Shedding of soluble epidermal growth factor receptor (sEGFR) is mediated by a metalloprotease/fibronectin/integrin axis and inhibited by cetuximab.

Soluble epidermal growth factor receptor (sEGFR) is a circulating serum biomarker in cancer patients. Recent studies suggest that baseline serum sEGFR concentrations may predict responsiveness to EGFR-targeted therapy. Here, we demonstrate that sEGFR is generated through proteolytic cleavage of a cell surface precursor of an alternately spliced EGF receptor isoform and that sEGFR binds to EGF with high affinity. Proteolytic cleavage is stimulated by an anti-α5/ß1 integrin antibody and 4-aminophenylmercuric acetate, and inhibited by fibronectin. Two FDA-approved therapeutic anti-EGFR antibodies also inhibit shedding of sEGFR, thus implicating the cell surface precursor of sEGFR as a competing target for anti-EGFR antibodies in human tissues. These observations parallel trastuzumab regulation of HER2 shedding and have implications for patient stratification in future clinical trials of EGFR-targeted antibodies.

Programmed cell death 4 inhibits breast cancer cell invasion by increasing tissue inhibitor of metalloproteinases-2 expression.

High levels of the cyclooxygenase-2 (COX-2) protein have been associated with invasion and metastasis of breast tumors. Both prostaglandin E(2) (PGE(2)) and interleukin-8 (IL-8) have been shown to mediate the invasive activity of COX-2 in breast cancer cells. Here we expand these studies to determine how COX-2 uses PGE(2) and IL-8 to induce breast cancer cell invasion. We demonstrated that PGE(2) and IL-8 decreased the expression of the tumor suppressor protein Programmed Cell Death 4 (PDCD4). We hypothesized that suppression of PDCD4 expression is vital to the invasive activity of PGE(2) and IL-8. In MCF-7 cells overexpressing PDCD4 (MCF-7/PDCD4), PGE(2) and IL-8 failed to induce invasion, in contrast to the parental MCF-7 cells, thus indicating that PDCD4 blocks breast cancer cell invasion. MCF-7/PDCD4 cells produced higher levels of the Tissue Inhibitor of Metalloproteinases-2 (TIMP-2) than the parental cells. Silencing TIMP-2 mRNA in MCF-7/PDCD4 cells reversed the anti-invasive effects of PDCD4, allowing PGE(2) and IL-8 to induce the invasion of these cells. Here we report the novel findings that suppression of PDCD4 expression is vital for the invasive activity of COX-2 mediated by PGE(2) and IL-8, and that PDCD4 increases TIMP-2 expression to inhibit breast cancer cell invasion.

Shedding of epidermal growth factor receptor is a regulated process that occurs with overexpression in malignant cells.

Soluble isoforms of the epidermal growth factor receptor (sEGFR) previously have been identified in the conditioned culture media (CCM) of the vulvar adenocarcinoma cell line, A431 and within exosomes of the keratinocyte cell line HaCaT. Here, we report that the extracellular domain (ECD) of EGFR is shed from the cell surface of human carcinoma cell lines that express 7x10(5) receptors/cell or more. We purified this proteolytic isoform of EGFR (PI-sEGFR) from the CCM of MDA-MB-468 breast cancer cells. The amino acid sequence of PI-sEGFR was determined by reverse-phase HPLC nano-electrospray tandem mass spectrometry of peptides generated by trypsin, chymotrypsin or GluC digestion. The PI-sEGFR protein is identical in amino acid sequence to the EGFR ECD. The release of PI-sEGFR from MDA-MB-468 cells is enhanced by phorbol 12-myristate 13-acetate, heat-inactivated fetal bovine serum, pervanadate, and EGFR ligands (i.e., EGF and TGF-alpha). In addition, 4-aminophenylmercuric acetate, an activator of metalloproteases, increased PI-sEGFR levels in the CCM of MDA-MB-468 cells. Inhibitors of metalloproteases decreased the constitutive shedding of EGFR while the PMA-induced shedding was inhibited by metalloprotease inhibitors, by the two serine protease inhibitors leupeptin and 3,4-dichloroisocoumarin (DCI), and by the aspartyl inhibitor pepstatin. These results suggest that PI-sEGFR arises by proteolytic cleavage of EGFR via a mechanism that is regulated by both PKC- and phosphorylation-dependent pathways. Our results further suggest that when proteolytic shedding of EGFR does occur, it is correlated with a highly malignant phenotype.

TIMP-2 mediates the anti-invasive effects of the nitric oxide-releasing prodrug JS-K in breast cancer cells.

INTRODUCTION: Tumor invasion and metastasis remain a major cause of mortality in breast cancer patients. High concentrations of nitric oxide (NO) suppress tumor invasion and metastasis in vivo. NO prodrugs generate large amounts of NO upon metabolism by appropriate intracellular enzymes, and therefore could have potential in the prevention and therapy of metastatic breast cancer. METHODS: The present study was designed to determine the effects of the NO-releasing prodrug O2-(2,4-dinitrophenyl) 1- [(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) on breast cancer invasion and the mechanisms involved. MDA-MB-231, MDA-MB-231/F10, and MCF-7/COX-2 were the three breast cancer cell lines tested. NO levels were determined spectrophotometrically using a NO assay kit. Invasion and the expression of matrix metalloproteinases (MMPs) and tissue inhibitor of MMPs were determined using Matrigel invasion assays, an MMP array kit and ELISAs. The activity and expression of extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase mitogen-activated protein kinases were determined using western blot analyses. RESULTS: Under conditions by which JS-K was not cytotoxic, JS-K significantly decreased (P < 0.05) the invasiveness of breast cancer cells across the Matrigel basement membrane, which was directly correlated with NO production. JS-43-126, a non-NO-releasing analog of JS-K, had no effect on NO levels or invasion. JS-K increased (P < 0.05) TIMP-2 production, and blocking TIMP-2 activity with a neutralizing antibody significantly increased (P < 0.05) the invasive activity of JS-K-treated cells across Matrigel. JS-K decreased p38 activity, whereas the activity and the expression of extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase were unaffected. CONCLUSION: We report the novel findings that JS-K inhibits breast cancer invasion across the Matrigel basement membrane, and NO production is vital for this activity. Upregulation of TIMP-2 production is one mechanism by which JS-K mediates its anti-invasive effects. JS-K and other NO prodrugs may represent an innovative biological approach in the prevention and treatment of metastatic breast cancer.

Cyclooxygenase-2 uses the protein kinase C/ interleukin-8/urokinase-type plasminogen activator pathway to increase the invasiveness of breast cancer cells.

Cyclooxygenase-2 (COX-2) increases breast cancer cell invasion. Expression of various pro-angiogenic and pro-invasive factors has been correlated with high expression of COX-2. However, whether these factors are essential to COX-2-mediated breast cancer invasion, and the mechanisms by which COX-2 increases the expression of these factors are unknown. Our microarray results indicate that higher COX-2 expression was associated with increased levels of interleukin-8 (IL-8), a key factor in breast cancer invasion and metastasis. COX-2 overexpressing cells (MCF-7/COX-2), generated by transfecting COX-2-encoding plasmids into the poorly invasive MCF-7 breast cancer cells, were more invasive and produced higher IL-8 levels than the parental cells. To investigate the role of IL-8 in COX-2-mediated invasion, MCF-7 parental cells were incubated with IL-8. Exogenous IL-8 increased the invasiveness of MCF-7 cells. IL-8 is one pathway by which COX-2 mediates breast cancer invasion. Protein kinase A (PKA) and protein kinase C (PKC) are activated by COX-2 and are involved in IL-8 regulation. Inhibition of PKC, not PKA, decreased IL-8 production and invasion in MCF-7/COX-2 cells. Activation of PKC, not PKA, increased IL-8 production and invasion in MCF-7 cells. Thus, the invasive effects of COX-2 are mediated by PKC, not PKA. Activity of the urokinase-type plasminogen activator (uPA) was increased in MCF-7 cells by COX-2 overexpression or by the addition of a PKC activator or by IL-8. Inhibition of PKC decreased uPA activity in MCF-7/COX-2 cells. Furthermore, inhibition of uPA activity decreased the invasiveness of MCF-7/COX-2 cells, indicating that uPA was essential to COX-2-mediated invasion. Herein we demonstrate for the first time a detailed mechanism by which COX-2 increases breast cancer invasion: the PKC/IL-8/uPA pathway.

Programmed cell death 4 inhibits leptin-induced breast cancer cell invasion.

Obesity is a significant risk factor for post-menopausal women to develop and die from breast cancer. Leptin, an adipokine is produced in high levels in obese individuals, and its receptor is overexpressed in breast tumors and lymph node metastases. Previously, we demonstrated that leptin stimulates breast cancer cell invasion, which is correlated with breast cancer metastasis. Programmed cell death 4 (PDCD4) has been shown to block cancer cell invasion. However, whether PDCD4 blocks leptin-induced breast cancer cell invasion is not known. Here, we report the novel findings that leptin failed to induce invasion in MCF-7 breast cancer cells overexpressing PDCD4 (MCF-7/PDCD4). Tissue inhibitor of metalloproteinase-2 (TIMP-2) was essential to the anti-invasive effect of PDCD4, as leptin stimulated the invasion of MCF-7/PDCD4 cells pretreated with TIMP-2 siRNA. Furthermore, TIMP-2 knockdown allowed leptin to augment phosphorylation of extracellular signal-regulated kinases 1,2 and signal transducer and activator of transcription 3, but not that of Jun N-terminal kinases. These data indicate that PDCD4 utilizes TIMP-2 to exert its anti-invasive effect by suppressing leptin-induced activation of extracellular signal-regulated kinases 1,2 and signal transducer and activator of transcription 3. Novel therapeutic strategies aiming at enhancing PDCD4 expression in breast tumors may be able to stop obesity-related breast tumor progression and prolong the life of patients.

JS-K, a nitric oxide-releasing prodrug, induces breast cancer cell death while sparing normal mammary epithelial cells.

Targeted therapy with reduced side effects is a major goal in cancer research. We investigated the effects of JS-K, a nitric oxide (NO) prodrug designed to release high levels of NO when suitably activated, on human breast cancer cell lines, on non-transformed human MCF-10A mammary cells, and on normal human mammary epithelial cells (HMECs). Cell viability assay, flow cytometry, electron microscopy, and Western blot analysis were used to study the effects of JS-K on breast cancer and on mammary epithelial cells. After a 3-day incubation, the IC50s of JS-K against the breast cancer cells ranged from 0.8 to 3 µM. However, JS-K decreased the viability of the MCF-10A cells by only 20% at 10-µM concentration, and HMECs were unaffected by 10 µM JS-K. Flow cytometry indicated that JS-K increased the percentages of breast cancer cells under-going apoptosis. Interestingly, flow cytometry indicated that JS-K increased acidic vesicle organelle formation in breast cancer cells, suggesting that JS-K induced autophagy in breast cancer cells. Electron microscopy confirmed that JS-K-treated breast cancer cells underwent autophagic cell death. Western blot analysis showed that JS-K induced the expression of microtubule light chain 3-II, another autophagy marker, in breast cancer cells. However, JS-K did not induce apoptosis or autophagy in normal human mammary epithelial cells. These data indicate that JS-K selectively induces programmed cell death in breast cancer cells while sparing normal mammary epithelial cells under the same conditions. The selective anti-tumor activity of JS-K warrants its further investigation in breast tumors.

Thioaptamer conjugated liposomes for tumor vasculature targeting.

Recent developments in multi-functional nanoparticles offer a great potential for targeted delivery of therapeutic compounds and imaging contrast agents to specific cell types, in turn, enhancing therapeutic effect and minimizing side effects. Despite the promise, site specific delivery carriers have not been translated into clinical reality. In this study, we have developed long circulating liposomes with the outer surface decorated with thioated oligonucleotide aptamer (thioaptamer) against E-selectin (ESTA) and evaluated the targeting efficacy and PK parameters. In vitro targeting studies using Human Umbilical Cord Vein Endothelial Cell (HUVEC) demonstrated efficient and rapid uptake of the ESTA conjugated liposomes (ESTA-lip). In vivo, the intravenous administration of ESTA-lip resulted in their accumulation at the tumor vasculature of breast tumor xenografts without shortening the circulation half-life. The study presented here represents an exemplary use of thioaptamer and liposome and opens the door to testing various combinations of thioaptamer and nanocarriers that can be constructed to target multiple cancer types and tumor components for delivery of both therapeutics and imaging agent.

Identification of thioaptamer ligand against E-selectin: potential application for inflamed vasculature targeting.

Active targeting of a drug carrier to a specific target site is crucial to provide a safe and efficient delivery of therapeutics and imaging contrast agents. E-selectin expression is induced on the endothelial cell surface of vessels in response to inflammatory stimuli but is absent in the normal vessels. Thus, E-selectin is an attractive molecular target, and high affinity ligands for E-selectin could be powerful tools for the delivery of therapeutics and/or imaging agents to inflamed vessels. In this study, we identified a thiophosphate modified aptamer (thioaptamer, TA) against E-selectin (ESTA-1) by employing a two-step selection strategy: a recombinant protein-based TA binding selection from a combinatorial library followed by a cell-based TA binding selection using E-selectin expressing human microvascular endothelial cells. ESTA-1 selectively bound to E-selectin with nanomolar binding affinity (K(D) = 47 nM) while exhibiting minimal cross reactivity to P- and L-selectin. Furthermore, ESTA-1 binding to E-selectin on the endothelial cells markedly antagonized the adhesion (over 75% inhibition) of sLe(x) positive HL-60 cells at nanomolar concentration. ESTA-1 also bound specifically to the inflamed tumor-associated vasculature of human carcinomas derived from breast, ovarian, and skin but not to normal organs, and this binding was highly associated with the E-selectin expression level. Similarly, intravenously injected ESTA-1 demonstrated distinct binding to the tumor vasculature in a breast cancer xenograft model. Together, our data substantiates the discovery of a thioaptamer (ESTA-1) that binds to E-selectin with high affinity and specificity, thereby highlighting the potential application of ESTA-1 for E-selectin targeted delivery.

Silencing kinase-interacting stathmin gene enhances erlotinib sensitivity by inhibiting Ser¹° p27 phosphorylation in epidermal growth factor receptor-expressing breast cancer.

The epidermal growth factor receptor (EGFR) signaling pathway has emerged as a promising target for cancer therapy. EGFR tyrosine kinase inhibitors (TKI) such as erlotinib have been approved for cancer treatment but have shown very limited activity in breast cancer patients. Clarifying the molecular mechanism underlying resistance to EGFR TKIs could lead to more effective treatment against breast cancer. We previously reported that the sensitivity of breast cancer cells to erlotinib is partially dependent on p27 and that cytoplasmic localization of p27 is associated with erlotinib resistance. In the present study, we found that erlotinib induces p27 phosphorylation at Ser¹° (S10), and S10 p27 phosphorylation leads to erlotinib resistance in EGFR-expressing breast cancer. Inhibiting S10 phosphorylation of p27 by knocking down human kinase-interacting stathmin (KIS), a nuclear protein that can phosphorylate p27 at S10, led to p27 accumulation in the nucleus and enhanced erlotinib-mediated cytotoxicity. Further, in vivo KIS gene silencing enhanced the antitumor activity of erlotinib in an orthotopic breast cancer xenograft model. KIS depletion also enhanced erlotinib sensitivity in erlotinib-resistant EGFR-expressing triple-negative breast cancer cells. Our study provides a rationale for the development of combinations of erlotinib with KIS inhibition to overcome EGFR TKI resistance in EGFR-expressing breast cancer.

Sustained small interfering RNA delivery by mesoporous silicon particles.

RNA interference (RNAi) is a powerful approach for silencing genes associated with a variety of pathologic conditions; however, in vivo RNAi delivery has remained a major challenge due to lack of safe, efficient, and sustained systemic delivery. Here, we report on a novel approach to overcome these limitations using a multistage vector composed of mesoporous silicon particles (stage 1 microparticles, S1MP) loaded with neutral nanoliposomes (dioleoyl phosphatidylcholine, DOPC) containing small interfering RNA (siRNA) targeted against the EphA2 oncoprotein, which is overexpressed in most cancers, including ovarian. Our delivery methods resulted in sustained EphA2 gene silencing for at least 3 weeks in two independent orthotopic mouse models of ovarian cancer following a single i.v. administration of S1MP loaded with EphA2-siRNA-DOPC. Furthermore, a single administration of S1MP loaded with-EphA2-siRNA-DOPC substantially reduced tumor burden, angiogenesis, and cell proliferation compared with a noncoding control siRNA alone (SKOV3ip1, 54%; HeyA8, 57%), with no significant changes in serum chemistries or in proinflammatory cytokines. In summary, we have provided the first in vivo therapeutic validation of a novel, multistage siRNA delivery system for sustained gene silencing with broad applicability to pathologies beyond ovarian neoplasms.

Leptin utilizes Jun N-terminal kinases to stimulate the invasion of MCF-7 breast cancer cells.

In breast tumors, high levels of leptin have been associated with increased incidence of breast cancer metastasis. Breast cancer metastasis is directly associated with breast cancer cell invasion. However, whether leptin could augment breast cancer cell invasion is not known. Here we showed that leptin increased the invasiveness and the matrix metallo-proteinase-2 (MMP-2) activity of the MCF-7 breast cancer cell line. Leptin stimulated the phosphorylation of extracellular signals regulated kinases, signal transducers and activators of transcription 3 and Jun N-terminal kinases (JNK); however, only inhibition of JNK decreased leptin-mediated activation of MMP-2. Furthermore, inhibition of JNK suppressed leptin-mediated breast cancer cell invasion. Here we report the novel findings that leptin increased invasion of breast cancer cells by activating JNK, resulting in increased MMP-2 activity.