Integrin α6ß4 Confers Doxorubicin Resistance in Cancer Cells by Suppressing Caspase-3-Mediated Apoptosis: Involvement of N-Glycans on ß4 Integrin Subunit.

Drug resistance is a major obstacle to successful cancer treatment. Therefore, it is essential to understand the molecular mechanisms underlying drug resistance to develop successful therapeutic strategies. α6ß4 integrin confers resistance to apoptosis and regulates the survival of cancer cells; however, it remains unclear whether α6ß4 integrin is directly involved in chemoresistance. Here, we show that α6ß4 integrin promotes doxorubicin resistance by decreasing caspase-3-mediated apoptosis. We found that the overexpression of α6ß4 integrin by the ß4 integrin gene rendered MDA-MB435S and Panc-1 cells more resistant to doxorubicin than control cells. The acquired resistance to doxorubicin by α6ß4 integrin expression was abolished by the deletion of the cytoplasmic signal domain in ß4 integrin. Similar results were found in MDA-MB435S and Panc-1 cells when N-glycan-defective ß4 integrin mutants were overexpressed or bisecting GlcNAc residues were increased on ß4 integrin by the co-expression of N-acetylglucosaminyltransferase III with ß4 integrin. The abrogation of α6ß4 integrin-mediated resistance to doxorubicin was accompanied by reduced cell viability and an increased caspase-3 activation. Taken together, our results clearly suggest that α6ß4 integrin signaling plays a key role in the doxorubicin resistance of cancer cells, and N-glycans on ß4 integrin are involved in the regulation of cancer cells.

Phosphorylated Osteopontin Secreted from Cancer Cells Induces Cancer Cell Motility.

Osteopontin (OPN) plays a pivotal role in cancer cell invasion and metastasis. Although OPN has a large number of phosphorylation sites, the functional significance of OPN phosphorylation in cancer cell motility remains unclear. In this study, we attempted to investigate whether phosphorylated OPN secreted from cancer cells affect cancer cell migration. Quantitative PCR and Western blot analyses revealed that MDA-MB435S, A549, and H460 cells highly expressed OPN, whereas the OPN expression levels in H358, MIAPaca-2, and Panc-1 cells were quite low or were not detected. Compared with the cancer cell lines with a low OPN expression, the high OPN-expressing cancer cell lines displayed a higher cell migration, and the cell migration was suppressed by the anti-OPN antibody. This was confirmed by the OPN overexpression in H358 cancer cells with a low endogenous OPN. Phos-tag ELISA showed that phosphorylated OPN was abundant in the cell culture media of A549 and H460 cells, but not in those of MDA-MB435S cells. Moreover, the A549 and H460 cell culture media, as well as the MDA-MB435S cell culture media with a kinase treatment increased cancer cell motility, both of which were abrogated by phosphatase treatment or anti-OPN antibodies. These results suggest that phosphorylated OPN secreted from cancer cells regulates cancer cell motility.

αvß3 Integrin induces partial EMT independent of TGF-ß signaling.

Epithelial-mesenchymal transition (EMT) plays a pivotal role for tumor progression. Recent studies have revealed the existence of distinct intermediate states in EMT (partial EMT); however, the mechanisms underlying partial EMT are not fully understood. Here, we demonstrate that αvß3 integrin induces partial EMT, which is characterized by acquiring mesenchymal phenotypes while retaining epithelial markers. We found αvß3 integrin to be associated with poor survival in patients with lung adenocarcinoma. Moreover, αvß3 integrin-induced partial EMT promoted migration, invasion, tumorigenesis, stemness, and metastasis of lung cancer cells in a TGF-ß-independent fashion. Additionally, TGF-ß1 promoted EMT progression synergistically with αvß3 integrin, while a TGF-ß signaling inhibitor showed no effect on αvß3 integrin-induced partial EMT. Meanwhile, the microRNA-200 family abolished the αvß3 integrin-induced partial EMT by suppressing αvß3 integrin cell surface expression. These findings indicate that αvß3 integrin is a key inducer of partial EMT, and highlight a new mechanism for cancer progression.

Biological role of site-specific O-glycosylation in cell adhesion activity and phosphorylation of osteopontin.

Osteopontin (OPN) is an extracellular glycosylated phosphoprotein that promotes cell adhesion by interacting with several integrin receptors. We previously reported that an OPN mutant lacking five O-glycosylation sites (Thr134/Thr138/Thr143/Thr147/Thr152) in the threonine/proline-rich region increased cell adhesion activity and phosphorylation compared with the wild type. However, the role of O-glycosylation in cell adhesion activity and phosphorylation of OPN remains to be clarified. Here, we show that site-specific O-glycosylation in the threonine/proline-rich region of OPN affects its cell adhesion activity and phosphorylation independently and/or synergistically. Using site-directed mutagenesis, we found that OPN mutants with substitution sets of Thr134/Thr138 or Thr143/Thr147/Thr152 had decreased and increased cell adhesion activity, respectively. In contrast, the introduction of a single mutation into the O-glycosylation sites had no effect on OPN cell adhesion activity. An adhesion assay using function-blocking antibodies against αvß3 and ß1 integrins, as well as αvß3 integrin-overexpressing A549 cells, revealed that site-specific O-glycosylation affected the association of OPN with the two integrins. Phosphorylation analyses using phos-tag and LC-MS/MS indicated that phosphorylation levels and sites were influenced by the O-glycosylation status, although the number of O-glycosylation sites was not correlated with the phosphorylation level in OPN. Furthermore, a correlation analysis between phosphorylation level and cell adhesion activity in OPN mutants with the site-specific O-glycosylation showed that they were not always correlated. These results provide conclusive evidence of a novel regulatory mechanism of cell adhesion activity and phosphorylation of OPN by site-specific O-glycosylation.

ß4-Integrin/PI3K Signaling Promotes Tumor Progression through the Galectin-3-N-Glycan Complex.

Malignant transformation is associated with aberrant N-glycosylation, but the role of protein N-glycosylation in cancer progression remains poorly defined. ß4-integrin is a major carrier of N-glycans and is associated with poor prognosis, tumorigenesis, and metastasis. Here, N-glycosylation of ß4-integrin contributes to the activation of signaling pathways that promote ß4-dependent tumor development and progression. Increased expression of ß1,6GlcNAc-branched N-glycans was found to be colocalized with ß4-integrin in human cutaneous squamous cell carcinoma tissues, and that the ß1,6GlcNAc residue was abundant on ß4-integrin in transformed keratinocytes. Interruption of ß1,6GlcNAc-branching formation on ß4-integrin with the introduction of bisecting GlcNAc by N-acetylglucosaminyltransferase III overexpression was correlated with suppression of cancer cell migration and tumorigenesis. N-Glycan deletion on ß4-integrin impaired ß4-dependent cancer cell migration, invasion, and growth in vitro and diminished tumorigenesis and proliferation in vivo The reduced abilities of ß4-integrin were accompanied with decreased phosphoinositol-3 kinase (PI3K)/Akt signals and were restored by the overexpression of the constitutively active p110 PI3K subunit. Binding of galectin-3 to ß4-integrin via ß1,6GlcNAc-branched N-glycans promoted ß4-integrin-mediated cancer cell adhesion and migration. In contrast, a neutralizing antibody against galectin-3 attenuated ß4-integrin N-glycan-mediated PI3K activation and inhibited the ability of ß4-integrin to promote cell motility. Furthermore, galectin-3 knockdown by shRNA suppressed ß4-integrin N-glycan-mediated tumorigenesis. These findings provide a novel role for N-glycosylation of ß4-integrin in tumor development and progression, and the regulatory mechanism for ß4-integrin/PI3K signaling via the galectin-3-N-glycan complex.Implications:N-Glycosylation of ß4-integrin plays a functional role in promoting tumor development and progression through PI3K activation via the galectin-3-N-glycan complex. Mol Cancer Res; 16(6); 1024-34. ©2018 AACR.

Roles of Integrin α6ß4 Glycosylation in Cancer.

Malignant transformation is accompanied with aberrant glycosylation of proteins. Such changes in glycan structure also occur in the integrins, which are a large family of cell surface receptors for the extracellular matrix and play key roles in tumor progression. There is now increasing evidence that glycosylation of integrins affects cellular signaling and interaction with the extracellular matrix, receptor tyrosine kinases, and galectins, thereby regulating cell adhesion, motility, growth, and survival. Integrin α6ß4 is a receptor for laminin-332 and the increased expression level is correlated with malignant progression and poor survival in various types of cancers. Recent studies have revealed that integrin α6ß4 plays central roles in tumorigenesis and the metastatic process. In this review, we summarize our current understanding of the molecular mechanisms of tumor progression driven by integrin α6ß4 and also discuss the modification of glycans on integrin ß4 subunit to address the important roles of glycan in integrin-mediated tumor progression.

RNase activity of sialic acid-binding lectin from bullfrog eggs drives antitumor effect via the activation of p38 MAPK to caspase-3/7 signaling pathway in human breast cancer cells.

Sialic acid-binding lectin obtained from bullfrog eggs (SBL) induces cell death in cancer cells but not in normal cells. This antitumor effect is mediated through its ribonuclease (RNase) activity. However, the underlying molecular mechanisms remain unclear. We found that the p38 mitogen-activated protein kinase (MAPK) signaling pathway was activated when SBL induced cell death in three human breast cancer cell lines: SK-BR-3, MCF-7, and MDA­MB231. The suppression of p38 MAPK phosphorylation by a p38 MAPK inhibitor as well as short interference RNA knockdown of p38 MAPK expression significantly decreased cell death and increased the cell viability of SBL-treated MDA­MB231 cells. H103A, an SBL mutant lacking in RNase activity, showed decreased SBL-induced cell death compared with native SBL. However, the loss of RNase activity of SBL had no effect on its internalization into cells. The H103A mutant also displayed decreased phosphorylation of p38 MAPK. Moreover, SBL promoted caspase­3/7 activation followed by a cleavage of poly (ADP-ribose)-polymerase, whereas the SBL mutant, H103A, lost this ability. The SBL-induced caspase­3/7 activation was suppressed by the p38 MAPK inhibitor, SB203580, as well as pan-caspase inhibitor, zVAD-fmk. In the presence of zVAD-fmk, the SBL-induced cell death was decreased. In addition, the cell viability of SBL-treated MDA­MB231 cells recovered by zVAD-fmk treatment. Taken together, our results suggest that the RNase activity of SBL leads to breast cancer cell death through the activation of p38 MAPK followed by the activation of caspase­3/7.

Increased cerebrospinal fluid osteopontin levels and its involvement in macrophage infiltration in neuromyelitis optica.

BACKGROUND: Neuromyelitis optica (NMO) is an inflammatory disease of the central nervous system that predominantly affects the optic nerves and spinal cord. Although NMO has long been considered a subtype of multiple sclerosis (MS), the effects of interferon-ß treatment are different between NMO and MS. Recent findings of NMO-IgG suggest that NMO could be a distinct disease rather than a subtype of MS. However, the underlying molecular mechanism of NMO pathology remains poorly understood. METHODS: OPN in the cerebrospinal fluid and brain of patients with NMO and with MS, as well as of patients with other neurologic disease/idiopathic other neurologic disease was examined using Western blotting, ELISA, immunohistochemistry and Boyden chamber. RESULTS: Here we show that osteopontin is significantly increased in the cerebrospinal fluid of NMO patients compared with MS patients. Immunohistochemical analyses revealed that osteopontin was markedly elevated in the cerebral white matter of NMO patients and produced by astrocytes, neurons, and oligodendroglia as well as infiltrating macrophages. We also demonstrate that the interaction of the cerebrospinal fluid osteopontin in NMO patients with integrin αvß3 promoted macrophage chemotaxis by activating phosphoinositide 3-kinase and MEK1/2 signaling pathways. CONCLUSION: These results indicate that osteopontin is involved in NMO pathology. GENERAL SIGNIFICANCE: Thus therapeutic strategies that target osteopontin signaling may be useful to treat NMO.

Sialic acid-binding lectin (leczyme) induces apoptosis to malignant mesothelioma and exerts synergistic antitumor effects with TRAIL.

Malignant mesothelioma is a highly aggressive tumor with poor prognosis. An effective drug for treatment of malignant mesothelioma is greatly needed. Sialic acid-binding lectin (SBL) isolated from oocytes of Rana catesbeiana is a multifunctional protein which has lectin activity, ribonuclease activity and antitumor activity, so it could be developed as a new type of anticancer drug. The validity of SBL for treatment of malignant mesothelioma was assessed using three malignant mesotheliomas and a non-malignant mesothlial cell line. Effectiveness of combinatorial treatment of SBL and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) was also elucidated and characterized. SBL induced tumor-selective cytotoxicity that was attributed to induction of apoptosis. Combinatorial treatment of SBL and TRAIL showed synergistic apoptosis-inducing effect. Additional experiments revealed that Bid was the mediating molecule for the synergistic effect in SBL and TRAIL. These results suggested that SBL could be a promising candidate for the therapeutics for malignant mesothelioma. Furthermore, the combinatorial treatment of SBL and TRAIL could be an effective regimen against malignant mesothelioma.

Involvement of ER stress in apoptosis induced by sialic acid-binding lectin (leczyme) from bullfrog eggs.

Sialic-acid binding lectin (SBL) isolated from bullfrog (Rana catesbeiana) oocytes is a multifunctional protein which has lectin activity, ribonuclease activity and cancer-selective antitumor activity. It has been reported that SBL induces apoptosis accompanied by rigid mitochondrial perturbation, which indicates mediation of the intrinsic pathway. However, the mechanism of the antitumor effect of SBL has not been fully elucidated. We report, here, that ER stress is evoked in SBL-treated cells. We show that caspase-4, an initiator caspase of ER stress-mediated apoptosis was activated, and inhibition of caspase-4 resulted in significant attenuation of apoptosis induced by SBL. We analyzed the precise mechanism of activation of the caspase cascade induced by SBL, and found that caspase-9 and -4 are activated upstream of activation of caspase-8. Further study revealed that SBL induces the mitochondrial and ER stress-mediated pathways independently. It is noteworthy that SBL can induce cancer-selective apoptosis by multiple apoptotic signaling pathways, and it can serve as a candidate molecule for anticancer drugs in a novel field.

Sialic acid-binding lectin (leczyme) induces caspase-dependent apoptosis-mediated mitochondrial perturbation in Jurkat cells.

Sialic acid binding lectin (SBL) isolated from Rana catesbeiana oocytes is a multifunctional protein which has lectin activity, ribonuclease activity and antitumor activity. However, the mechanism of antitumor effects of SBL is unclear to date and the validity for human leukemia cells has not been fully studied. We report here that SBL shows cytotoxicity for some human leukemia cell lines including multidrug-resistant (MDR) cells. The precise mechanisms of SBL-induced apoptotic signals were analyzed by combinational usage of specific caspase inhibitors and the mitochondrial membrane depolarization detector JC-1. It was demonstrated that SBL causes mitochondrial perturbation and the apoptotic signal is amplified by caspases and cell death is executed in a caspase-dependent manner. The efficacy of this combinational usage was shown for the first time, to distinguish the apoptotic pathway in detail. SBL selectively kills tumor cells, is able to exhibit cytotoxicity regardless of P-glycoprotein expression and has potential as an alternative to conventional DNA-damaging anticancer drugs.

Polymerized laminin-332 matrix supports rapid and tight adhesion of keratinocytes, suppressing cell migration.

Laminin-332 (α3ß3γ2) (Lm332) supports the stable anchoring of basal keratinocytes to the epidermal basement membrane, while it functions as a motility factor for wound healing and cancer invasion. To understand these contrasting activities of Lm332, we investigated Lm332 matrices deposited by normal human keratinocytes and other Lm332-expressing cell lines. All types of the cells efficiently deposited Lm332 on the culture plates in specific patterns. On the contrary, laminins containing laminin ß1 and/or γ1 chains, such as Lm511 and Lm311, were not deposited on the culture plates even if secreted into culture medium. The Lm332 deposition was not inhibited by function-blocking antibodies to the α3 and α6 integrins but was inhibited by sodium selenate, suggesting that sulfated glycosaminoglycans on cell surface, e.g. heparan sulfate proteoglycans, might be involved in the process. HEK293 cells overexpressing exogenous Lm332 (Lm332-HEK) almost exclusively deposited Lm332 on the plates. The deposited Lm332 matrix showed a mesh-like network structure as analyzed by electron microscopy, suggesting that Lm332 was highly polymerized. When biological activity was analyzed, the Lm332 matrix rather suppressed the migration of keratinocytes as compared with purified Lm332, which highly promoted the cell migration. The Lm332 matrix supported adhesion of keratinocytes much more strongly and stably than purified Lm332. Integrin α3ß1 bound to the Lm332 matrix at a three times higher level than purified Lm332. Normal keratinocytes prominently showed integrin α6ß4-containing, hemidesmosome-like structures on the Lm332 matrix but not on the purified one. These results indicate that the polymerized Lm332 matrix supports stable cell adhesion by interacting with both integrin α6ß4 and α3ß1, whereas unassembled soluble Lm332 supports cell migration.