Osteopontin is a proximal effector of leptin-mediated non-alcoholic steatohepatitis (NASH) fibrosis.

INTRODUCTION: Liver fibrosis develops when hepatic stellate cells (HSC) are activated into collagen-producing myofibroblasts. In non-alcoholic steatohepatitis (NASH), the adipokine leptin is upregulated, and promotes liver fibrosis by directly activating HSC via the hedgehog pathway. We reported that hedgehog-regulated osteopontin (OPN) plays a key role in promoting liver fibrosis. Herein, we evaluated if OPN mediates leptin-profibrogenic effects in NASH. METHODS: Leptin-deficient (ob/ob) and wild-type (WT) mice were fed control or methionine-choline deficient (MCD) diet. Liver tissues were assessed by Sirius-red, OPN and αSMA IHC, and qRT-PCR for fibrogenic genes. In vitro, HSC with stable OPN (or control) knockdown were treated with recombinant (r)leptin and OPN-neutralizing or sham-aptamers. HSC response to OPN loss was assessed by wound healing assay. OPN-aptamers were also added to precision-cut liver slices (PCLS), and administered to MCD-fed WT (leptin-intact) mice to determine if OPN neutralization abrogated fibrogenesis. RESULTS: MCD-fed WT mice developed NASH-fibrosis, upregulated OPN, and accumulated αSMA+ cells. Conversely, MCD-fed ob/ob mice developed less fibrosis and accumulated fewer αSMA+ and OPN+ cells. In vitro, leptin-treated HSC upregulated OPN, αSMA, collagen 1α1 and TGFß mRNA by nearly 3-fold, but this effect was blunted by OPN loss. Inhibition of PI3K and transduction of dominant negative-Akt abrogated leptin-mediated OPN induction, while constitutive active-Akt upregulated OPN. Finally, OPN neutralization reduced leptin-mediated fibrogenesis in both PCLS and MCD-fed mice. CONCLUSION: OPN overexpression in NASH enhances leptin-mediated fibrogenesis via PI3K/Akt. OPN neutralization significantly reduces NASH fibrosis, reinforcing the potential utility of targeting OPN in the treatment of patients with advanced NASH.

Alcohol inhibits osteopontin-dependent transforming growth factor-ß1 expression in human mesenchymal stem cells.

Alcohol (EtOH) intoxication is a risk factor for increased morbidity and mortality with traumatic injuries, in part through inhibition of bone fracture healing. Animal models have shown that EtOH decreases fracture callus volume, diameter, and biomechanical strength. Transforming growth factor ß1 (TGF-ß1) and osteopontin (OPN) play important roles in bone remodeling and fracture healing. Mesenchymal stem cells (MSC) reside in bone and are recruited to fracture sites for the healing process. Resident MSC are critical for fracture healing and function as a source of TGF-ß1 induced by local OPN, which acts through the transcription factor myeloid zinc finger 1 (MZF1). The molecular mechanisms responsible for the effect of EtOH on fracture healing are still incompletely understood, and this study investigated the role of EtOH in affecting OPN-dependent TGF-ß1 expression in MSC. We have demonstrated that EtOH inhibits OPN-induced TGF-ß1 protein expression, decreases MZF1-dependent TGF-ß1 transcription and MZF1 transcription, and blocks OPN-induced MZF1 phosphorylation. We also found that PKA signaling enhances OPN-induced TGF-ß1 expression. Last, we showed that EtOH exposure reduces the TGF-ß1 protein levels in mouse fracture callus. We conclude that EtOH acts in a novel mechanism by interfering directly with the OPN-MZF1-TGF-ß1 signaling pathway in MSC.

Components of Hospital Perioperative Infrastructure Can Overcome the Weekend Effect in Urgent General Surgery Procedures.

OBJECTIVE: We hypothesized that perioperative hospital resources could overcome the "weekend effect" (WE) in patients undergoing emergent/urgent surgeries. SUMMARY BACKGROUND DATA: The WE is the observation that surgeon-independent patient outcomes are worse on the weekend compared with weekdays. The WE is often explained by differences in staffing and resources resulting in variation in care between the week and weekend. METHODS: Emergent/urgent surgeries were identified using the Healthcare Cost and Utilization Project State Inpatient Database (Florida) from 2007 to 2011 and linked to the American Hospital Association (AHA) Annual Survey Database to determine hospital level characteristics. Extended median length of stay (LOS) on the weekend compared with the weekdays (after controlling for hospital, year, and procedure type) was selected as a surrogate for WE. RESULTS: Included were 126,666 patients at 166 hospitals. A total of 17 hospitals overcame the WE during the study period. Logistic regression, controlling for patient characteristics, identified full adoption of electronic medical records (OR 4.74), home health program (OR 2.37), pain management program [odds ratio (OR) 1.48)], increased registered nurse-to-bed ratio (OR 1.44), and inpatient physical rehabilitation (OR 1.03) as resources that were predictors for overcoming the WE. The prevalence of these factors in hospitals exhibiting the WE for all 5 years of the study period were compared with those hospitals that overcame the WE (P < 0.001). CONCLUSIONS: Specific hospital resources can overcome the WE seen in urgent general surgery procedures. Improved hospital perioperative infrastructure represents an important target for overcoming disparities in surgical care.

Osteopontin is up-regulated in chronic hepatitis C and is associated with cellular permissiveness for hepatitis C virus replication.

OPN (osteopontin)) is a Hh (Hedgehog)-regulated cytokine that is up-regulated during chronic liver injury and directly promotes fibrosis. We have reported that Hh signalling enhances viral permissiveness and replication in HCV (hepatitis C virus)-infected cells. Hence we hypothesized that OPN directly promotes HCV replication, and that targeting OPN could be beneficial in HCV. In the present study, we compared the expression of OPN mRNA and protein in HCV (JFH1)-infected Huh7 and Huh7.5 cells, and evaluated whether modulating OPN levels using exogenous OPN ligands (up-regulate OPN) or OPN-specific RNA-aptamers (neutralize OPN) leads to changes in HCV expression. Sera and livers from patients with chronic HCV were analysed to determine whether OPN levels were associated with disease severity or response to therapy. Compared with Huh7 cells, Huh7.5 cells support higher levels of HCV replication (15-fold) and expressed significantly more OPN mRNA (30-fold) and protein. Treating Huh7 cells with OPN ligands led to a dose-related increase in HCV (15-fold) and OPN (8-fold) mRNA. Conversely, treating Huh7.5 cells with OPN-specific RNA aptamers inhibited HCV RNA and protein by >50% and repressed OPN mRNA to basal levels. Liver OPN expression was significantly higher (3-fold) in patients with advanced fibrosis. Serum OPN positively correlated with fibrosis-stage (P=0.009), but negatively correlated with ETBCR (end-of-treatment biochemical response), ETVR (end-of-treatment virological response), SBCR (sustained biochemical response) and SVR (sustained virological response) (P=0.007). The OPN fibrosis score (serum OPN and presence of fibrosis ≥F2) may be a predictor of SVR. In conclusion, OPN is up-regulated in the liver and serum of patients with chronic hepatitis C, and supports increased viral replication. OPN neutralization may be a novel therapeutic strategy in chronic hepatitis C.

Examining surgeon stress in robotic and laparoscopic surgery.

Robotic surgery may decrease surgeon stress compared to laparoscopic. To evaluate intraoperative surgeon stress, we measured salivary alpha-amylase and cortisol. We hypothesized robotic elicited lower increases in surgeon salivary amylase and cortisol than laparoscopic. Surgical faculty (n = 7) performing laparoscopic and robotic operations participated. Demographics: age, years in practice, time using laparoscopic vs robotic, comfort level and enthusiasm for each. Operative data included operative time, WRVU (surgical "effort"), resident year. Saliva was collected using passive drool collection system at beginning, middle and end of each case; amylase and cortisol measured using ELISA. Standard values were created using 7-minute exercise (HIIT), collecting saliva pre- and post-workout. Linear regression and Student's t test used for statistical analysis; p values < 0.05 were significant. Ninety-four cases (56 robotic, 38 laparoscopic) were collected (April-October 2022). Standardized change in amylase was 8.4 ± 4.5 (p < 0.001). Among operations, raw maximum amylase change in laparoscopic and robotic was 23.4 ± 11.5 and 22.2 ± 13.4; raw maximum cortisol change was 44.21 ± 46.57 and 53.21 ± 50.36, respectively. Values normalized to individual surgeon HIIT response, WRVU, and operative time, showing 40% decrease in amylase in robotic: 0.095 ± 0.12, vs laparoscopic: 0.164 ± 0.16 (p < 0.02). Normalized change in cortisol was: laparoscopic 0.30 ± 0.44, robotic 0.22 ± 0.4 (p = NS). On linear regression (p < 0.001), surgeons comfortable with complex laparoscopic cases had lower change in normalized amylase (p < 0.01); comfort with complex robotic was not significant. Robotic may be less physiologically stressful, eliciting less increase in salivary amylase than laparoscopic. Comfort with complex laparoscopic decreased stress in robotic, suggesting laparoscopic experience is valuable prior to robotic.

NKT-associated hedgehog and osteopontin drive fibrogenesis in non-alcoholic fatty liver disease.

OBJECTIVE: Immune responses are important in dictating non-alcoholic steatohepatitis (NASH) outcome. We previously reported that upregulation of hedgehog (Hh) and osteopontin (OPN) occurs in NASH, that Hh-regulated accumulation of natural killer T (NKT) cells promotes hepatic stellate cell (HSC) activation, and that cirrhotic livers harbour large numbers of NKT cells. DESIGN: The hypothesis that activated NKT cells drive fibrogenesis during NASH was evaluated by assessing if NKT depletion protects against NASH fibrosis; identifying the NKT-associated fibrogenic factors; and correlating plasma levels of the NKT cell-associated factor OPN with fibrosis severity in mice and humans. RESULTS: When fed methionine-choline-deficient (MCD) diets for 8 weeks, wild type (WT) mice exhibited Hh pathway activation, enhanced OPN expression, and NASH-fibrosis. Ja18-/- and CD1d-/- mice which lack NKT cells had significantly attenuated Hh and OPN expression and dramatically less fibrosis. Liver mononuclear cells (LMNCs) from MCD diet fed WT mice contained activated NKT cells, generated Hh and OPN, and stimulated HSCs to become myofibroblasts; neutralising these factors abrogated the fibrogenic actions of WT LMNCs. LMNCs from NKT-cell-deficient mice were deficient in fibrogenic factors, failing to activate collagen gene expression in HSCs. Human NASH livers with advanced fibrosis contained more OPN and Hh protein than those with early fibrosis. Plasma levels of OPN mirrored hepatic OPN expression and correlated with fibrosis severity. CONCLUSION: Hepatic NKT cells drive production of OPN and Hh ligands that promote fibrogenesis during NASH. Associated increases in plasma levels of OPN may provide a biomarker of NASH fibrosis.

RNA Aptamer Targeting of Adam8 in Cancer Growth and Metastasis.

Cancer progression depends on an accumulation of metastasis-supporting physiological changes, which are regulated by cell-signaling molecules. In this regard, a disintegrin and metalloproteinase 8 (Adam8) is a transmembrane glycoprotein that is selectively expressed and induced by a variety of inflammatory stimuli. In this study, we identified Adam8 as a sox2-dependent protein expressed in MDA-MB-231 breast cancer cells when cocultured with mesenchymal-stem-cell-derived myofibroblast-like cancer-associated fibroblasts (myCAF). We have previously found that myCAF-induced cancer stemness is required for the maintenance of the myCAF phenotype, suggesting that the initiation and maintenance of the myCAF phenotype require distinct cell-signaling crosstalk pathways between cancer cells and myCAF. Adam8 was identified as a candidate secreted protein induced by myCAF-mediated cancer stemness. Adam8 has a known sheddase function against which we developed an RNA aptamer, namely, Adam8-Apt1-26nt. The Adam8-Apt1-26nt-mediated blockade of the extracellular soluble Adam8 metalloproteinase domain abolishes the previously initiated myCAF phenotype, or, termed differently, blocks the maintenance of the myCAF phenotype. Consequently, cancer stemness is significantly decreased. Xenograft models show that Adam8-Apt-1-26nt administration is associated with decreased tumor growth and metastasis, while flow cytometric analyses demonstrate a significantly decreased fraction of myCAF after Adam8-Apt-1-26nt treatment. The role of soluble Adam8 in the maintenance of the myCAF phenotype has not been previously characterized. Our study suggests that the signal pathways for the induction or initiation of the myCAF phenotype may be distinct from those involved with the maintenance of the myCAF phenotype.

Osteopontin regulates epithelial mesenchymal transition-associated growth of hepatocellular cancer in a mouse xenograft model.

OBJECTIVE: To determine the efficacy of osteopontin (OPN) targeting in hepatocellular cancer (HCC). SUMMARY/BACKGROUND: OPN is associated with HCC growth and metastasis and represents a unique therapeutic target. METHODS: OPN and epithelial-mesenchymal transition (EMT) markers, α-smooth muscle actin (SMA), vimentin, and tenascin-c, were measured in archived human HCC tissues from metastatic (n = 4) and nonmetastatic (n = 4) settings. Additional studies utilized human Sk-Hep-1 (high OPN expression) and Hep3b (low OPN expression) HCC cells. An RNA aptamer (APT) that avidly binds (Kd = 18 nM; t1/2 = 7 hours) and ablates OPN binding was developed. Adhesion, migration/invasion, and EMT markers were determined with APT or a mutant control aptamer (Mu-APT). RFP-Luc-Sk-Hep-1 were implanted into NOD-scid mice livers and followed by using bioluminescence imaging. After verification of tumor growth, at week 3, APT (0.5 mg/kg; n = 4) or Mu-APT (0.5 mg/kg; n = 4) was injected q48h. When mice were killed at week 8, tumor cells were reisolated and assayed for EMT markers. RESULTS: OPN and EMT markers were significantly increased in the metastatic cohort. APT inhibited Sk-Hep-1 adhesion and migration/invasion by 5- and 4-fold, respectively. APT significantly decreased EMT protein markers, SMA, vimentin, and tenascin-c. In contrast, APT did not alter Hep3B adhesion, or migration/invasion. EMT markers were slightly decreased. In the in vivo model, at weeks 6 to 8, APT inhibited HCC growth by more than 10-fold. SMA, vimentin, and tenascin-c mRNAs were decreased by 60%, 40%, and 49%, respectively, in RFP-positive Sk-Hep-1 recovered by fluorescence-activated cell sorting (P < 0.04 vs Mu-APT for all). CONCLUSIONS: APT targeting of OPN significantly decreases EMT and tumor growth of HCC.

Osteopontin is induced by hedgehog pathway activation and promotes fibrosis progression in nonalcoholic steatohepatitis.

UNLABELLED: Nonalcoholic steatohepatitis (NASH) is a leading cause of cirrhosis. Recently, we showed that NASH-related cirrhosis is associated with Hedgehog (Hh) pathway activation. The gene encoding osteopontin (OPN), a profibrogenic extracellular matrix protein and cytokine, is a direct transcriptional target of the Hh pathway. Thus, we hypothesize that Hh signaling induces OPN to promote liver fibrosis in NASH. Hepatic OPN expression and liver fibrosis were analyzed in wild-type (WT) mice, Patched-deficient (Ptc(+/-) ) (overly active Hh signaling) mice, and OPN-deficient mice before and after feeding methionine and choline-deficient (MCD) diets to induce NASH-related fibrosis. Hepatic OPN was also quantified in human NASH and nondiseased livers. Hh signaling was manipulated in cultured liver cells to assess direct effects on OPN expression, and hepatic stellate cells (HSCs) were cultured in medium with different OPN activities to determine effects on HSC phenotype. When fed MCD diets, Ptc(+/-) mice expressed more OPN and developed worse liver fibrosis (P < 0.05) than WT mice, whereas OPN-deficient mice exhibited reduced fibrosis (P < 0.05). In NASH patients, OPN was significantly up-regulated and correlated with Hh pathway activity and fibrosis stage. During NASH, ductular cells strongly expressed OPN. In cultured HSCs, SAG (an Hh agonist) up-regulated, whereas cyclopamine (an Hh antagonist) repressed OPN expression (P < 0.005). Cholangiocyte-derived OPN and recombinant OPN promoted fibrogenic responses in HSCs (P < 0.05); neutralizing OPN with RNA aptamers attenuated this (P < 0.05). CONCLUSION: OPN is Hh-regulated and directly promotes profibrogenic responses. OPN induction correlates with Hh pathway activity and fibrosis stage. Therefore, OPN inhibition may be beneficial in NASH.

Tumor: Stroma Interaction and Cancer.

The understanding of how normal cells transform into tumor cells and progress to invasive cancer and metastases continues to evolve. The tumor mass is comprised of a heterogeneous population of cells that include recruited host immune cells, stromal cells, matrix components, and endothelial cells. This tumor microenvironment plays a fundamental role in the acquisition of hallmark traits, and has been the intense focus of current research. A key regulatory mechanism triggered by these tumor-stroma interactions includes processes that resemble epithelial-mesenchymal transition, a physiologic program that allows a polarized epithelial cell to undergo biochemical and cellular changes and adopt mesenchymal cell characteristics. These cellular adaptations facilitate enhanced migratory capacity, invasiveness, elevated resistance to apoptosis, and greatly increased production of ECM components. Indeed, it has been postulated that cancer cells undergo epithelial-mesenchymal transition to invade and metastasize.In the following discussion, the physiology of chronic inflammation, wound healing, fibrosis, and tumor invasion will be explored. The key regulatory cytokines, transforming growth factor ß and osteopontin, and their roles in cancer metastasis will be highlighted.

Maintaining Myofibroblastic-Like Cancer-Associated Fibroblasts by Cancer Stemness Signal Transduction Feedback Loop.

BACKGROUND: Myofibroblast-like cancer-associated fibroblasts (myCAF) in the tumor microenvironment (TME) promote cancer stemness, growth, and metastasis. Cancer cell-derived osteopontin (OPN) has been reported as a biomarker related to malignant cancer growth. In this study, we confirm that cancer cell stemness is required for the maintenance of an OPN-induced myCAF phenotype. METHODS: MDA-MB-231 or HepG2 cells and Sox2 knockout variants were co-cultured with human mesenchymal stem cells (MSC). In selected instances, the OPN bioactivity inhibitor OPN-R3 aptamer (APT), OPN-R3 mutant aptamer (MuAPT), or cancer cell stemness inhibitor BBI-608 were added separately. MDA-MB-231 cancer stemness and myCAF markers were quantified by real-time PCR. Stemness-lacking cancer cell mice models were created to confirm that stemness is required for the maintenance of the OPN-induced myCAF phenotype in vivo. RESULTS: In an MDA-MB-231 co-culture system, myCAF and stemness markers increased. Osteopontin and stemness blockade in this co-culture system decreased both myCAF and stemness marker expression, but OPN blockade after 72 hours had no effect. In contrast, when BBI608 was added at 72 hours, myCAF markers were abated after 36-hour treatment. Replacing wildtype with MDA-MB-231(-/-sox2) in co-cultures at 72 hours decreased myCAF marker expression to baseline despite the Western blot confirming the presence of OPN. Conversely, replacing MDA-MB-231(-/-sox2) cells with wildtype increased myCAF marker expression to a level equivalent to the MDA-MB-231+MSC co-culture system. In vivo osteopontin blockade diminished stemness and myCAF marker expression and stemness lacking cancer cell models, indicated by decreasing myCAF presence. Experiments were repeated in a HepG2 cell line with identical results. CONCLUSIONS: Cancer and myCAF crosstalk increases myCAF maintenance and cancer cell stemness. In this study using human breast and liver cancer cell lines, maintenance of the OPN-induced myCAF phenotype also requires cancer stemness. This indicates that the myCAF phenotype requires two distinct signaling pathways: initiation and maintenance.

Osteopontin promotes CCL5-mesenchymal stromal cell-mediated breast cancer metastasis.

The interaction between cancer and its local microenvironment can determine properties of growth and metastasis. A critical component of the tumor microenvironment in this context is the cancer-associated fibroblast (CAF), which can promote tumor growth, angiogenesis and metastasis. It has been hypothesized that CAF may be derived from mesenchymal stromal cells (MSC), derived from local or distant sources. However, the signaling mechanisms by which tumors and MSCs interact to promote CAF-dependent cancer growth are largely unknown. In this study with in vitro and in vivo models using MDA-MB231 human breast cancer cells, we demonstrate that tumor-derived osteopontin (OPN) induces MSC production of CCL5; the mechanism involves OPN binding to integrin cell surface receptors and activator protein-1 c-jun homodimer transactivation. In a murine xenograft model, concomitant inoculation of MSC with MDA-MB231 cells induces: (i) significantly increased growth and metastasis of MB231 cells and (ii) increased MSC migration to metastatic sites in lung and liver; this mechanism is both OPN and CCL5 dependent. MSCs retrieved from sites of metastases exhibit OPN-dependent expression of the CAF markers, α-smooth muscle actin, tenascin-c, CXCL12 (or stromal cell-derived factor 1) and fibroblast-specific protein-1 and the matrix metalloproteinases (MMP)-2 and MMP-9. Based upon these results, we propose that tumor-derived OPN promotes tumor progression via the transformation of MSC into CAF.

Osteopontin and protein kinase C regulate PDLIM2 activation and STAT1 ubiquitination in LPS-treated murine macrophages.

The molecular pathways regulating signal transducer and activator of transcription 1 (STAT1) levels in states of inflammation are incompletely understood. The suppressor of cytokine signaling, protein inhibitor of STAT, and SHP-1/2 tyrosine phosphatases ultimately regulate activity of STAT molecules. However, these mechanisms do not degrade STAT proteins. In this regard, using a murine macrophage model of LPS stimulation, we previously demonstrated that osteopontin (OPN) increased STAT1 ubiquitination and 26 S proteasome degradation via the ubiquitin E3 ligase, PDLIM2. In this study, we further characterize OPN-dependent activation of PDLIM2 in a model of LPS-stimulated RAW264.7 murine macrophages. We identify serine 137 as a protein kinase C-phosphorylation site in PDLIM2 that is required for ubiquitination of STAT1. PDLIM2 phosphorylation requires OPN expression. Using phospho-mutants and phospho-mimetic constructs of PDLIM2, our in vivo and in vitro ubiquitination studies confirm the role of PDLIM2 in formation and degradation of Ub-STAT1. The functional consequences of PDLIM2-mediated STAT1 degradation were confirmed using an IFN-γ-regulated transcription factor STAT1α reporter construct and chromatin immunoprecipitation assay for the inducible nitric-oxide synthase promoter. In a murine cecal ligation and puncture model of sepsis in wild-type and OPN (-/-) animals, OPN was necessary for PDLIM2 serine phosphorylation and STAT1 ubiquitination in bone marrow macrophages. We conclude that OPN and PDLIM2 are important regulators of STAT1-mediated inflammatory responses.

EF1A1-actin interactions alter mRNA stability to determine differential osteopontin expression in HepG2 and Hep3B cells.

Cancer progression depends on an accumulation of metastasis-supporting physiological changes which are regulated by cell signaling molecules. One such molecule, osteopontin (OPN), is a secreted phosphoprotein which mediates increased cellular migratory and invasive behavior, increased metastasis, protection from apoptosis, promotion of colony formation and 3D growth ability, induction of tumor-associated inflammatory cells, and induction of expression of angiogenic factors. Studies show that OPN expression is controlled by complex regulatory pathways at the transcriptional level in several cancers, but the molecular mechanisms which determine expression of OPN in HCC are largely unknown. In HepG2 and Hep3B tumor cell lines that differentially express OPN mRNA and protein, we identify elongation translation factor-1A1 (EF1A1) to be the trans-acting factor regulating differential OPN mRNA stability between HepG2 and Hep3B cell lines and characterize its interactions with G- and F-actin. EF1A1 binds to the OPN 5'-UTR to regulate OPN mRNA half-life. EF1A1 binds to actin in Hep3B cells. Pharmacologic manipulation to increase the G:F actin ratio in Hep3B increases OPN mRNA half-life and protein expression with simultaneous decrease in EF1A1 binding to OPN 5'-UTR. The converse findings were noted in HepG2 cells. Overall, our results suggest that EF1A1 regulation of OPN mRNA stability is actin dependent. EF1A1 has not been previously identified as a regulatory factor in OPN expression in cancer.

RNA aptamer blockade of osteopontin inhibits growth and metastasis of MDA-MB231 breast cancer cells.

Osteopontin (OPN) is a secreted phosphoprotein which mediates tumorigenesis, local growth, and metastasis in a variety of cancers. It is a potential therapeutic target for the regulation of cancer metastasis. RNA aptamer technology targeting OPN may represent a clinically viable therapy. In this study, we characterize the critical sequence of an RNA aptamer, termed OPN-R3, directed against human OPN. It has a K(d) of 18 nmol/l and binds specifically to human OPN as determined by RNA electrophoretic mobility assays. In MDA-MB231 human breast cancer cells examined under fluorescence microscopy, OPN-R3 ablates cell surface binding of OPN to its cell surface CD44 and alpha(v)beta(3) integrin receptors. Critical enzymatic components of the OPN signal transduction pathways, PI3K, JNK1/2, Src and Akt, and mediators of extracellular matrix degradation, matrix metalloproteinase 2 (MMP2) and uroplasminogen activator (uPA), are significantly decreased following exposure to OPN-R3. OPN-R3 inhibits MDA-MB231 in vitro adhesion, migration, and invasion characteristics by 60, 50, and 65%, respectively. In an in vivo xenograft model of breast cancer, OPN-R3 significantly decreases local progression and distant metastases. On the basis of this "proof-of-concept" study, we conclude that RNA aptamer targeting of OPN has biologically relevance for modifying tumor growth and metastasis.

Thrombin-cleaved COOH(-) terminal osteopontin peptide binds with cyclophilin C to CD147 in murine breast cancer cells.

Osteopontin is a glycoprotein that has been linked to metastatic function in breast, lung, and prostate cancers. However, the mechanism by which osteopontin acts to induce metastatic properties is largely unknown. One intriguing feature of osteopontin is the presence of a conserved thrombin cleavage site that is COOH-terminal from a well-characterized RGD domain. Although the COOH-terminal fragment may bind to cell surface CD44 receptors, little is known about the COOH-terminal osteopontin fragment. In the current study, we use the murine mammary epithelial tumor cell lines 4T1 and 4T07; these cells are thioguanine-resistant sublines derived from the parental population of 410.4 cells from Balb/cfC3H mice. Using flow cytometry and Forster resonance energy transfer, we show that the COOH-terminal fragment of osteopontin binds with another marker of metastatic function (cyclophilin C or rotamase) to the CD147 cell surface glycoprotein (also known as extracellular matrix metalloproteinase inducer), to activate Akt1/2 and matrix metalloproteinase-2. In in vitro assays, thrombin cleavage of osteopontin to generate short COOH-terminal osteopontin in the presence of cyclophilin C increases migration and invasion of both 4T07 and 4T1 cells. This interaction between osteopontin peptide and cyclophilin C has not been previously described but assigns a heretofore unknown function for the thrombin-cleaved osteopontin COOH-terminal fragment.

Myeloid zinc finger-1 regulates expression of cancer-associated fibroblast and cancer stemness profiles in breast cancer.

BACKGROUND: Osteopontin acts thru myeloid zinc finger-1 and transforming growth factor-ß to drive the adoption of a cancer-associated fibroblast phenotype by local mesenchymal stem cells. Cancer-associated fibroblasts increase cancer cell stemness. METHODS: Mesenchymal stem cells were exposed to osteopontin or were cocultured with MB231 human breast cancer cells (high osteopontin producer) in the presence or absence of aptamer (inactivates extracellular osteopontin). Myeloid zinc finger-1 phosphorylation sites were identified, and phosphomutants of T134 (SCAN domain) and S453 (zinc finger DNA binding domain) were constructed. Transforming growth factor-ß F and cancer-associated fibroblast markers (smooth muscle actin, vimentin, and tenascin-c) were measured in mesenchymal stem cells. In MB231, stemness markers Sox2, Nanog, and Oct4 were measured. RESULTS: Mesenchymal stem cells plus osteopontin increased transforming growth factor-ß and cancer-associated fibroblast markers (P < .05 vs mesenchymal stem cells alone); this was abolished by aptamer inactivation of osteopontin. In mesenchymal stem cells transfected with phosphoresistant myeloid zinc finger-1, osteopontin did not increase cancer-associated fibroblast markers or transforming growth factor-ß. In contrast, phosphomimetic myeloid zinc finger-1 increased cancer-associated fibroblast markers and transforming growth factor-ß (P < .05 vs mesenchymal stem cells alone). In mesenchymal stem cells plus MB231, MB231 stemness markers were increased (P < .05 vs MB231 alone). In MB231 plus mesenchymal stem cells expressing phosphoresistant myeloid zinc finger-1, MB231 stemness markers were not increased in comparison with MB231 plus mesenchymal stem cells. CONCLUSION: Myeloid zinc finger-1 phosphorylation in mesenchymal stem cells drives the osteopontin-mediated cancer-associated fibroblast phenotype, which then increases the cancer cell stemness profile.