Acquisition Of Metastatic Potential Research Articles

The Comparison of Serum Exosome Protein Profile in Diagnosis of NSCLC Patients.

A thorough study of the exosomal proteomic cargo may enable the identification of proteins that play an important role in cancer development. The aim of this study was to compare the protein profiles of the serum exosomes derived from non-small lung cancer (NSCLC) patients and healthy volunteers (control) using the high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS) method to identify potentially new diagnostic and/or prognostic protein biomarkers. Proteins exclusively identified in NSCLC and control groups were analyzed using several bioinformatic tools and platforms (FunRich, Vesiclepedia, STRING, and TIMER2.0) to find key protein hubs involved in NSCLC progression and the acquisition of metastatic potential. This analysis revealed 150 NSCLC proteins, which are significantly involved in osmoregulation, cell-cell adhesion, cell motility, and differentiation. Among them, 3 proteins: Interleukin-34 (IL-34), HLA class II histocompatibility antigen, DM alpha chain (HLA-DMA), and HLA class II histocompatibility antigen, DO beta chain (HLA-DOB) were shown to be significantly involved in the cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) infiltration processes. Additionally, detected proteins were analyzed according to the presence of lymph node metastasis, showing that differences in frequency of detection of protein FAM166B, killer cell immunoglobulin-like receptor 2DL1, and olfactory receptor 52R1 correlate with the N feature according to the TNM Classification of Malignant Tumors. These results prove their involvement in NSCLC lymph node spread and metastasis. However, this study requires further investigation.

Single-cell RNA sequencing reveals a pro-metastatic subpopulation and the driver transcription factor NFE2L1 in ovarian cancer cells.

Although cytoreductive surgery followed by adjuvant chemotherapy is effective as a standard treatment for early-stage ovarian cancer, the majority of ovarian cancer cases are diagnosed at the advanced stages with dissemination to the peritoneal cavity, leading to a poor prognosis. Therefore, it is crucial to understand the cellular and molecular mechanisms underlying metastasis and identify novel therapeutic targets. In this study, we aimed to elucidate the mechanisms underlying gene expression alterations during the acquisition of metastatic potential and characterize the metastatic subpopulations within ovarian cancer cells. We conducted single-cell RNA sequencing of two human ovarian cancer cell lines: SKOV-3 and SKOV-3-13, a highly metastatic subclone of SKOV-3. Suppression of NFE2L1 expression was performed through siRNA-mediated knockdown and CRISPR-Cas9-mediated knockout. Clustering and pseudotime trajectory analysis revealed pro-metastatic subpopulation within these cells. Furthermore, gene set enrichment analysis and prognosis analysis indicated that NFE2L1 could be a key transcription factor in the acquisition of metastasis potential. Inhibition of NFE2L1 significantly reduced migration and viability of both cells. In addition, NFE2L1 knockout cells exhibited significantly reduced tumor growth in a mouse xenograft model, recapitulating in silico and in vitro results. The results presented in this study deepen our understanding of the molecular pathogenesis of ovarian cancer metastasis with the ultimate goal of developing treatments targeting pro-metastatic subclones prior to metastasis.

Abstract 5551: Cancer associated PSA-glycoforms as prostate cancer markers

Abstract Changes in protein glycosylation have been observed in several cancers, where they affect cellular growth behavior, invasiveness and acquisition of metastatic potential. Such changes are not random and have been found to associate with cancer aggressiveness. Thus, detection of protein glycosylation may offer novel opportunities for cancer biomarker development. Since prostate-specific antigen (PSA) is glycosylated, assessing different PSA-glycoforms may provide valuable diagnostic and prognostic information. We recently established a novel in situ proximity ligation-based method for the detection of different protein-glycoforms in tissue sections. This method utilizes protein-specific antibody and glycan-binding lectins. We have optimized this method for the detection of different PSA-glycoforms in formalin-fixed paraffin-embedded prostatic tumor samples. We found that PSA is, indeed, differently glycosylated in prostate cancer (n=23) compared to benign prostate tissue (n=4). Two of the 25 studied lectins showed prominent staining only in cancer tissues (p<0.0001 for both), although the total PSA staining was stronger in benign tissues (p=0.006). We are currently validating these results in large, well-curated clinical cohorts, with intention to test whether PSA-glycoforms in tissue offer improved detection of clinically significant prostate cancer. These studies facilitate development of tools for identification of cancer-specific PSA-isoforms in serum samples. Detection of such isoforms is likely to provide independent diagnostic and prognostic information. Citation Format: Hannu Koistinen, Ruusu-Maaria Kovanen, Timo-Pekka Lehto, Antti Rannikko, Tuomas Mirtti. Cancer associated PSA-glycoforms as prostate cancer markers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5551.

Niclosamide inhibits epithelial-mesenchymal transition with apoptosis induction in BRAF/ NRAS mutated metastatic melanoma cells

Malignant melanoma is considered a deadly aggressive form of skin cancer that frequently metastasizes to various distal organs, which harbors mutations of the BRAF or NRAS which occur in 30 to 50% of melanoma patients. The growth factors secreted by melanoma cells contribute to tumor angiogenesis with the acquisition of metastatic potential by epithelial-mesenchymal transition (EMT) and drive melanoma growth toward a more aggressive form. Niclosamide (NCL) is an FDA-approved anthelmintic drug and is reported to have strong anti-cancer properties against various solid and liquid tumors. Its role in BRAF or NRAS mutated cells is unknown. In this context, we uncovered the role of NCL in impeding malignant metastatic melanoma in vitro in SK-MEL-2 and SK-MEL-28 cell lines. We found that NCL induces significant ROS generation and apoptosis through a series of molecular mechanisms, such as depolarization of mitochondrial membrane potential, arresting the cell cycle at the sub G1 phase with a significant increase in the DNA cleavage via topoisomerase II in both cell lines. We also found that NCL potently inhibited metastasis, which was examined by scratch wound assay, Additionally, we found that NCL inhibits the most important markers involved in the EMT signaling cascade that are stimulated by TGF-β such as N-cadherin, Snail, Slug, Vimentin, α-SMA and p-Smad 2/3. This work provides useful insights into the mechanism of NCL in BRAF/NRAF mutant melanoma cells via inhibition of molecular signaling events involved in EMT signaling, and apoptosis induction.

HPV-Related Cervical Cancer and Extracellular Vesicles

Cervical cancer is the fourth most common type of cancer in females worldwide. Infection with a human papillomavirus is crucial to the etiopathogenesis of cervical cancer. The natural trajectory of HPV infection comprises HPV acquisition, HPV persistence versus clearance, and progression to precancer and invasive cancer. The majority of HPV infections are cleared and controlled by the immune system within 2 years, but some infections may become quiescent or undetectable. The persistence of high-risk HPV infection for a longer period of time enhances the risk of malignant transformation of infected cells; however, the mechanisms responsible for the persistence of infection are not yet well-understood. It is estimated that 10-15% of infections do persist, and the local microenvironment is now recognized as an important cofactor promoting infection maintenance. Extracellular vesicles (EVs) are small membrane vesicles derived from both normal cells and cancer cells. EVs contain various proteins, such as cytoskeletal proteins, adhesion molecules, heat shock proteins, major histocompatibility complex, and membrane fusion proteins. EVs derived from HPV-infected cells also contain viral proteins and nucleic acids. These biologically active molecules are transferred via EVs to target cells, constituting a kind of cell-to-cell communication. The viral components incorporated into EVs are transmitted independently of the production of infectious virions. This mode of transfer makes EVs a perfect vector for viruses and their components. EVs participate in both physiological and pathological conditions; they have also been identified as one of the mediators involved in cancer metastasis. This review discusses the potential role of EVs in remodeling the cervical cancer microenvironment which may be crucial to tumor development and the acquisition of metastatic potential. EVs are promising as potential biomarkers in cervical cancer.

Increased ATG5 Expression Predicts Poor Prognosis and Promotes EMT in Cervical Carcinoma.

Cervical cancer has the second-highest incidence and mortality of female malignancy. The major causes of mortality in patients with cervical cancer are invasion and metastasis. The epithelial–mesenchymal transition (EMT) process plays a major role in the acquisition of metastatic potential and motility. Autophagy-related genes (ARGs) are implicated in the EMT process, and autophagy exerts a dual function in EMT management at different phases of tumor progression. However, the role of specific ARGs during the EMT process has not yet been reported in cervical cancer. Based on the data from the Cancer Genome Atlas (TCGA) cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) sequencing database, we performed the prognosis analysis for those ARGs obtained from the Human Autophagy database. ATG5 was identified as the only important harmful marker influencing survival of cervical cancer patients by univariate Cox regression (HR 1.7; 95% CI: 1.0–2.8, p = 0.047), and the 5-years survival rate for the high- and low-ATG5 expression groups was 0.486 (0.375–0.631) and 0.782 (0.708–0.863), respectively. TCGA CESC methylation data showed that eight methylation sites of ATG5 could also be significantly associated with the overall survival (OS) of cervical cancer patients. Single-sample gene-set enrichment and gene functional enrichment results showed that ATG5 was correlated with some cancer-related pathways, such as phagocytosis-related genes, endocytosis-related genes, immune-related genes, EMT score, and some EMT signature-related genes. Next, cell migration and invasion assay and Western blot were applied to detect the function of ATG5 in EMT of cervical cancer. In cervical cancer cells, ATG5 knockdown resulted in attenuation of migration and invasion. The functional study showed that knockdown of ATG5 could reverse EMT process by P-ERK, P-NFκBp65, P-mTOR pathways, and so on. In conclusion, the present study implies that ATG5 was a major contributor to EMT regulation and poor prognosis in cervical cancer.

Epigenetically associated CCL20 upregulation correlates with esophageal cancer progression and immune disorder

Chemokines have distinct effects on tumor progression by affecting cancer immunity and tumorigenesis. However, the characteristic chemokine profiles and their roles in immune cell recruitment and cancer cell biology are not entirely understood in esophageal cancer. Here, we scrutinized chemokine's expression profiles in independent esophageal cancer cohorts and identified the elevated CCL20 as a risk factor to predict patients' prognosis regardless of histology subtypes. Enhanced CCL20 expression was also associated with the acquisition of metastatic potential. Mechanistically, the upregulation of CCL20 in tumor cells was associated with promoter hypomethylation. Furthermore, by analyzing single-cell RNA sequencing data of a mouse model mimicking human ESCC development, we observed an imbalance among CD4+ T subtypes in the tumor microenvironment, namely Ccr6+ Th17 and Treg cells infiltration alongside the elevated Ccl20 expression in abnormal epithelial cells during the tumorigenic process. Together, these results reveal that hypomethylation-induced CCL20 promotes esophageal cancer progression and immune disorder. Targeting CCL20 might be a promising therapeutic approach in esophageal cancer.

PTCH1 regulates anchorage-independent growth and bone invasion of non-small cell lung cancer cells.

Acquisition of metastatic potential by cancer cells is related to cancer stemness and anchorage-independent growth. The onset and progression of cancer are known to involve Hedgehog (HH) signaling that is activated by the binding of HH to the Patched 1 (PTCH1) receptor. However, the functions and mechanisms of action of PTCH1 in the context of bone metastasis remain to be elucidated. In this study, lentivirally-delivered shRNA was used to deplete PTCH1 levels, which resulted in the inhibition of spherical colony formation by the human non-small cell lung cancer (NSCLC) cell line; this suggested that PTCH1 promotes anchorage-independent growth. Concordantly, knockdown of PTCH1 resulted in significantly reduced migration and invasion of NSCLC cells; this was accompanied by the downregulation of MMP7 and SOX2. PTCH1 knockdown resulted in decreased bone destruction and osteoclastogenesis in a mouse bone metastasis model. These results indicate that PTCH1 may be an important regulator of bone invasion, and strongly suggest that knockdown of PTCH1 may decrease the anchorage-independent growth and metastatic potential of NSCLC.

THE ROLE AND MECHANISMS OF EPITHELIAL-MESENCHYMAL TRANSITION IN THE PROGRESSION OF MELANOMA

Despite the achievements of modern medicine in the diagnosis and treatment of oncological diseases, skin melanoma remains one of the leading causes of death worldwide: every third case of melanoma ends in death. As you know, one of the main causes of death is the high incidence of melanoma progression. It is important to note that the mechanisms of melanoma progression are diverse and the rapidly developing area of drug therapy for tumors requires a deep understanding of their characteristics. This is primarily due to the fact that these processes lead to the formation of special, minor tumor clones with stem properties. They are highly resistant to therapy. The latter is the mainobstacle to effective treatment of melanoma patients. The epithelial-mesenchymal transition (EMT) plays a leading role in the acquisition of metastatic potential by melanoma cells. An important distinguishing feature of EMT is a change in the level of expression of transmembrane glycoproteins involved in cell adhesion. With EMT, both a decrease in the level of E-cadherin and an increase in the expression of N-cadherin are observed. Such a switch in different classes of adhesion molecules leads to the fact that melanoma cells lose contact with neighboring keratinocytes and begin to interact with fibroblasts and endothelial cells. The key regulator in EMT induction in melanoma is the Notch1 signaling pathway, which accelerates N-cadherin expression when activated. In addition, EMT also regulates many other pathways – RAS/RAF/MEK/ERK, PI3K/AKT/mTOR, Wnt/β-catenin, the dysregulation of which is associated with the development of drug resistance in melanoma. The analysis was carried out in the article of modern literature data on the importance of EMT in carcinogenesis and prognosis of melanoma. The modern mechanisms of EMT, currently known prognostic factors, as well as potential therapeutic targets that affect EMT and, accordingly, inhibit the process of metastasis, are described in detail.

Abstract B30: The histone demethylase PHF8 epigenetically regulates the TGFbeta pathway to promote melanoma metastasis

Abstract The specific contribution of epigenetic dysregulation to the metastatic process remains understudied. Through a meta-analysis of gene expression datasets followed by a functional mini-screen, we identified PHF8, a histone demethylase of the Jumonji C proteins family, as a novel prometastatic factor in melanoma. Loss- and gain-of-function approaches demonstrate that PHF8 promotes cell invasion without affecting proliferation in vitro, and increases dissemination but not tumor growth in vivo, thus strengthening its specific contribution to the acquisition of metastatic potential. PHF8 demethylase activity is required for its proinvasive role. Transcriptomic and epigenomic analyses revealed that PHF8 orchestrates molecular changes that directly control the TGFβ signaling pathway and, as a direct consequence, melanoma invasion. Citation Format: Rana Moubarak, Ana De Pablos-Aragoneses, Vanessa Ortiz-Barahona, Richard Von Itter, Yixiao Gong, Michael Gowen, Sebastian Stefan, Diana Argibay, Eleazar De Miera-Vega, Igor Dolgalev, Elena Sokolova, Farbod Darvishian, Aristotelis Tsirigos, Iman Osman, Eva Hernando. The histone demethylase PHF8 epigenetically regulates the TGFbeta pathway to promote melanoma metastasis [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B30.

TrkB-Induced Inhibition of R-SMAD/SMAD4 Activation is Essential for TGF-β-Mediated Tumor Suppressor Activity.

TrkB-mediated activation of the IL6/JAK2/STAT3 signaling pathway is associated with the induction of the epithelial–mesenchymal transition (EMT) program and the acquisition of metastatic potential by tumors. Conversely, the transforming of growth factor-β (TGF-β) is implicated in tumor suppression through the canonical SMAD-dependent signaling pathway. Hence, TrkB could play a role in disrupting the potent TGF-β-mediated growth inhibition, a concept that has not been fully explored. Here, we identified TrkB to be a crucial regulator of the TGF-β signaling pathway as it inhibits the TGF-β-mediated tumor suppression and the activation of TrkB kinase. We further show that the interactions between TrkB and SMADs inhibit TGF-β-mediated R-SMAD/SMAD4 complex formation and suppress TGF-β-induced nuclear translocation and target gene expression. Additionally, the knockdown of TrkB restored the tumor inhibitory activity of TGF-β signaling. These observations suggest that interactions between TrkB and SMADs are critical for the inhibition of TGF-β tumor suppressor activity in cancer cells.

In Vivo Evidence for Voltage-Gated Sodium Channel Expression in Carcinomas and Potentiation of Metastasis.

A wide body of evidence suggests that voltage-gated sodium channels (VGSCs) are expressed de novo in several human carcinomas where channel activity promotes a variety of cellular behaviours integral to the metastatic cascade. These include directional motility (including galvanotaxis), pH balance, extracellular proteolysis, and invasion. Contrary to the substantial in vitro data, however, evidence for VGSC involvement in the cancer process in vivo is limited. Here, we critically assess, for the first time, the available in vivo evidence, hierarchically from mRNA level to emerging clinical aspects, including protein-level studies, electrolyte content, animal tests, and clinical imaging. The evidence strongly suggests that different VGSC subtypes (mainly Nav1.5 and Nav1.7) are expressed de novo in human carcinoma tissues and generally parallel the situation in vitro. Consistent with this, tissue electrolyte (sodium) levels, quantified by clinical imaging, are significantly higher in cancer vs. matched non-cancer tissues. These are early events in the acquisition of metastatic potential by the cancer cells. Taken together, the multi-faceted evidence suggests that the VGSC expression has clinical (diagnostic and therapeutic) potential as a prognostic marker, as well as an anti-metastatic target. The distinct advantages offered by the VGSC include especially (1) its embryonic nature, demonstrated most clearly for the predominant neonatal Nav1.5 expression in breast and colon cancer, and (2) the specifically druggable persistent current that VGSCs develop under hypoxic conditions, as in growing tumours, which promotes invasiveness and metastasis.

Dual-strand tumor suppressor miR-193b-3p and -5p inhibit malignant phenotypes of lung cancer by suppressing their common targets

Emerging studies suggest that microRNAs (miRNAs) play multiple roles in cancer malignancy, including proliferation and acquisition of metastatic potential. Differentially expressed miRNAs responsible for the malignancy of lung cancer were searched by miRNA microarray using a previously established brain metastatic lung cancer model. Twenty-five miRNAs were down-regulated in brain metastatic lung cancer cells. Among those, miR-193b-3p and -5p were chosen for further studies. Their function in metastatic potential and proliferation was examined using Transwell invasion, wound healing, and colony forming assays. The underlying mechanism of tumor-suppressor miR-193b-3p and -5p was explored using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), Western blot, Argonaute 2-RNA immunoprecipitation (Ago2-RIP), and reporter assays. Both strands of miR-193b were down-regulated in brain metastatic lung cancer cells and in tissues from lung cancer patients. Overexpression of miR-193b-3p and -5p inhibited invasive and migratory activities and diminished clonogenic ability. Conversely, inhibition of miR-193b-3p or -5p increased the metastatic potential and colony forming ability. Cyclin D1 (CCND1), Ajuba LIM Protein (AJUBA), and heart development protein with EGF like domains 1 (HEG1) were identified as common target genes of miR-193b-3p and -5p. A reporter assay and an Ago2-RIP experiment showed that both miRNAs directly bind to the 3′ untranslated region (3′UTR) of the target mRNA. Knockdown of target gene reduced the proliferative and metastatic potential of primary and metastatic lung cancer cells. Our results demonstrate miR-193b is a dual-strand tumor suppressor and a novel therapeutic target for lung cancer.

Mitochondrial functions and melatonin: a tour of the reproductive cancers.

Cancers of the reproductive organs have a strong association with mitochondrial defects, and a deeper understanding of the role of this organelle in preneoplastic-neoplastic changes is important to determine the appropriate therapeutic intervention. Mitochondria are involved in events during cancer development, including metabolic and oxidative status, acquisition of metastatic potential, resistance to chemotherapy, apoptosis, and others. Because of their origin from melatonin-producing bacteria, mitochondria are speculated to produce melatonin and its derivatives at high levels; in addition, exogenously administered melatonin accumulates in the mitochondria against a concentration gradient. Melatonin is transported into tumor cell by GLUT/SLC2A and/or by the PEPT1/2 transporters, and plays beneficial roles in mitochondrial homeostasis, such as influencing oxidative phosphorylation and electron flux, ATP synthesis, bioenergetics, calcium influx, and mitochondrial permeability transition pore. Moreover, melatonin promotes mitochondrial homeostasis by regulating nuclear DNA and mtDNA transcriptional activities. This review focuses on the main functions of melatonin on mitochondrial processes, and reviews from a mechanistic standpoint, how mitochondrial crosstalk evolved in ovarian, endometrial, cervical, breast, and prostate cancers relative to melatonin's known actions. We put emphasis on signaling pathways whereby melatonin interferes within cancer-cell mitochondria after its administration. Depending on subtype and intratumor metabolic heterogeneity, melatonin seems to be helpful in promoting apoptosis, anti-proliferation, pro-oxidation, metabolic shifting, inhibiting neovasculogenesis and controlling inflammation, and restoration of chemosensitivity. This results in attenuation of development, progression, and metastatic potential of reproductive cancers, in addition to lowering the risk of recurrence and improving the life quality of patients.

Androgen receptor-regulated miRNA-193a-3p targets AJUBA to promote prostate cancer cell migration.

Background Dysregulation of microRNA (miRNA) expression is implicated in cancer development and progression by modulating oncogenes or tumor suppressors at the post-transcriptional level. Methods To investigate the role of miRNAs in prostate cancer (PCa) progression, we performed small RNA-sequencing (RNA-seq) analysis in androgen-dependent LNCaP cells and LNCaP-derived castration-resistant prostate cancer (CRPC) C4-2B cells. For functional validation, we specifically investigated miR-193a-3p, which is highly upregulated in C4-2B cells and modulated by the androgen receptor (AR). We elucidated the role of miR-193a-3p and its downstream target gene in PCa cell migration using biochemical approaches. Results We identified a subset of differentially expressed miRNAs in C4-2B cells compared to LNCaP cells. Computational analysis shows that the targets of upregulated miRNAs are significantly associated with downregulated protein-coding mRNAs in C4-2B cells. Gene Ontology analysis further reveals that these downregulated mRNAs are significantly enriched in cell-cell adhesion functions. Downregulation of these miRNA-targeted genes may change PCa cell motility resulting in the acquisition of metastatic potential. We then focus on miR-193a-3p and demonstrate overexpression of miR-193a-3p increases cell migration through downregulating its target AJUBA. AJUBA is a LIM domain protein and contributes to the formation and stability of cadherin-mediated cell-cell adhesion. Loss of AJUBA enhances PCa migration and downregulation of AJUBA expression is observed in metastatic PCa tumors. Conclusions Our results suggest a novel AR/miR-193a-3p/AJUBA pathway implicated in PCa progression. MiR-193a-3p is a potential therapeutic target for metastatic PCa.

Abstract P2-13-01: Differential protein expression in primary breast cancer tumors spreading to liver or elsewhere

Abstract Breast cancer (BC) outcome is determined mainly by its ability to spread to distant sites, since this is the lethal phase of the disease. Metastasizing cells have to acquire various molecular aberrations in order to go through the metastatic cascade and gain organ tropism. These are probably reflected at the primary tumor. This study was aimed to identify a set of abnormally expressed proteins in primary human BC tumors, which spread preferentially to the liver or elsewhere. Methods: We collected archival paraffin embedded primary BC samples from 48 patients who were divided into 3 groups: 10 who developed liver metastases (A), 20 with metastases to other sites (B) and 18 with no BC recurrence (C). Tissue microarray (TMA) were constructed, sliced and subjected to immuno-histochemical staining. A pre-defined panel of 11 proteins was selected, based on published data that were available at the time of study initiation. Level of expression (coded by IRS scoring) and the intra/extracellular location of each protein was determined by an expert pathologist and compared between the three groups. The panel included: cell-cell interaction proteins (CDH1, BIGH3, MMP14, CD44s, Galectin-3), transcription factors (FRA-1, c-Jun, GATA-3, TP53), an inflammatory chemokine (CCL5), cell signaling protein (Wnt-5a). Results: Metastatic tropism to the liver was studied by comparing results between A and B. When similar, A and B were combined and compared with C group. CDH1 protein expression was significantly reduced in the cytoplasm in groups A and B in comparison to C (p=0.004)and over-expressed in the membrane (p<0.001) in both. Hence, cytoplasmic CDH1was higher in group A than B (p=0.03). In group A only, a trend of BIGH3 over-expression was noted, though it did not reach significance. No further differences were found between A versus B. MMP14 was over-expressed both in the membrane and cytoplasm (p=0.001) of A and B, in comparison with C. CD44s was over-expressed in the membrane in A&B versus C (p<0.001). Galectin-3 was over-expressed in the cytoplasm (p=0.005) and down regulated in the nucleus (p=0.015) in both metastatic groups. CCL5 (RANTES) level was significantly reduced in the cytoplasm in A & B (p<0.001) while significantly over-expressed in the nuclei (p=0.005). c-Jun (part of AP1 complex) was over-expressed in both A and B (p=0.021). On the other hand, levels of FRA-1, localized in both cytoplasm and nucleus in C, were reduced in both compartments in A & B (p=0.029). No difference was noted between the 3 groups in level and localization of TP53, GATA-3 and Wnt-5a. Conclusions: Assessment of the expression of proteins in tumor cells should include protein localization besides its level of expression. Both CDH1 and BIGH3 seem to be over-expressed in tumors that spread preferentially to the liver. No significant changes in pattern of expression of the other proteins studied could be correlated with metastatic propensity to the liver. Compartmental translocation of various proteins is correlated with the acquisition of metastatic potential, such as CDH1 and CCL5 which presented a shift from nucleus to cytoplasm, and Galectin-3 and CCL5, where translocation from cytoplasm to membrane were observed in both metastatic groups. Citation Format: Goldberg H, Fell R, Apel-Sarid L. Differential protein expression in primary breast cancer tumors spreading to liver or elsewhere [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-13-01.

Abstract 4164: Synergism between NEDD9 overexpression and loss of PTEN and INK4A/ARF in melanoma tumorigenesis

Abstract The fact that melanoma, the most aggressive form of skin cancer, is driven by multiple combinations of genetic lesions has supported the use of genetically engineered mouse (GEM) models in validating the etiological and biological roles of oncogenes and tumor suppressors. Most of the currently studied mouse melanoma models are driven by RAS/RAF/MAPK activation via activating RAS or RAF mutations in combination with loss of INK4A/ARF or PTEN; therefore, BRAF/NRAS wild-type melanoma remains less well characterized and the overall survival of patients with metastatic BRAF/NRAS wild-type melanoma remains poor. In this study, we investigated the hypothesis that NEDD9 could synergize with the loss of PTEN and INK4A/ARF in melanoma tumorigenesis. This hypothesis is based on our observation of an inverse correlation between PTEN expression and levels of NEDD9 gene expression in human melanoma samples. NEDD9 (neural precursor derived, developmentally downregulated gene 9) was previously identified by us as a target of a recurrent focal amplification, associated with acquisition of metastatic potential in the RAS-Ink/Arf model. We have shown that NEDD9 is up-regulated in 35∼50% of metastatic melanomas and enhances proliferation and invasion. First, we demonstrated that NEDD9 cooperated with PTEN loss in Ink4a/Arf-/- melanocytes to enhance cell proliferation, anchorage independent growth, and invasion in vitro. Additionally, when melanocytes were injected into nude mice either subcutaneously or intravenously, NEDD9 overexpression facilitated increased tumor growth and lung seeding, respectively. Further analysis showed that NEDD9 expression correlated with increased phosphorylation of ERK, SFKs (Src family kinases), AKT2, and STAT3. Of these pathways we have demonstrated through the use of small molecule inhibitors that both ERK and AKT2 signaling are required for the enhanced anchorage independent growth observed in NEDD9 overexpressing melanocytes. In addition, we generated a mouse model based on loss of PTEN and INK4A/ARF with or without NEDD9 upregulation. To do this, we generated a tet-inducible NEDD9 allele that carries a doxycycline-responsive, melanocyte-targeted NEDD9. These mice were crossed with a strain containing floxed PTEN and INK4A/ARF in combination with a melanocyte-targeted CreERT2. The development of cutaneous and ocular melanomas in these mice was enhanced by NEDD9 induction. This study will generate a body of knowledge for the in vivo roles of NEDD9 in melanoma tumorigenesis and identify signaling pathways that could be therapeutically targeted to treat patients with BRAF/NRAS wild-type melanoma Citation Format: Kristen S. Hill, Xue Wang, Youngchul Kim, Jane L. Messina, Minjung Kim. Synergism between NEDD9 overexpression and loss of PTEN and INK4A/ARF in melanoma tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4164.

Characterization of preneoplastic and neoplastic rat mesothelial cell lines: the involvement of TETs, DNMTs, and 5-hydroxymethylcytosine.

Malignant mesothelioma (MM) is one of the worst cancers in terms of clinical outcome, urging the need to establish and characterize new preclinical tools for investigation of the tumorigenic process, improvement of early diagnosis and evaluation of new therapeutic strategies. For these purposes, we characterized a collection of 27 cell lines established from F344 rats, after 136 to 415 days of induction with crocidolite asbestos administered intraperitoneally. Four mesotheliomas were distinguished from 23 preneoplastic mesothelial cell lines (PN) according to their propensity to generate tumors after orthotopic transplantation into syngeneic rats, their growth pattern, and the expression profile of three genes. PN cell lines were further discriminated into groups / subgroups according to morphology in culture and the expression profiles of 14 additional genes. This approach was completed by analysis of positive and negative immunohistochemical MM markers in the four tumors, of karyotype alterations in the most aggressive MM cell line in comparison with a PN epithelioid cell line, and of human normal mesothelial and mesothelioma cells and a tissue array. Our results showed that both the rat and human MM cell lines shared in common a dramatic decrease in the relative expression of Cdkn2a and of epigenetic regulators, in comparison with PN and normal human mesothelial cells, respectively. In particular, we identified the involvement of the relative expression of the Ten-Eleven Translocation (TET) family of dioxygenases and Dnmt3a in relation to the 5-hydroxymethylcytosine level in malignant transformation and the acquisition of metastatic potential.

Snail-induced epithelial-to-mesenchymal transition of MCF-7 breast cancer cells: systems analysis of molecular changes and their effect on radiation and drug sensitivity.

BackgroundEpithelial-to-mesenchymal transition (EMT) has been associated with the acquisition of metastatic potential and the resistance of cancer cells to therapeutic treatments. MCF-7 breast cancer cells engineered to constitutively express the zinc-finger transcriptional repressor gene Snail (MCF-7-Snail cells) have been previously shown to display morphological and molecular changes characteristic of EMT. We report here the results of a comprehensive systems level molecular analysis of changes in global patterns of gene expression and levels of glutathione and reactive oxygen species (ROS) in MCF-7-Snail cells and the consequence of these changes on the sensitivity of cells to radiation treatment and therapeutic drugs.MethodsSnail-induced changes in global patterns of gene expression were identified by microarray profiling using the Affymetrix platform (U133 Plus 2.0). The resulting data were processed and analyzed by a variety of system level analytical methods. Levels of ROS and glutathione (GSH) were determined by fluorescent and luminescence assays, and nuclear levels of NF-κB protein were determined by an ELISA based method. The sensitivity of cells to ionizing radiation and anticancer drugs was determined using a resazurin-based cell cytotoxicity assay.ResultsConstitutive ectopic expression of Snail in epithelial-like, luminal A-type MCF-7 cells induced significant changes in the expression of >7600 genes including gene and miRNA regulators of EMT. Mesenchymal-like MCF-7-Snail cells acquired molecular profiles characteristic of triple-negative, claudin-low breast cancer cells, and displayed increased sensitivity to radiation treatment, and increased, decreased or no change in sensitivity to a variety of anticancer drugs. Elevated ROS levels in MCF-7-Snail cells were unexpectedly not positively correlated with NF-κB activity.ConclusionsEctopic expression of Snail in MCF-7 cells resulted in morphological and molecular changes previously associated with EMT. The results underscore the complexity and cell-type dependent nature of the EMT process and indicate that EMT is not necessarily predictive of decreased resistance to radiation and drug-based therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2274-5) contains supplementary material, which is available to authorized users.

Hepatitis C virus core protein interacts with Snail and histone deacetylases to promote the metastasis of hepatocellular carcinoma.

Downregulation of E-cadherin by the transcriptional repressor Snail is associated with acquisition of metastatic potential. Although hepatitis C virus (HCV) core protein has been implicated in hepatocarcinogenesis, it is unclear whether Snail is involved in HCV core-induced dysregulation of E-cadherin. Herein, we investigated the mechanism by which HCV core induces E-cadherin repression and the role of Snail in HCV core-mediated invasiveness and metastasis. We found that HCV infection, especially HCV core expression, effectively induced the epithelial-mesenchymal transition (EMT) in hepatoma cells by repressing E-cadherin. HCV core interacted with Snail and enhanced its binding to the E-box in the promoter region of E-cadherin, leading to decreased E-cadherin promoter activity. We found that HCV core, Snail, and the histone deacetylases HDAC1/HDAC2 formed a co-repressor complex at the E-cadherin promoter. Moreover, HCV core was shown to stabilize Snail through activation of the PI3K/Akt/GSK3β pathway. Silencing Snail expression restored E-cadherin expression and inhibited HCV core-promoted tumor growth and distant lung metastasis in vivo. Collectively, these results demonstrated that HCV core induced EMT by interacting with the transcriptional repressor complex Snail/HDACs at the E-cadherin promoter, which led to E-cadherin repression and increased invasiveness of hepatoma cells. These findings increase understanding of factors regulating metastasis in hepatoma and may ultimately lead to the development of novel treatment strategies for HCV-associated hepatocellular carcinoma.