Chimeric galectin-3 and collagens: Biomarkers and potential therapeutic targets in fibroproliferative diseases.

Fibrosis, stiffening and scarring of an organ/tissue due to genetic abnormalities, environmental factors, infection, and/or injury, is responsible for > 40% of all deaths in the industrialized world, and to date, there is no cure for it despite extensive research and numerous clinical trials. Several biomarkers have been identified, but no effective therapeutic targets are available. Human galectin-3 is a chimeric gene product formed by the fusion of the internal domain of the collagen alpha gene [N-terminal domain (ND)] at the 5'-end of galectin-1 [C-terminal domain (CRD)] that appeared during evolution together with vertebrates. Due to the overlapping structural similarities between collagen and galectin-3 and their shared susceptibility to cleavage by matrix metalloproteases to generate circulating collagen-like peptides, this review will discuss present knowledge on the role of collagen and galectin-3 as biomarkers of fibrosis. We will also highlight the need for transformative approaches targeting both the ND and CRD domains of galectin-3, since glycoconjugate binding by the CRD is triggered by ND-mediated oligomerization and the therapies targeted only at the CRD have so far achieved limited success.

MYH9 binds to dNTPs via deoxyribose moiety and plays an important role in DNA synthesis.

The accepted notion of dNTP transport following cytoplasmic biosynthesis is 'facilitated diffusion'; however, whether this alone is sufficient for moving dNTPs for DNA synthesis remains an open question. The data presented here show that the MYH9 gene encoded heavy chain of non-muscle myosin IIA binds dNTPs potentially serving as a 'reservoir'. Pull-down assays showed that MYH9 present in the cytoplasmic, mitochondrial and nuclear compartments bind to DNA and this interaction is inhibited by dNTPs and 2-deoxyribose-5-phosphate (dRP) suggesting that MYH9-DNA binding is mediated via pentose sugar recognition. Direct dNTP-MYH9 binding was demonstrated by ELISA and a novel PCR-based method, which showed that all dNTPs bind to MYH9 with varying efficiencies. Cellular thermal shift assays showed that MYH9 thermal stability is enhanced by dNTPs. MYH9 siRNA transfection or treatment with myosin II selective inhibitors ML7 or blebbistatin decreased cell proliferation compared to controls. EdU labeling and cell cycle analysis by flow cytometry confirmed MYH9 siRNA and myosin II inhibitors decreased progression to S-phase with accumulation of cells in G0/G1 phase. Taken together, our data suggest a novel role for MYH9 in dNTP binding and DNA synthesis.

Migration and proliferation of cancer cells in culture are differentially affected by molecular size of modified citrus pectin.

While chemically and thermally modified citrus pectin (MCP) has already been studied for health benefits, it is unknown how size-fractionated oligo- and polysaccharides differentially affect cancer cell behavior. We produced thermally MCP and fractionated it by molecular size to evaluate the effect these polymers have on cancer cells. MCP30/10 (between 30 and 10 kDa) had more esterified homogalacturonans (HG) and fewer rhamnogalacturonans (RG-I) than MCP and MCP30 (higher than 30 kDa), while MCP10/3 (between 10 and 3 kDa) showed higher amounts of type I arabinogalactans (AGI) and lower amounts of RG-I. MCP3 (smaller than 3 kDa) presented less esterified HG and the lowest amount of AGI and RG-I. Our data indicate that the enrichment of de-esterified HG oligomers and the AGI and RG-I depletions in MCP3, or the increase of AGI and loss of RGI in MCP30/10, enhance the anticancer behaviors by inhibiting migration, aggregation, and proliferation of cancer cells.

Galectin-3 and cancer stemness.

Over the last few decades galectin-3, a carbohydrate binding protein, with affinity for N-acetyllactosamine residues, has been unique due to the regulatory roles it performs in processes associated with tumor progression and metastasis such as cell proliferation, homotypic/heterotypic aggregation, dynamic cellular transformation, migration and invasion, survival and apoptosis. Structure-function association of galectin-3 reveals that it consists of a short amino terminal motif, which regulates its nuclear-cytoplasmic shuttling; a collagen α-like domain, susceptible to cleavage by matrix metalloproteases and prostate specific antigen; accountable for its oligomerization and lattice formation, and a carbohydrate-recognition/binding domain containing the anti-death motif of the Bcl2 protein family. This structural complexity permits galectin-3 to associate with numerous molecules utilizing protein-protein and/or protein-carbohydrate interactions in the extra-cellular as well as intracellular milieu and regulate diverse signaling pathways, a number of which appear directed towards epithelial-mesenchymal transition and cancer stemness. Self-renewal, differentiation, long-term culturing and drug-resistance potential characterize cancer stem cells (CSCs), a small cell subpopulation within the tumor that is thought to be accountable for heterogeneity, recurrence and metastasis of tumors. Despite the fact that association of galectin-3 to the tumor stemness phenomenon is still in its infancy, there is sufficient direct evidence of its regulatory roles in CSC-associated phenotypes and signaling pathways. In this review, we have highlighted the available data on galectin-3 regulated functions pertinent to cancer stemness and explored the opportunities of its exploitation as a CSC marker and a therapeutic target.

Cancer Self-Defense: An Immune Stealth.

The hurdles in realizing successful cancer immunotherapy stem from the fact that cancer patients are either refractory to immune response and/or develop resistance. Here, we propose that these phenomena are due, in part, to the deployment/secretion of a "decoy flare," for example, anomalous cancer-associated antigens by the tumor cells. The cancer secretome, which resembles the parent cell make-up, is composed of soluble macromolecules (proteins, glycans, lipids, DNAs, RNAs, etc.) and insoluble vesicles (exosomes), thus hindering cancer detection/recognition by immunotherapeutic agents, resulting in a "cancer-stealth" effect. Immunotherapy, or any treatment that relies on antigens' expression/function, could be improved by the understanding of the properties of the cancer secretome, as its clinical evaluation may change the therapeutic landscape. Cancer Res; 77(20); 5441-4. ©2017 AACR.

The influence of PSA autoantibodies in prostate cancer patients: a prospective clinical study-II.

The U.S. Preventive Services Task Force (USPSTF) has recommended against PSA-based screening for prostate cancer due to potential possibilities of false-results. Since no alternative test is available to replace it, we have initiated a trial with the purpose of establishing whether Galectin-3 (Gal-3) serum level and/or the patients' immune response to PSA and Gal-3 antigens could complement the PSA test as diagnostic tools for prostate cancer patients. A blind, prospective, single institution, pilot study was conducted. A total of 95 men were recruited and classified into 5 different groups: healthy controls (Group1), newly diagnosed patients (Group2), no recurrence after local therapy (Group3), rising PSA after local therapy (Group4), and metastatic patients (Group5). The primary endpoints were the levels of serum PSA, PSA autoantibodies (AAPSA), Gal-3, and Gal-3 autoantibodies (AAGal-3). Data were analyzed by Spearman's rank correlation (rho) and least squares linear regression modeling. The expression levels of PSA, AAPSA, Gal-3, and AAGal-3 were determined in both healthy controls and prostate cancer patients. Negative correlations were observed between PSA and AAPSA levels among all 95 men combined (rho = -0.321, P = 0.0021; fitted slope -0.288, P = 0.0048), and in metastatic patients (rho = -0.472, P = 0.0413; fitted slope -1.145, P = 0.0061). We suggest an association between PSA and AAPSA, whereby the AAPSA may alter PSA levels. It provides a novel outlook for prostate cancer diagnosis, and should serve as a basis for an all-inclusive diagnostic trial centering on patients with metastasis.

Positive associations between galectin-3 and PSA levels in prostate cancer patients: a prospective clinical study-I.

Galectin-3 (Gal-3), an oncogenic pro-inflammatory protein, has been suggested as a possible complementary diagnostic candidate to prostate specific antigen (PSA) blood test for prostate cancer patients. The presence of the proteins in the circulation (biomarkers) may elicit an intrinsic humoral immune reaction by generating autoantibodies, which consequently could alter the detection levels. Here, we report the associations of the two prostate cancer biomarkers, Gal-3 and PSA in patients at different clinical states of prostate cancer while taking into account the autoantibody levels. A blind, prospective, single institution, pilot study was conducted. A total of 95 men were classified into 5 groups: healthy controls (Group1), newly diagnosed patients (Group2), no recurrence after local therapy (Group3), rising PSA after local therapy (Group4), and metastatic patients (Group5). Gal-3 and PSA level were divided by their respective autoantibodies, which yielded relative PSA and relative Gal-3 levels. After the adjustments, Spearman's rank correlations and linear regression modeling revealed the positive associations between relative Gal-3 and relative PSA levels among all 95 men combined (rho = 0.446, P < 0.0001; fitted slope 0.448, P < 0.0001), in Group2 (rho = 0.616, P = 0.0050; fitted slope 0.438, P =0.0011), and Group3 (rho = 0.484, P = 0.0360; fitted slope 0.470, P = 0.0187). The data show positive associations of relative Gal-3 and relative PSA levels in prostate cancer patients, notably at early clinical time course. Allowing for the influence of autoantibodies, Gal-3 level might be considered as a potential biomarker since it is positively associated with PSA level.

Galectin-3 in bone tumor microenvironment: a beacon for individual skeletal metastasis management.

The skeleton is frequently a secondary growth site of disseminated cancers, often leading to painful and devastating clinical outcomes. Metastatic cancer distorts bone marrow homeostasis through tumor-derived factors, which shapes different bone tumor microenvironments depending on the tumor cells' origin. Here, we propose a novel insight on tumor-secreted Galectin-3 (Gal-3) that controls the induction of an inflammatory cascade, differentiation of osteoblasts, osteoclasts, and bone marrow cells, resulting in bone destruction and therapeutic failure. In the approaching era of personalized medicine, the current treatment modalities targeting bone metastatic environments are provided to the patient with limited consideration of the cancer cells' origin. Our new outlook suggests delivering individual tumor microenvironment treatments based on the expression level/activity/functionality of tumor-derived factors, rather than utilizing a commonly shared therapeutic umbrella. The notion of "Gal-3-associated bone remodeling" could be the first step toward a specific personalized therapy for each cancer type generating a different bone niche in patients afflicted with non-curable bone metastasis.

Galectin-3 Cleavage Alters Bone Remodeling: Different Outcomes in Breast and Prostate Cancer Skeletal Metastasis.

Management of bone metastasis remains clinically challenging and requires the identification of new molecular target(s) that can be therapeutically exploited to improve patient outcome. Galectin-3 (Gal-3) has been implicated as a secreted factor that alters the bone microenvironment. Proteolytic cleavage of Gal-3 may also contribute to malignant cellular behaviors, but has not been addressed in cancer metastasis. Here, we report that Gal-3 modulates the osteolytic bone tumor microenvironment in the presence of RANKL. Gal-3 was localized on the osteoclast cell surface, and its suppression by RNAi or a specific antagonist markedly inhibited osteoclast differentiation markers, including tartrate-resistant acid phosphatase, and reduced the number of mature osteoclasts. Structurally, the 158-175 amino acid sequence in the carbohydrate recognition domain (CRD) of Gal-3 was responsible for augmented osteoclastogenesis. During osteoclast maturation, Gal-3 interacted and colocalized with myosin-2A along the surface of cell-cell fusion. Pathologically, bone metastatic cancers expressed and released an intact form of Gal-3, mainly detected in breast cancer bone metastases, as well as a cleaved form, more abundant in prostate cancer bone metastases. Secreted intact Gal-3 interacted with myosin-2A, leading to osteoclastogenesis, whereas a shift to cleaved Gal-3 attenuated the enhancement in osteoclast differentiation. Thus, our studies demonstrate that Gal-3 shapes the bone tumor microenvironment through distinct roles contingent on its cleavage status, and highlight Gal-3 targeting through the CRD as a potential therapeutic strategy for mitigating osteolytic bone remodeling in the metastatic niche.

Extracellular galectin-3 programs multidrug resistance through Na+/K+-ATPase and P-glycoprotein signaling.

Galectin-3 (Gal-3, LGALS3) is a pleotropic versatile, 29-35 kDa chimeric gene product, and involved in diverse physiological and pathological processes, including cell growth, homeostasis, apoptosis, pre-mRNA splicing, cell-cell and cell-matrix adhesion, cellular polarity, motility, adhesion, activation, differentiation, transformation, signaling, regulation of innate/adaptive immunity, and angiogenesis. In multiple diseases, it was found that the level of circulating Gal-3 is markedly elevated, suggesting that Gal-3-dependent function is mediated by specific interaction with yet an unknown ubiquitous cell-surface protein. Recently, we showed that Gal-3 attenuated drug-induced apoptosis, which is one of the mechanisms underlying multidrug resistance (MDR). Here, we document that MDR could be mediated by Gal-3 interaction with the house-keeping gene product e.g., Na+/K+-ATPase, and P-glycoprotein (P-gp). Gal-3 interacts with Na+/K+-ATPase and induces the phosphorylation of P-gp. We also find that Gal-3 binds P-gp and enhances its ATPase activity. Furthermore Gal-3 antagonist suppresses this interaction and results in a decrease of the phosphorylation and the ATPase activity of P-gp, leading to an increased sensitivity to doxorubicin-mediated cell death. Taken together, these findings may explain the reported roles of Gal-3 in diverse diseases and suggest that a combined therapy of inhibitors of Na+/K+-ATPase and Gal-3, and a disease specific drug(s) might be superior to a single therapeutic modality.

Galectin-3 leads to attenuation of apoptosis through Bax heterodimerization in human thyroid carcinoma cells.

Cancer cells survive escaping normal apoptosis and the blocks in apoptosis that keep cancer cells alive are promising candidates for targeted therapy. Galectin-3 (Gal-3) is, a member of the lectin family, which is involved in cell growth, adhesion, proliferation and apoptosis. It remains elusive to understand the role of Gal-3 on apoptosis in thyroid carcinoma cells. Here, we report that Gal-3 heterodimerizes Bax, mediated by the carbohydrate recognition domain (CRD) of Gal-3, leading to anti-apoptotic characteristic. Gal-3/Bax interaction was suppressed by an antagonist of Gal-3, in which in turn cells became sensitive to apoptosis. The data presented here highlight that Gal-3 is involved in the anti-apoptosis of thyroid carcinoma cells. Thus, it suggests that targeting Gal-3 may lead to an improved therapeutic modality for thyroid cancer.

Galectin-3 inhibits osteoblast differentiation through notch signaling.

Patients with bone cancer metastasis suffer from unbearable pain and bone fractures due to bone remodeling. This is caused by tumor cells that disturb the bone microenvironment. Here, we have investigated the role of tumor-secreted sugar-binding protein, i.e., galectin-3, on osteoblast differentiation and report that it downregulates the expression of osteoblast differentiation markers, e.g., RUNX2, SP7, ALPL, COL1A1, IBSP, and BGLAP, of treated human fetal osteoblast (hFOB) cells. Co-culturing of hFOB cells with human breast cancer BT-549 and prostate cancer LNCaP cells harboring galectin-3 has resulted in inhibition of osteoblast differentiation by the secreted galectin-3 into culture medium. The inhibitory effect of galectin-3 was found to be through its binding to Notch1 in a sugar-dependent manner that has led to accelerated Notch1 cleavage and activation of Notch signaling. Taken together, our findings show that soluble galectin-3 in the bone microenvironment niche regulates bone remodeling through Notch signaling, suggesting a novel bone metastasis therapeutic target.

Autocrine motility factor promotes HER2 cleavage and signaling in breast cancer cells.

Trastuzumab (Herceptin) is an effective targeted therapy in HER2-overexpressing human breast carcinoma. However, many HER2-positive patients initially or eventually become resistant to this treatment, so elucidating mechanisms of trastuzumab resistance that emerge in breast carcinoma cells is clinically important. Here, we show that autocrine motility factor (AMF) binds to HER2 and induces cleavage to the ectodomain-deleted and constitutively active form p95HER2. Mechanistic investigations indicated that interaction of AMF with HER2 triggers HER2 phosphorylation and metalloprotease-mediated ectodomain shedding, activating phosphoinositide-3-kinase (PI3K) and mitogen-activated protein kinase signaling and ablating the ability of trastuzumab to inhibit breast carcinoma cell growth. Furthermore, we found that HER2 expression and AMF secretion were inversely related in breast carcinoma cells. On the basis of this evidence that AMF may contribute to HER2-mediated breast cancer progression, our findings suggest that AMF-HER2 interaction might be a novel target for therapeutic management of patients with breast cancer, whose disease is resistant to trastuzumab.

Cleavage of galectin-3 by matrix metalloproteases induces angiogenesis in breast cancer.

Galectin-3 cleavage is related to progression of human breast and prostate cancer and is partly responsible for tumor growth, angiogenesis and apoptosis resistance in mouse models. A functional polymorphism in galectin-3 gene, determining its susceptibility to cleavage by matrix metalloproteinases (MMPs)-2/-9 is related to racial disparity in breast cancer incidence in Asian and Caucasian women. The purpose of our study is to evaluate (i) if cleavage of galectin-3 could be related to angiogenesis during the progression of human breast cancer, (ii) the role of cleaved galectin-3 in induction of angiogenesis and (iii) determination of the galectin-3 domain responsible for induction of angiogenic response. Galectin-3 null breast cancer cells BT-459 were transfected with either cleavable full-length galectin-3 or its fragmented peptides. Chemotaxis, chemoinvasion, heterotypic aggregation, epithelial-endothelial cell interactions and angiogenesis were compared to noncleavable galectin-3. BT-549-H(64) cells harboring cleavable galectin-3 exhibited increased chemotaxis, invasion and interactions with endothelial cells resulting in angiogenesis and 3D morphogenesis compared to BT-549-P(64) cells harboring noncleavable galectin-3. BT-549-H(64) cells induced increased migration and phosphorylation of focal adhesion kinase in migrating endothelial cells. Endothelial cells cocultured with BT-549 cells transfected with galectin-3 peptides indicate that amino acids 1-62 and 33-250 stimulate migration and morphogenesis of endothelial cells. Immunohistochemical analysis of blood vessel density and galectin-3 cleavage in a breast cancer progression tissue array support the in vitro findings. We conclude that the cleavage of the N terminus of galectin-3 followed by its release in the tumor microenvironment in part leads to breast cancer angiogenesis and progression.

Phosphoglucose isomerase/autocrine motility factor mediates epithelial and mesenchymal phenotype conversions in breast cancer.

Phosphoglucose isomerase/autocrine motility factor (PGI/AMF) is a housekeeping gene product/cytokine that catalyzes a step in glycolysis and gluconeogenesis, and acts as a multifunctional cytokine associated with aggressive tumors. PGI/AMF has been correlated significantly with breast cancer progression and poor prognosis in breast cancer. We show here that ectopic expression of PGI/AMF induced epithelial-to-mesenchymal transition (EMT) in MCF10A normal human breast epithelial cells, and inhibition of PGI/AMF expression triggered mesenchymal-to-epithelial transition (MET) in aggressive mesenchymal-type human breast cancer MDA-MB-231 cells. EMT in MCF10A cells was shown by morphologic changes and loss of E-cadherin/beta-catenin-mediated cell-cell adhesion, which is concomitant with the induction of the E-cadherin transcriptional repressor Snail and proteosome-dependent degradation of beta-catenin protein. Molecular analysis showed that PGI/AMF suppressed epithelial marker expressions and enhanced mesenchymal marker expressions. Silencing of PGI/AMF expression by RNA interference in MDA-MB-231 cells induced the reverse processes of EMT including altered cell shape, gain of epithelial marker, and reduction of mesenchymal marker, e.g., MET. Taken together, the results show the involvement of PGI/AMF in both EMT and MET: overexpression of PGI/AMF induces EMT in normal breast epithelial cells and reduction of PGI/AMF expression led to MET in aggressive breast cancer cells. These results suggest for the first time that PGI/AMF is a key gene to both EMT in the initiating step of cancer metastasis and MET in the later stage of metastasis during breast cancer progression.

Regulation of prostate cancer progression by galectin-3.

Galectin-3, a beta-galactoside-binding protein, has been implicated in a variety of biological functions including cell proliferation, apoptosis, angiogenesis, tumor progression, and metastasis. The present study was undertaken to understand the role of galectin-3 in the progression of prostate cancer. Immunohistochemical analysis of galectin-3 expression revealed that galectin-3 was cleaved during the progression of prostate cancer. Galectin-3 knockdown by small interfering RNA (siRNA) was associated with reduced cell migration, invasion, cell proliferation, anchorage-independent colony formation, and tumor growth in the prostates of nude mice. Galectin-3 knockdown in human prostate cancer PC3 cells led to cell-cycle arrest at G(1) phase, up-regulation of nuclear p21, and hypophosphorylation of the retinoblastoma tumor suppressor protein (pRb), with no effect on cyclin D1, cyclin E, cyclin-dependent kinases (CDK2 and CDK4), and p27 protein expression levels. The data obtained here implicate galectin-3 in prostate cancer progression and suggest that galectin-3 may serve as both a diagnostic marker and therapeutic target for future disease treatments.

Racial disparity in breast cancer and functional germ line mutation in galectin-3 (rs4644): a pilot study.

For reasons largely unknown, Caucasian women are at a significantly higher risk of developing breast cancer than Asian women. Over a decade ago, mutations in BRCA1/2 were identified as genetic risk factors; however, the discovery of additional breast cancer genes and genes contributing to racial disparities are lacking. We report a functional germline mutation (polymorphism) in the galectin-3 gene at position 191 (rs4644) substituting proline with histidine (P64H), which results in susceptibility to matrix metalloproteinase cleavage and acquisition of resistance to drug-induced apoptosis. This substitution correlates with incidence of breast cancer and racial disparity. Genotype analysis of 338 Caucasian (194 disease free and 144 breast cancer patients) and 140 Asian (79 disease free and 61 breast cancer patients) women showed that the allele homozygous for H64 exists in disease free Caucasian and Asian women at a frequency of 12% and 5%, respectively, versus 37% and 82% in breast cancer patients. The data indicate that H/H allele is associated with increased breast cancer risk in both races. The data implicate galectin-3 H(64) in breast cancer and explain, in part, the noted racial disparity, thus providing a novel target for diagnosis and treatment.

Galectin-3 cleavage: a novel surrogate marker for matrix metalloproteinase activity in growing breast cancers.

Failed therapies directed against matrix metalloproteinases (MMP) in cancer patients may be attributed, in part, to lack of diagnostic tools to differentiate between pro-MMPs and active MMPs, which indicate whether a treatment is efficacious or not. Because galectin-3 is cleavable in vitro by MMPs, we have developed differential antibodies recognizing its cleaved and noncleaved forms and tested their clinical utilization as a surrogate diagnostic marker for the presence of active MMPs in growing breast cancers. Wild-type and cleavage-resistant galectin-3 were constructed and expressed in galectin-3-null human breast carcinoma cells (BT-549). Tumorigenic and angiogenic potential of the clones was studied by injections into nude mice. MMP-2, MMP-9, full-length, and cleaved galectin-3 were localized in the xenografts by immunohistochemical analysis of paraffin-embedded sections using specific antibodies. Activities of MMP-2/9 were corroborated by in situ zymography on frozen tissue sections. Galectin-3 cleavage was shown in vivo by differential antibody staining and colocalized with predicted active MMPs both in mouse xenografts and human breast cancer specimens. In situ zymography validated these results. In addition, BT-549 cells harboring noncleavable galectin-3 showed reduced tumor growth and angiogenesis compared with the wild-type. We conclude that galectin-3 cleavage is an active process during tumor progression and could be used as a simple, rapid, and reliable surrogate marker for the activities of MMPs in growing breast cancers.

Down-regulation of phosphoglucose isomerase/autocrine motility factor expression sensitizes human fibrosarcoma cells to oxidative stress leading to cellular senescence.

Phosphoglucose isomerase/autocrine motility factor (PGI/AMF) is a housekeeping gene product present in all cells, is an essential enzyme of catabolic glycolysis and anabolic gluconeogenesis, and regulates tumor cell growth and metastasis. Because glycolytic enzyme up-regulation of expression contributes to glycolytic flux, leading to increased of cell growth and a resistance to cellular stress of normal fibroblasts whereas down-regulation of PGI/AMF leads to mesenchymal-to-epithelial transition in tumor cells, we examined the involvement of PGI/AMF in overcoming cellular senescence in cancer cells. PGI/AMF cellular expression in HT1080 human fibrosarcoma was down-regulated by small interfering RNA methodology, which resulted in an increased sensitivity to oxidative stress and oxidative stress-induced cellular senescence. Signaling analysis revealed that the senescence pathway involving p21 cyclin-dependent kinase inhibitor was up-regulated in PGI/AMF knockdown cells and that superoxide dismutase is the upstream regulator protein of p21-mediated cellular senescence. A specific inhibitor of PGI/AMF induced cellular senescence and p21 expression in tumor cells exposed to an oxidative stress environment. Taken together, the results presented here suggest that PGI/AMF is involved in oxidative stress-induced cellular senescence and should bring novel insights into the control of cellular growth leading to a new methodology for cancer treatment.

Down-regulation of phosphoglucose isomerase/autocrine motility factor results in mesenchymal-to-epithelial transition of human lung fibrosarcoma cells.

Phosphoglucose isomerase (PGI) is one of the glycolytic enzymes and is a multifunctional enzyme that functions in glucose metabolism inside the cell while acting as a cytokine outside the cell, with properties that include autocrine motility factor (AMF) regulating tumor cell motility. Although there are many studies indicating that PGI/AMF has been implicated in progression of metastasis, no direct studies of the significance of exogenous PGI/AMF on tumor progression have been reported. Here, we report on the mesenchymal-to-epithelial transition (MET), which is the reverse phenomenon of the epithelial-to-mesenchymal transition that is associated with loss of cell polarity, loss of epithelia markers, and enhancement of cell motility essential for tumor cell invasion and metastasis. Mesenchymal human fibrosarcoma HT1080 cells, which have naturally high levels of endogenous and exogenous PGI/AMF, were stably transfected with PGI/AMF small interfering RNA (siRNA). The siRNA targeting human PGI/AMF down-regulated the endogenous PGI/AMF expression and completely extinguished the secretion of PGI/AMF in a human fibrosarcoma HT1080, whereas the control siRNA showed no effects. The PGI/AMF siRNA caused cells to change shape dramatically and inhibited cell motility and invasion markedly. Suppression of PGI/AMF led to a contact-dependent inhibition of cell growth. Those PGI/AMF siRNA-transfected cells showed epithelial phenotype. Furthermore, tumor cells with PGI/AMF deficiency lost their abilities to form tumor mass. This study identifies that MET in HT1080 human lung fibrosarcoma cells was initiated by down-regulation of the housekeeping gene product/cytokine PGI/AMF, and the results depicted here suggest a novel therapeutic target/modality for mesenchymal cancers.