Feasibility of cisplatin/5-fluorouracil and panitumumab in Japanese patients with squamous cell carcinoma of the head and neck.

OBJECTIVE: In Japan, cisplatin/5-fluorouracil 80/800 (cisplatin 80 mg/m2, 5-fluorouracil 800 mg/m2) is widely used to treat recurrent/metastatic squamous cell carcinoma of the head and neck, whereas cisplatin/5-fluorouracil 100/1000 (1000 mg/m2/24 h by continuous intravenous infusion on Days 1-4 plus cisplatin 100 mg/m2 on Day 1 in 3-week cycles) is the standard treatment in Europe and North America. METHODS: We prospectively evaluated the feasibility of cisplatin/5-fluorouracil 100/1000 in Japanese patients enrolled in the global Phase 3 study of panitumumab 9 mg/kg combined with cisplatin/5-fluorouracil 100/1000 (Arm 1) versus cisplatin/5-fluorouracil 100/1000 alone (Arm 2). RESULTS: Twenty Japanese patients were enrolled and received treatment (Arm 1, n=13; Arm 2, n=7). Grade 3/4 adverse events included neutropenia, hypomagnesemia, stomatitis, hyponatremia, paronychia, febrile neutropenia, decreased appetite and hypokalemia. There were no fatal adverse events. Median overall survival was not estimable in Arm 1 and 15.4 months in Arm 2. Median progression-free survival was 6.9 months in Arm 1 and 5.7 months in Arm 2. The median number of infusions (cycles) of cisplatin was 5 in Arm 1 and 4 in Arm 2; the median number of infusions (cycles) of 5-fluorouracil was 6 in both arms. The mean administered dose for cisplatin was 93.6 mg/m2 in Arm 1 and 97.2 mg/m2 in Arm 2, and 3732.6 and 3880 mg/m2 in Arm 1 and Arm 2, respectively, for 5-fluorouracil. CONCLUSIONS: These results suggested that cisplatin/5-fluorouracil 100/1000 was feasible for recurrent/metastatic squamous cell carcinoma of the head and neck in Japanese patients.

Panitumumab in Japanese patients with unresectable colorectal cancer: a post-marketing surveillance study of 3085 patients.

OBJECTIVE: Panitumumab was approved in Japan in April 2010 for the treatment of Kirsten rat sarcoma-2 virus oncogene wild-type unresectable and recurrent colorectal cancer. We conducted a post-marketing surveillance study to evaluate the safety and effectiveness of panitumumab. METHODS: After panitumumab was commercially available in Japan, all patients to be treated with panitumumab were enrolled. Data on baseline characteristics, treatment outcome, and incidence and severity of adverse drug reactions were collected. RESULTS: In total, 3091 patients were registered. In the safety analysis set (n = 3085), panitumumab was administered as monotherapy (40.7%) or combination therapy (59.4%). The median treatment duration was 113 days (range: 1-559 days), and 451 (14.6%) patients received panitumumab for ≥10 months. The overall incidence rate of adverse drug reactions was 84.1%, and the most common adverse drug reaction was skin disorders (78.4%). The incidence rates (all grades) of interstitial lung disease, infusion reaction, electrolyte abnormalities and cardiac disorders were 1.3% (mortality rate: 0.6%), 1.5, 19.3 and 0.2%, respectively. The median survival time of patients treated with panitumumab monotherapy as the third-line, or later, therapy was 10.3 months. CONCLUSION: This post-marketing survey in clinical practice confirmed the safety and effectiveness of panitumumab. The benefit/risk balance for panitumumab in Japanese patients with unresectable colorectal cancer remains favorable.

A prospective, multicenter, clinical study of duodenoscope contamination after reprocessing.

OBJECTIVE: Several clinical procedures utilize duodenoscopes, which are processed for reuse after the procedures are completed. However, infection outbreaks due to improper duodenoscope processing occur frequently. To address this, we aimed to assess the contamination rates of duodenoscopes after reprocessing in nonoutbreak settings. DESIGN AND SETTING: Prospective study in 16 clinical sites in the United States. METHODS: We sampled and cultured reprocessed duodenoscopes following the FDA/CDC/ASM guideline; "Duodenoscope Surveillance Sampling and Culturing - Reducing the Risks of Infection." High-concern (HC) organisms were those highly associated with disease, including gram-negative rods, Staphylococcus aureus, Staphylococcus lugdunensis, ß-hemolytic Streptococcus, Enterococcus spp, and yeasts. We evaluated duodenoscopes with ≥1 CFU of organisms after reprocessing. The reprocessing environments were also sampled and cultured. RESULTS: We assessed 859 newer-model (NM) duodenoscopes (TJF-Q180V) and 850 older-model (OM) duodenoscopes (TJF-160F/VF); of these, 35 NM samples (4.1%) and 56 OM samples (6.6%) were contaminated with HC organisms. We detected and classified the HC organisms as gastrointestinal (45.4%), human origin (16.7%), environmental (24.1%), waterborne (13.0%), and unidentified (0.9%). CONCLUSIONS: We detected an overall HC contamination rate of 5.3% in nonoutbreak settings. Although the relationship between endoscopic contamination and the occurrence of infections remains unclear, attempts should continue to be made to further reduce contamination rates. Additional improvements to the manufacturer's instructions for use, human factors during the reprocessing procedure, ongoing training programs, cleanliness of reprocessing environments, and the design of the distal end of the duodenoscope should be considered.

Matrix metalloproteinases (MMPs) regulate fibrin-invasive activity via MT1-MMP-dependent and -independent processes.

Cross-linked fibrin is deposited in tissues surrounding wounds, inflammatory sites, or tumors and serves not only as a supporting substratum for trafficking cells, but also as a structural barrier to invasion. While the plasminogen activator-plasminogen axis provides cells with a powerful fibrinolytic system, plasminogen-deleted animals use alternate proteolytic processes that allow fibrin invasion to proceed normally. Using fibroblasts recovered from wild-type or gene-deleted mice, invasion of three-dimensional fibrin gels proceeded in a matrix metalloproteinase (MMP)-dependent fashion. Consistent with earlier studies supporting a singular role for the membrane-anchored MMP, MT1-MMP, in fibrin-invasive events, fibroblasts from MT1-MMP-null mice displayed an early defect in invasion. However, MT1-MMP-deleted fibroblasts circumvented this early deficiency and exhibited compensatory fibrin-invasive activity. The MT1-MMP-independent process was sensitive to MMP inhibitors that target membrane-anchored MMPs, and further studies identified MT2-MMP and MT3-MMP, but not MT4-MMP, as alternate pro-invasive factors. Given the widespread distribution of MT1-, 2-, and 3-MMP in normal and neoplastic cells, these data identify a subset of membrane-anchored MMPs that operate in an autonomous fashion to drive fibrin-invasive activity.

The role of MT2-MMP in cancer progression.

The role of MT2-MMP in cancer progression remains to be elucidated in spite of many reports on MT1-MMP. Using a human fibrosarcoma cell, HT1080 and a human gastric cancer cell, TMK-1, endogenous expression of MT1-MMP or MT2-MMP was suppressed by siRNA induction to examine the influence of cancer progression in vitro and in vivo. In HT1080 cells, positive both in MT1-MMP and MT2-MMP, the migration as well as the invasion was impaired by MT1-MMP or MT2-MMP suppression. Also cell proliferation in three dimensional (3D) condition was inhibited by MT1-MMP or MT2-MMP suppression and tumor growth in the nude mice transplanted with tumor cells were reduced either MT1-MMP or MT2-MMP suppression with a prolongation of survival time in vivo. MT2-MMP suppression induces more inhibitory effects on 3D proliferation and in vivo tumor growth than MT1-MMP. On the other hand, TMK-1 cells, negative in MT1-MMP and MMP-2 but positive in MT2-MMP, all the migratory, invasive, and 3D proliferative activities in TMK-1 are decreased only by MT2-MMP suppression. These results indicate MT2-MMP might be involved in the cancer progression more than or equal to MT1-MMP independently of MMP-2 and MT1-MMP.

Stroma-derived matrix metalloproteinase (MMP)-2 promotes membrane type 1-MMP-dependent tumor growth in mice.

Matrix metalloproteinase-2 (MMP-2) is a stroma-derived MMP belonging to the type IV collagenase family. It is believed to mediate tumor cell behavior by degrading deposits of type IV collagen, a major component of the basement membrane. The membrane type 1-MMP (MT1-MMP) is a highly potent activator of MMP-2 and is expressed in many tumor and stromal cells. However, the roles played by stromal MMP-2 in tumor progression in vivo remain poorly understood. We established a colon epithelial cell line from an Mt1-mmp(-/-) mouse strain and transfected these cells with an inducible expression system for MT1-MMP (MT1rev cells). Following s.c. implantation into Mmp-2(+/+) mice and induction of MT1-MMP expression, MT1rev cells grew rapidly, whereas they grew very slowly in Mmp-2(-/-) mice, even in the presence of MT1-MMP. This MT1-MMP-dependent tumor growth of MT1rev cells was enhanced in Mmp-2(-/-) mice as long as MMP-2 was supplied via transfection or coimplantation of MMP-2-positive fibroblasts. MT1rev cells cultured in vitro in a three-dimensional collagen gel matrix also required the MT1-MMP/MMP-2 axis for rapid proliferation. MT1rev cells deposit type IV collagen primarily at the cell-collagen interface, and these deposits seem scarce at sites of invasion and proliferation. These data suggest that cooperation between stroma-derived MMP-2 and tumor-derived MT1-MMP may play a role in tumor invasion and proliferation via remodeling of the tumor-associated basement membrane. To our knowledge, this is the first study demonstrating that MT1-MMP-dependent tumor growth in vivo requires stromal-derived MMP-2. It also suggests that MMP-2 represents a potential target for tumor therapeutics.

Competitive disruption of the tumor-promoting function of membrane type 1 matrix metalloproteinase/matrix metalloproteinase-14 in vivo.

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a potent modulator of the pericellular environment and promotes tumor cell invasion and proliferation in many types of tumor. The activation of proMMP-2 and processing of collagen I by MT1-MMP have been thought to be important for its tumor-promoting function. These activities can be inhibited by mutant forms of MT1-MMP lacking the catalytic domain. However, the effect of such dominant-negative mutants has never been evaluated in vivo. Various mutants lacking the catalytic domain (dCAT) were prepared and confirmed to inhibit MT1-MMP activity in human fibrosarcoma HT1080 cells, and tumor cells expressing these mutants were implanted s.c. into nude mice to monitor tumor formation. Only the membrane-anchored form of a dCAT construct through the transmembrane domain [dCAT(1)] showed potent antitumor activity not only in HT1080 cells but also in gastric carcinoma MKN28 and MKN45 cells expressing MT1-MMP. A soluble form of dCAT lacking the transmembrane domain did not show such activity. The expression of dCAT(1) in MKN28 or MKN45 further prevented the metastatic spread of tumor cells into the peritoneal cavity; however, dCAT(1) showed no effect against TMK-1, another gastric carcinoma cell line expressing no MT1-MMP. It is of note that the tumorigenicity of TMK-1 cells enhanced by MT1-MMP overexpression was, in turn, canceled by the additional expression of dCAT(1). Thus, MT1-MMP expressed in tumor cells seems to play a pivotal role in tumor growth in mice. The results also suggest new possibilities to abrogate the tumor-promoting function of MT1-MMP other than the conventional protease inhibitor-based approach.

Absence of mechanical allodynia and Abeta-fiber sprouting after sciatic nerve injury in mice lacking membrane-type 5 matrix metalloproteinase.

Matrix metalloproteinases (MMPs) are a family of endopeptidases that degrade extracellular matrix components. Membrane-type 5 MMP (MT5-MMP/MMP-24) was identified as neuron-specific, and is believed to contribute to neuronal circuit formation and plasticity. To elucidate its function in vivo, we have generated mice lacking MT5-MMP by gene targeting. MT5-MMP-deficient mice were born without obvious morphological abnormalities. No apparent histological defects were observed in the nervous system either. However, MT5-MMP-deficient mice did not develop neuropathic pain with mechanical allodynia after sciatic nerve injury, though responses to acute noxious stimuli were normal. Neuropathic pain induced by peripheral nerve lesions is known to accompany structural reorganization of the nervous system. Intraneural injection of cholera toxin B subunit, a transganglionic tracer, into the injured sciatic nerve of wild-type mice revealed that the myelinated Abeta-fiber primary afferents sprouted from laminae III-VI of the dorsal horn of the spinal cord and invaded lamina II. However, no such sprouting and invasion of Abeta-fibers were observed in MT5-MMP-deficient mice. These findings suggest that MT5-MMP is essential for the development of mechanical allodynia and plays an important role in neuronal plasticity in this mouse model.

MT-MMPs play pivotal roles in cancer dissemination.

Matrix metalloproteinases (MMPs), a family of zinc-binding endopeptidases, play important roles in cancer proliferation and dissemination, and may be further associated with other diseases. In particular, membrane-type MMPs (MT-MMPs) are crucial for cancer cell invasion. In this report, we summarize the current views on the role of MT-MMPs in cancer dissemination. The regulated and restricted degradation of the extracellular matrix (ECM) surrounding the tumor surface is a trigger event for cell protrusion and invasion. This is thought to be primarily organized by MT-MMPs, since a shift in balance between cell adhesion molecules, ECM and proteolysis at the focal cell surface may result in conditions especially suitable for cancer cells to progress and invade the ECM. To resolve the physiological mechanisms of cancer invasion and migration, molecular milieu surrounding the MT-MMPs expressed on tumor cell surfaces should be further examined for each cell type, which may consequently provide a novel clinical tool to regulate cancer behavior.

Molecular dissection of the structural machinery underlying the tissue-invasive activity of membrane type-1 matrix metalloproteinase.

Membrane type-1 matrix metalloproteinase (MT1-MMP) drives cell invasion through three-dimensional (3-D) extracellular matrix (ECM) barriers dominated by type I collagen or fibrin. Based largely on analyses of its impact on cell function under two-dimensional culture conditions, MT1-MMP is categorized as a multifunctional molecule with 1) a structurally distinct, N-terminal catalytic domain; 2) a C-terminal hemopexin domain that regulates substrate recognition as well as conformation; and 3) a type I transmembrane domain whose cytosolic tail controls protease trafficking and signaling cascades. The MT1-MMP domains that subserve cell trafficking through 3-D ECM barriers in vitro or in vivo, however, remain largely undefined. Herein, we demonstrate that collagen-invasive activity is not confined strictly to the catalytic, hemopexin, transmembrane, or cytosolic domain sequences of MT1-MMP. Indeed, even a secreted collagenase supports invasion when tethered to the cell surface in the absence of the MT1-MMP hemopexin, transmembrane, and cytosolic tail domains. By contrast, the ability of MT1-MMP to support fibrin-invasive activity diverges from collagenolytic potential, and alternatively, it requires the specific participation of MT-MMP catalytic and hemopexin domains. Hence, the tissue-invasive properties of MT1-MMP are unexpectedly embedded within distinct, but parsimonious, sequences that serve to tether the requisite matrix-degradative activity to the surface of migrating cells.

Establishment of an MT4-MMP-deficient mouse strain representing an efficient tracking system for MT4-MMP/MMP-17 expression in vivo using beta-galactosidase.

The biological functions of membrane-type 4 matrix metalloproteinase (MT4-MMP/MMP-17) are poorly understood because of the lack of a sensitive system for tracking its expression in vivo. We established a mutant mouse strain (Mt4-mmp(-/-)) in which Mt4-mmp was replaced with a reporter gene encoding beta-galactosidase (LacZ). Mt4-mmp(-/-) mice had normal gestations, and no apparent defects in growth, life span and fertility. Using LacZ as a marker, we were able to monitor the expression and promoter activity of Mt4-mmp for the first time in vivo. The tissue distribution of Mt4-mmp mRNA correlated with LacZ expression, and we showed that Mt4-mmp is expressed primarily in cerebrum, lung, spleen, intestine and uterus. We identified LacZ-positive neurons in the cerebrum, smooth muscle cells in the intestine and uterus, and macrophages located in the lung alveolar or intraperitoneal space. Contrary to the reported role of MT4-MMP as a tumor necrosis factor-alpha (TNF-alpha) sheddase, the lipopolysaccharide (LPS)-induced release of TNF-alpha from Mt4-mmp(-/-)macrophages was similar to that in wild-type cells, and expression of Mt4-mmp mRNA was repressed following LPS stimulation. Thus, we have established a mutant mouse strain for analyzing the physiological functions of MT4-MMP, which also serves as a sensitive system for monitoring and tracking the expression of MT4-MMP in vivo.

Crosstalk between neovessels and mural cells directs the site-specific expression of MT1-MMP to endothelial tip cells.

The membrane-anchored matrix metalloproteinase MT1-MMP (also known as Mmp14) plays a key role in the angiogenic process, but the mechanisms underlying its spatiotemporal regulation in the in vivo setting have not been defined. Using whole-mount immunohistochemical analysis and the lacZ gene inserted into the Mmp14 gene, we demonstrate that MT1-MMP vascular expression in vivo is confined largely to the sprouting tip of neocapillary structures where endothelial cell proliferation and collagen degradation are coordinately localized. During angiogenesis in vitro, wherein endothelial cells are stimulated to undergo neovessel formation in the presence or absence of accessory mural cells, site-specific MT1-MMP expression is shown to be controlled by crosstalk between endothelial cells and vascular smooth muscle cells (VSMC). When vessel maturation induced by VSMCs is inhibited by introducing a soluble form of the receptor tyrosine kinase Tek, MT1-MMP distribution is no longer restricted to the endothelial tip cells, but instead distributes throughout the neovessel network in vitro as well as ex vivo. Taken together, these data demonstrate that vascular maturation coordinated by endothelial cell/mural cell interactions redirects MT1-MMP expression to the neovessel tip where the protease regulates matrix remodeling at the leading edge of the developing vasculature.

Negative regulation of osteoclastogenesis by ectodomain shedding of receptor activator of NF-kappaB ligand.

Receptor activator of NF-kappaB ligand (RANKL) is a transmembrane glycoprotein that has an essential role in the development of osteoclasts. The extracellular portion of RANKL is cleaved proteolytically to produce soluble RANKL, but definite RANKL sheddase(s) and the physiologic function of RANKL shedding have not yet been determined. In the present study, we found that matrix metalloproteinase (MMP) 14 and a disintegrin and metalloproteinase (ADAM) 10 have strong RANKL shedding activity. In Western blot analysis, soluble RANKL was detected as two different molecular weight products, and RNA interference of MMP14 and ADAM10 resulted in a reduction of both the lower and higher molecular weight products. Suppression of MMP14 in primary osteoblasts increased membrane-bound RANKL and promoted osteoclastogenesis in cocultures with macrophages. Soluble RANKL produced by osteoblasts from MMP14-deficient mice was markedly reduced, and their osteoclastogenic activity was promoted, consistent with the findings of increased osteoclastogenesis in vivo. RANKL shedding is an important process that down-regulates local osteoclastogenesis.

Roles of pericellular proteolysis by membrane type-1 matrix metalloproteinase in cancer invasion and angiogenesis.

Behavior of cancer cells is profoundly affected by their microenvironment, which is often controlled by pericellular proteolysis or the processing of protein components, including extracellular matrices, growth factors, cytokines, receptors, cell adhesion molecules, and so on. Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteases responsible for the proteolytic events in the extracellular milieu. Among the multiple MMPs expressed in a wide range of tumors, membrane type-1 MMP (MT1-MMP), which is expressed especially in tumor cells with significant invasive properties, is thought to be particularly important for pericellular proteolysis. Recent studies have elucidated in part how MT1-MMP is regulated biologically for the promotion of invasion by tumors or for angiogenesis by endothelial cells. Understanding of the proteolysis by, and the regulation of MT1-MMP, which probably promotes cell invasion, could provide a therapeutic hint as to how to block or delay the progression of cancer.

Role of pericellular proteolysis by membrane-type 1 matrix metalloproteinase in cancer invasion and angiogenesis.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is an integral membrane proteinase that is frequently expressed in malignant cancer cells and has potent invasion-promoting activity. When expressed on the cell surface, MT1-MMP degrades the extracellular matrix (ECM) barrier adjacent to the cells to maintain the migration route to traverse the tissue. But MT1-MMP is not just an enzyme that degrades ECM. MT1-MMP also introduces limited cleavage into proteins at the cell-ECM interspaces and converts their functions. The target molecules are ECM components, cell adhesion molecules, and latent forms of MMPs. Through these processing events MT1-MMP modulates the migratory and invasive behavior of the cells.

Clusterin, an abundant serum factor, is a possible negative regulator of MT6-MMP/MMP-25 produced by neutrophils.

MT6-MMP/MMP-25 is the latest member of the membrane-type matrix metalloproteinase (MT-MMP) subgroup in the MMP family and is expressed in neutrophils and some brain tumors. The proteolytic activity of MT6-MMP has been studied using recombinant catalytic fragments and shown to degrade several components of the extracellular matrix. However, the activity is possibly modulated further by the C-terminal hemopexin-like domain, because some MMPs are known to interact with other proteins through this domain. To explore the possible function of this domain, we purified a recombinant MT6-MMP with the hemopexin-like domain as a soluble form using a Madin-Darby canine kidney cell line as a producer. Mature and soluble MT6-MMP processed at the furin motif was purified as a 45-kDa protein together with a 46-kDa protein having a single cleavage in the hemopexin-like domain. Interestingly, 73- and 70-kDa proteins were co-purified with the soluble MT6-MMP by forming stable complexes. They were identified as clusterin, a major component of serum, by N-terminal amino acid sequencing. MT1-MMP that also has a hemopexin-like domain did not form a complex with clusterin. MT6-MMP forming a complex with clusterin was detected in human neutrophils as well. The enzyme activity of the soluble MT6-MMP was inactive in the clusterin complex. Purified clusterin was inhibitory against the activity of soluble MT6-MMP. On the other hand, it had no effect on the activities of MMP-2 and soluble MT1-MMP. Because clusterin is an abundant protein in the body fluid in tissues, it may act as a negative regulator of MT6-MMP in vivo.

CD44 directs membrane-type 1 matrix metalloproteinase to lamellipodia by associating with its hemopexin-like domain.

Membrane-type 1 matrix metalloproteinase (MT1- MMP) localizes at the front of migrating cells and degrades the extracellular matrix barrier during cancer invasion. However, it is poorly understood how the polarized distribution of MT1-MMP at the migration front is regulated. Here, we demonstrate that MT1-MMP forms a complex with CD44H via the hemopexin-like (PEX) domain. A mutant MT1-MMP lacking the PEX domain failed to bind CD44H and did not localize at the lamellipodia. The cytoplasmic tail of CD44H, which comprises interfaces that associate with the actin cytoskeleton, was important for its localization at lamellipodia. Overexpression of a CD44H mutant lacking the cytoplasmic tail also prevented MT1-MMP from localizing at the lamellipodia. Modulation of F-actin with cytochalasin D revealed that both CD44H and MT1-MMP co-localize closely with the actin cytoskeleton, dependent on the cytoplasmic tail of CD44H. Thus, CD44H appears to act as a linker that connects MT1-MMP to the actin cytoskeleton and to play a role in directing MT1-MMP to the migration front. The PEX domain of MT1-MMP was indispensable in promoting cell migration and CD44H shedding.