells. sHA8k induced caspase-3, -8, -9 activation, up-regulation of Fas, Fas-L, FADD, DR4, DR5 and E-cadherin. sHA8k downregulated CD44, RHAMM, bcl-2, phospho(p)-Akt, pGSK3β, pβcatenin(ser552), snail and Twist expression. Effect of sHA8k were attenuated by angiogenic HA fragments or overexpression of m-Akt and downregulation of CD44 and RHAMM mimicked sHA8k effects. sHA significantly inhibited 253J-Lung xenograft growth. The majority of the animals did not form palpable tumors at 50-mg/kg dose. No weight loss or serum and organ toxicity was observed in sHA treated animals. Biochemical analysis of tumors also showed the same alterations in Akt and EMT pathways as observed cell culture. CONCLUSION: This is the first study that shows sHA8k, a small molecular mass HYAL-1 inhibitor has potent antitumor activity. Support: R01 CA 72821-14 (VBL) Citation Format: Andre R. Jordan, Juan Chipollini, Luis Lopez, Travis Yates, Vinata Lokeshwar. Anti-tumor activity of sHA8k, a HYAL1 hyaluronidase inhibitor, in bladder cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2647. doi:10.1158/1538-7445.AM2015-2647