Chk1 suppression leads to a reduction in the enhanced radiation-induced invasive capability on breast cancer cells.

Radiation therapy is generally effective for treating breast cancers. However, approximately 30% of patients with breast cancer experience occasional post-treatment local and distant metastasis. Low-dose (0.5 Gy) irradiation is a risk factor that promotes the invasiveness of breast cancers. Although an inhibitor of checkpoint kinase 1 (Chk1) suppresses the growth and motility of breast cancer cell lines, no study has investigated the effects of the combined use of a Chk1 inhibitor and radiation on cancer metastasis. Here, we addressed this question by treating the human breast cancer cell line MDA-MB-231 (in vitro) and mouse mammary tumor cell line 4 T1 (in vitro and in vivo) with γ-irradiation and the Chk1 inhibitor PD407824. Low-dose γ-irradiation promoted invasiveness, which was suppressed by PD407824. Comprehensive gene expression analysis revealed that low-dose γ-irradiation upregulated the mRNA and protein levels of S100A4, the both of which were downregulated by PD407824. We conclude that PD407824 suppresses the expression of S100A4. As the result, γ-irradiation-induced cell invasiveness were inhibited.

Overexpression of S100A4 protects retinal ganglion cells against retinal ischemia-reperfusion injury in mice.

BACKGROUND: Glaucoma is characterized by the neurodegeneration of retinal ganglion cells (RGCs) and the optic nerve. Numerous studies have reported that S100A4 participates in the metastasis of tumor cells and nerve protection. This study was intended to explore the role of S100A4 on RGCs under retinal ischemia-reperfusion (I/R) injury in mice. METHODS: C57BL/6J mice were used to induce retinal I/R injury. The intravitreal administration of rAAV-EF1α-s100a4-EGFP-WPRE (rAAV-S100A4) or rAAV-EF1α-EGFP-WPRE-Pa was performed 4 weeks before I/R injury. Expression of S100A4 was detected by quantitative real-time PCR, immunofluorescence staining of retinal sections and western blot. Surviving RGCs were quantified using immunofluorescence staining. Staining of TUNEL was utilized to evaluate the apoptosis of retinal cells. Electroretinogram (ERG) was used to analyze retinal function. Expression of Akt, phospho-Akt, Bcl-2, and Bax were determined using western blotting to investigate the potential mechanisms of S100A4. RESULTS: Retinal S100A4 level had no statistical difference 7 days after I/R injury. The rAAV-S100A4 was clearly demonstrated by the green fluorescence protein in many layers of the retina after intravitreal injection and up-regulated the expression of S100A4. I/R injury resulted in an increase of the apoptosis of retinal cells and the reduction of surviving RGCs, however, overexpressed S100A4 inhibited the apoptosis of cells and a decrease of RGCs. ERG analysis showed a drop on amplitude of a-wave and b-wave was impeded to some extent by overexpressing of S100A4. Up-regulation of S100A4 raised the expression of phospho-Akt and reduced Bax expression. Nevertheless, there were no significant changes in the levels of Bcl-2 and total Akt. CONCLUSION: Our results indicate the neuroprotective effects of overexpressed S100A4 on RGCs by activating the Akt pathway and then inhibiting the apoptosis of cells after I/R injury. The use of S100A4 protein may be a novel therapeutic strategy for glaucoma.

S100A4 Silencing Facilitates Corneal Wound Healing After Alkali Burns by Promoting Autophagy via Blocking the PI3K/Akt/mTOR Signaling Pathway.

Purpose: This study investigated the role of S100 calcium binding protein A4 (S100A4) in corneal wound healing and the underlying mechanism of the S100A4-mediated PI3K/Akt/mammalian target of rapamycin (mTOR) pathway. Methods: The rabbit corneal alkali burn model was established in vivo. S100A4 expression, wound healing, inflammation, and autophagy in rabbit cornea after alkali burn were detected. The NaOH-treated rabbit corneal stromal cells (rCSCs) were transfected with overexpressed S100A4 or silencing S100A4 to examine the effect of S100A4 on corneal wound healing in vitro. The effect of S100A4 on cell viability, proliferation, migration, invasion, fibrosis, and autophagy of rCSCs after alkali burn was analyzed. Then the functional rescue experiments were carried out. The PI3K inhibitor, LY294002, was used to elucidate the PI3K/Akt/mTOR signaling pathway in rCSCs. Results: S100A4 silencing promoted rabbit corneal wound healing by inhibiting fibrosis and inflammation and promoting autophagy in alkali-burned cornea, corresponding to increased levels of LC3, Beclin 1, and Atg4B but lowered α-smooth muscle actin, TNF-ɑ, and p62 levels. Moreover, silencing S100A4 inhibited proliferation, migration, invasion, and fibrosis of NaOH-treated rCSCs and promoted the differentiation of rCSCs into corneal cells and the autophagy of damaged rCSCs. The inhibitory role of S100A4 in wound healing was achieved via activation of the PI3K/Akt/mTOR pathway. Conclusions: S100A4 silencing confers a promising effect on wound healing of alkali-burned cornea by blocking the PI3K/Akt/mTOR pathway, supporting the advancement of corneal gene therapies for wound healing.

Mts1 Up-regulation is Associated With Aggressive Pathological Features in Thyroid Cancer.

BACKGROUND/AIM: Thyroid cancer is the most common type of endocrine cancer and its incidence and mortality are increasing. However, few studies on the molecular factors related to its poor prognosis have been performed. The aim of our study was to identify a poor prognostic factor for thyroid cancer to reduce its overtreatment, recurrence, and mortality. MATERIALS AND METHODS: The present study is a retrospective study of 55 patients who were diagnosed with papillary thyroid cancer and operated in Korea from September 2013 to November 2015. RESULTS: Mts1 is a member of the S100 protein family and is involved in tumor progression and metastasis. Mts1 was highly expressed in patients with thyroid cancer and high Mts1 levels were related to poor prognoses such as lymph node metastasis. CONCLUSION: Mts1 is associated with aggressive pathological features in thyroid cancer, and may be a poor prognostic factor for thyroid cancer.

The Expression Level of S100A4 Protein Affects the Migration Activity of Breast Cancer Cells.

Reduced expression of metastatic marker protein S100A4 in triple-negative breast cancer cells MDA-MB-231 leads to a decrease in the migration ability of cells and increases the sensitivity of the modified cells to docetaxel therapy. Cells capable of migration differ from the immotile cells in the content of the S100A4 protein in the cell, and this difference persists after the treatment of cells with the agents that reduce the intracellular level of S100A4. The presence of exogenous S100A4 protein in culture medium reduces the content of this protein in breast cancer cells. The results of the study show that the ability of breast cancer cells to migrate depends on the S100A4 protein concentration in the cell.

S100a4 Is Secreted by Alternatively Activated Alveolar Macrophages and Promotes Activation of Lung Fibroblasts in Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease, characterized by damage of lung epithelial cells, excessive deposition of extracellular matrix in the lung interstitium, and enhanced activation and proliferation of fibroblasts. S100a4, also termed FSP-1 (fibroblast-specific protein-1), was previously considered as a marker of fibroblasts but recent findings in renal and liver fibrosis indicated that M2 macrophages are an important cellular source of S100a4. Thus, we hypothesized that also in pulmonary fibrosis, M2 macrophages produce and secrete S100a4, and that secreted S100a4 induces the proliferation and activation of fibroblasts. To prove this hypothesis, we comprehensively characterized two established mouse models of lung fibrosis: infection of IFN-γR-/- mice with MHV-68 and intratracheal application of bleomycin to C57BL/6 mice. We further provide in vitro data using primary macrophages and fibroblasts to investigate the mechanism by which S100A4 exerts its effects. Finally, we inhibit S100a4 in vivo in the bleomycin-induced lung fibrosis model by treatment with niclosamide. Our data suggest that S100a4 is produced and secreted by M2 polarized alveolar macrophages and enhances the proliferation and activation of lung fibroblasts. Inhibition of S100a4 might represent a potential therapeutic strategy for pulmonary fibrosis.

Autophagy contributes to hypoxia-induced epithelial to mesenchymal transition of endometrial epithelial cells in endometriosis.

Endometriosis is a benign gynecologic disorder, and presents with malignant characteristics, such as migration and invasion. Hypoxia has been implicated in triggering epithelial-mesenchymal transition (EMT). Hypoxia is also known to induce autophagy. However, the relationship between autophagy and EMT under hypoxia conditions in endometriosis remains unknown. In the present study, we found that the expression of hypoxia-inducible factor-1α (HIF-1α), microtubule associated protein light chain 3 (LC3), and mesenchymal cell marker vimentin was significantly higher in ectopic endometrium from patients with endometriosis, along with decreased expression of epithelial cell marker E-cadherin. After hypoxia treatment, endometrial epithelial cells exhibited enhanced migration and invasion abilities, as well as promoted autophagy and the EMT phenotype. Our analyses also show that HIF-1α was responsible for induction of autophagy. Moreover, inhibition of autophagy by chemical or genetic approaches suppressed hypoxia triggered EMT and reduced cell migration and invasion. Collectively, our findings identify that autophagy is critical for the migration and invasion of endometrial cells through the induction of EMT and indicate that inhibition of autophagy may be a novel useful strategy in the treatment of endometriosis.

Mutated MITF-E87R in Melanoma Enhances Tumor Progression via S100A4.

Melanoma, a melanocyte origin neoplasm, is the most lethal type of skin cancer, and incidence is increasing. Several familial and somatic mutations have been identified in the gene encoding the melanocyte lineage master regulator, MITF; however, the neoplastic mechanisms of these mutant MITF variants are mostly unknown. Here, by performing unbiased analysis of the transcriptomes in cells expressing mutant MITF, we identified calcium-binding protein S100A4 as a downstream target of MITF-E87R. By using wild-type and mutant MITF melanoma lines, we found that both endogenous wild-type and MITF-E87R variants occupy the S100A4 promoter. Remarkably, whereas wild-type MITF represses S100A4 expression, MITF-E87R activates its transcription. The opposite effects of wild-type and mutant MITF result in opposing cellular phenotypes, because MITF-E87R via S100A4 enhanced invasion and reduced adhesion in contrast to wild-type MITF activity. Finally, we found that melanoma patients with altered S100A4 expression have poor prognosis. These data show that a change in MITF transcriptional activity from repression to activation of S100A4 that results from a point mutation in MITF alters melanoma invasive ability. These data suggest new opportunities for diagnosis and treatment of metastatic melanoma.

Clusterin promotes growth and invasion of clear cell renal carcinoma cell by upregulation of S100A4 expression.

BACKGROUND AND OBJECTIVE: Clusterin promotes cell proliferation, motility and invasiveness in human renal cell carcinoma (RCC) cells but the underlying molecular mechanisms of this action are largely unknown. The aim of this study was to investigate the effects of clusterin on cancer cell growth, invasion and S100A4 expression and to determine the effects of clusterin on in vitro cell proliferation and migration and in vivo tumour growth in RCC cells. METHODS: We have established stable transfectants of highly invasive Caki-1 human RCC cells with expression of clusterin shRNA targeting clusterin (Caki-1/clusterin shRNA). We also established stable transfectants of 786-O human RCC cells with expression of clusterin cDNA plaismid (786-O/clusterin cDNA). Clusterin and S100A4 expression was detected by reverse transcription (RT) PCR and western blot assay; Caki-1/clusterin shRNA and 786-O/clusterin cDNA clones were subjected to in vitro-invasion assays. Cell viability and cell growth was assessed in MTT and clonogenic assay. Specific small interfering RNA was employed to down-regulate S100A4. The expression plasmid for S100A4 (pCMV-S100A4) was used to upregulate S100A4. Caki-1/clusterin shRNA clones were injected subcutaneously in nude mice to determine tumour growth and cancer cell invasiveness in vivo. Xenograft tumour tissues were assessed by immunohistochemistry and frozen tissues were used for the detection of S100A4 and clusterin. RESULTS: Overexpression of clusterin increased cell invasiveness; and targeting clusterin reduced cell invasiveness in vitro. This increase in cell invasiveness was mediated by S100A4. Targeting clusterin decreased cell proliferation and down-regulated cellular S100A4 levels in Caki-1 cells; Overexpression of clusterin increased cell proliferation and up-regulated cellular S100A4 levels in 786-O cells; Stable Caki-1/clusterin shRNA transfectants produced smaller xenograft tumours containing reduced S100A4 protein levels in vivo. Stable 786-O/clusterin cDNA transfectants produced larger xenograft tumours containing increased S100A4 protein levels in vivo. CONCLUSION: Our results indicate that clusterin promotes growth and invasion in RCC cells in vitro and in vivo through upregulation of S100A4; And targeting clusterin confers growth inhibitory and anti-invasive properties in RCC cells in vitro and in vivo through a down-regulation of S100A4. These findings provide the rationale for future oncostatic strategies aimed at suppressing clusterin-mediated signal transduction pathways as a novel therapeutic approach in human RCC.

Inversed Expression Patterns of S100A4 and E-Cadherin in Cervical Cancers: Implication in Epithelial-Mesenchymal Transition.

Cervical cancer/CC is the third commonest female malignancy worldwide. The aggressive growth and distal metastases are the leading causes of CC mortality, which is largely due to epithelial-mesenchymal transition/EMT. Fibroblast specific protein S100A4 promotes cancer metastasis and epithelial type cadherin/E-cadherin play pivotal roles in cell-cell and cell-extracellular matrix interaction. Therefore, the expression patterns of S100A4 and E-cadherin reflect statuses of EMT of carcinoma cells. However, S100A4 expression and its relevance with E-cadherin and HPV16 infection in cervical cancers remain unknown. This study aims to address the above issues using cervical cancer specimens. Immunohistochemistry reveals that the levels of mesenchymal marker S100A4 is upregulated (>++) in cervical adenocarcinomas/CACs (12/16; 75%) and squamous cell carcinomas/CSCCs (23/28; 82%) than that in noncancerous glandular epithelia/GE (0/12; 0%) and squamous epithelia/SE (0/12; 0%). Epithelial marker membranous E-cadherin is remarkably reduced on the surface of CAC and CSCC cells (P = 0.00; P = 0.00), especially those showing poorly differentiated phenotypes (P < 0.05) in comparison with their noncancerous counterparts. Correlative analyses revealed an inverse relationship between S100A4 and E-cadherin expression among the cervical cancer samples (P = 0.01, r = -0.38). S100A4 expression level in HPV16-infected group is higher than that in HPV16-free group (P = 0.02). These results suggest the close correlation of S100A4 upregulation with cervical cancer formation and HPV16 infection and E-cadherin reduction with the grades of CC dedifferentiation. The concurrent gain of S100A4 and loss of membrane E-cadherin suggest EMT tendency of CC cells and can be regarded as an unfavorable prognostic parameter of CC patients. Anat Rec, 300:2184-2191, 2017. © 2017 Wiley Periodicals, Inc.

Epigenetic silencing of miR-520c leads to induced S100A4 expression and its mediated colorectal cancer progression.

The S100 calcium-binding protein A4 (S100A4) induces epithelial mesenchymal transition, migration, invasion, angiogenesis and metastasis. Its induced expression in several cancer types correlates with poor prognosis. Apart from the functional and transcriptional regulatory aspects of S100A4, its post-transcriptional regulation is not yet clearly elucidated. In this study, we show that microRNAs (miR) miR-505-5p and miR-520c-3p target the 3'-UTR of S100A4 and inhibits its expression and its mediated migration and invasion. 5-Aza treatment significantly increased miR-520c-3p expression and reduced the S100A4 protein amounts. The upstream promoter region of miR-520c is hypermethylated irrespective of the metastasis status of colorectal cancer (CRC) patient tissues and in all analyzed CRC cell lines. Moreover, in a cohort of CRC patient specimen (n = 59), miR-520c-3p was significantly downregulated. miR-520c-3p stably expressing HCT116 cells showed a reduced metastasis formation in livers after implanting in mice spleen. Taken together, our findings demonstrate that S100A4 is post-transcriptionally regulated by tumor suppressor miRs, miR-505c-5p and miR-520c-3p, and particularly miR-520c-3p expression is epigenetically silenced in CRC.

Epithelial mesenchymal transition (EMT) and non-small cell lung cancer (NSCLC): a mutual association with airway disease.

NSCLC is a leading cause of morbidity and mortality worldwide. It includes adeno- and squamous cell carcinoma. In the background, COPD and smoking play a vital role in development of NSCLC. Local progression and metastasis of NSCLC has been associated with various mechanisms, but in particular by a process called epithelial mesenchymal transition (EMT), which is implicated in COPD pathogenesis. In this study, we have investigated whether expression of EGFR (activation marker) and S100A4, vimentin and N-cadherin (as EMT) is different both in central and leading edge of NSCLC and to what extent related to EMT activity of both small and large airways, stage and differentiation of NSCLC. We have investigated EMT biomarkers (S100A4, vimentin, and N-cadherin), an epithelial activation marker (EGFR) and a vascularity marker (Type-IV collagen) in surgically resected tissue from patients with NSCLC (adeno- and squamous cell carcinoma), and compared them with expression in the corresponding non-tumorous airways. EGFR, S100A4, vimentin, N-cadherin expression was higher in tumor cells located at the peripheral leading edge of NSCLC when compared with centrally located tumor cells of same subjects (P < 0.01). Type-IV collagen-expressing blood vessels were also more at the leading edge in comparison with central parts of NSCLC. EGFR and S100A4 expression was related to differentiation status (P < 0.05) and TNM stage (P < 0.05) of NSCLC. Moreover, EMT markers in the leading edge were significantly related to airway EMT activity, while peripheral edge vascularity of squamous cell carcinoma only was significantly related to large airway Rbm vascularity (P < 0.05). EGFR- and EMT-related protein expression was markedly high in the peripheral leading edge of NSCLCs and related to tumor characteristics associated with poor prognosis. The relationships between EMT-related tumor biomarker expression and those in the airway epithelium and Rbm provide a background for utility of airway changes in clinical settings.

Successful Consecutive Expansion of Limbal Explants Using a Biosafe Culture Medium under Feeder Layer-Free Conditions.

PURPOSE: Transplantation of in vitro cultured limbal epithelial stem cells (LESCs) is a treatment widely used for LESC deficiency. However, the number of limbal tissue donors is limited, and protocols for LESC cultivation often include compounds and/or feeder layers that can induce side effects and/or increase the cost of the culture procedure. We investigated the feasibility of obtaining more than one limbal primary culture (LPC) from the same biopsy using a culture medium in which several potentially harmful compounds were replaced at the same time by biosafe supplements, allowing the LESC cultivation without feeder layers. MATERIALS AND METHODS: We established feeder layer-free LPCs with three culture media: (1) a modified supplemental hormonal epithelial medium, containing potential harmful components (cholera toxin, dimethylsulfoxide, and fetal bovine serum [FBS]), (2) IOBA-FBS, a medium with FBS but with no other harmful supplements, and (3) IOBA-HS, similar to IOBA-FBS but with human serum instead of FBS. Additionally, the same limbal explant was consecutively cultured with IOBA-HS producing three cultures. LPCs were characterized by real-time reverse transcription polymerase chain reaction and/or immunofluorescence. RESULTS: LPCs cultured with the three media under feeder layer-free conditions showed cuboidal cells and no significant differences in the percentage of positive cells for limbal (ABCG2, p63, and K14) and corneal (K3, K12) proteins. Except for ABCG2, the relative mRNA expression of the LESC markers was significantly higher when IOBA-FBS or IOBA-HS was used. LPC1 showed characteristics similar to LPC0, while LPC2 cell morphology became elongated and the expression of some LESC markers was diminished. CONCLUSION: IOBA-HS enables the culturing of up to two biosafe homologous LPCs from one limbal tissue under feeder layer-free conditions. The routine use of this culture medium could improve both the biosafety and the number of available LPCs for potential clinical transplantation, as well as decrease the expense of the culture procedure.

Extracellular matrix 1 (ECM1) regulates the actin cytoskeletal architecture of aggressive breast cancer cells in part via S100A4 and Rho-family GTPases.

ECM1 overexpression is an independent predictor of poor prognosis in primary breast carcinomas, however the mechanisms by which ECM1 affects tumor progression have not been completely elucidated. ECM1 was silenced in the triple-negative breast cancer cell lines Hs578T and MDAMB231 using siRNA and the cells were evaluated for changes in morphology, migration, invasion and adhesion. Actin cytoskeleton alterations were evaluated by fluorescent staining and levels of activated Rho GTPases by pull down assays. ECM1 downregulation led to significantly diminished cell migration (p = 0.0005 for Hs578T and p = 0.02 for MDAMB231) and cell adhesion (p < 0.001 for Hs578T and p = 0.01 for MDAMB231). Cell invasion (matrigel) was reduced only in the Hs578T cells (p < 0.01). Silencing decreased the expression of the prometastatic molecules S100A4 and TGFßR2 in both cell lines and CD44 in Hs578T cells. ECM1-silenced cells also exhibited alterations in cell shape and showed bundles of F-actin across the cell (stress fibers) whereas NT-siRNA treated cells showed peripheral membrane ruffling. Downregulation of ECM1 was also associated with an increased F/G actin ratio, when compared to the cells transfected with NT siRNA (p < 0.001 for Hs578T and p < 0.00035 for MDAMB231) and a concomitant decline of activated Rho A in the Hs578T cells. Re-expression of S100A4 in ECM1-silenced cells rescued the phenotype in the Hs578T cells but not the MDAMB231 cells. We conclude that ECM1 is a key player in the metastatic process and regulates the actin cytoskeletal architecture of aggressive breast cancer cells at least in part via alterations in S100A4 and Rho A.