High mannose-binding Pseudomonas fluorescens lectin (PFL) downregulates cell surface integrin/EGFR and induces autophagy in gastric cancer cells

In the present study, we demonstrated that the high mannose binding bacterial lectin PFL induced rapid intracellular trafficking of both EGFR and ?2 integrin in MKN28 gastric cancer cells. The adherence strength of MKN28 cells to ECM was dramatically abolished by PFL treatment, correlating with the loss of cell surface integrins. Activated EGFR transiently modulates ?2 integrin cell surface expression and stimulates integrin trafficking via caveolae/raft-mediated endocytosis [9]. Furthermore, physical and functional association between integrins and EGFR also has been demonstrated [10, 11]. Such cross-talk between integrins and EGFR has been hypothesized to play a critical role in cancer progression [12]. It is still unclear whether PFL bound to glycans of integrin and EGFR independently, or to those of integrin/EGFR complexes. In either case, we assume that both molecules internalized by PFL would be eventually degraded, resulting in dysfunction of cell attachment and proliferation. Since high mannose EGFR has reported to represent a novel tumor-specific antigen [13], PFL would be useful as a cancer targeting material. Recently, ?1 integrin-silenced cells were reported to exhibit defective activation of EGFR signaling, leading to increased sensitivity to gefitinib [14]. Similarly, we observed that PFL enhanced the susceptibility of MKN28 cells to gefitinib, possibly due to EGFR downregulation. Moreover, enhanced drug sensitivity of other cancer cell lines in the presence of PFL also was observed. For instance, the 5-FU sensitivity of HT29 colon cancer cells was increased in the presence of 0.5 ?M PFL, a concentration that was not harmful to the cells, although the effect of PFL was limited compared with EGFR drugs. The mechanism behind this observation is yet to be clarified; however, it is plausible that PFL might activate endocytosis transiently during integrin/EGFR internalization, thereby facilitating uptake of extracellular substances.

Our data suggest that the internalized integrin/EGFR associated with PFL was degraded by autophagy, a mechanism that eliminates damaged proteins, aged proteins and organelles, or harmful aggregated proteins [15–17]. Expression of many autophagic proteins in MKN28 cells was elevated significantly by PFL treatment, but expression of activated apoptotic proteins was hardly detectable. Although we could not rule out the possibility that both autophagy and apoptosis might occur in PFL-treated cancer cells, because both phenomena are reported to cross-talk through shared molecules such as ATG5 by taking either the calpain-cleaved or non-cleaved form [18, 19], the present data indicate that PFL primarily induces autophagy.

Some plant lectins are reported to induce autophagy in cancer cells via a mitochondria-mediated pathway [20]. For example, legume lectin Concanavalin A (ConA), which recognizes high mannose N-glycans as well as monosaccharides including mannose, binds to cell membrane glycoproteins and is internalized and accumulates in mitochondria, resulting in mitochondrial dysfunction and induction of autophagy to degrade these mitochondria [21]. Polygonatum cyrtonema lectin, which belongs to the GNA-related lectin family, induces cancer cell autophagy by promoting the ROS-p38-p53 pathway [22]. By contrast, PFL-induced autophagy is mediated by integrin/EGFR internalization, a mechanism that has not been reported so far, although other surface glycoproteins with high mannose glycans might be involved in this process as well.

Following PFL-induced receptor endocytosis, the association of EGFR with vesicular trafficking proteins such as RAB7 was observed. RAB7, a member of small RAB GTPase family, is known as a common modulator in endocytosis and autophagy. It is involved not only in the maturation of endosomes and autophagosomes, but also in the trafficking of cargos along microtubules and in fusion with lysosomes [15, 23–25]. Furthermore, it is reported that functional RAB7 is required for the degradation of EGFR by the lysosome [26]. In the present study, we observed increased expression of RAB7, and that siRNA against RAB7 inhibited PFL-induced EGFR degradation, which suggest that RAB7 plays an important role in this degradation process. In addition, ATG9, a multipass transmembrane protein necessary for optimal autophagy [27], increased in expression in response to PFL treatment of MKN28 cells, and colocalized with the internalized EGFR. Mammalian ATG9 has been suggested to interact transiently with isolation membranes and autophagosomes, but its actual function is still controversial [28]. Although the detailed mechanism of receptor membrane trafficking remains be clarified, DNA microarray data suggest that multiple molecules, including autophagic proteins, membrane trafficking proteins, and motor proteins such as dynein, might be involved in PFL-mediated receptor degradation. The roles of autophagy are assumed to differ in different stages of cancer development [29]. For example, autophagy initially has a preventive role against cancer, but for developed cancer cells, it can be utilized to promote cancer cell survival. Although we have no direct evidence as to whether autophagy might contribute to PFL-induced cell death and tumor suppression, it would be plausible that PFL-induced autophagy ultimately affects cell survival, because it leads directly to defects in integrin and EGFR signaling that are crucial for cell adhesion to appropriate location, migration and proliferation.

Direct administration of PFL to subcutaneous tumors significantly reduced tumor growth in vivo. Other lectins, such as ESA-2, which is from the same lectin family, reportedly exhibit potent anti-cancer activity when administered intravenously or intraperitoneally in surface-bound form in Span 80 vesicles to mice bearing subcutaneous tumors [30]. Since this lectin family of proteins exhibit a characteristic stable structure, i.e., a domain swap structure with abundant ? sheets [3, 7], it would be advantageous to utilize them for clinical applications. Furthermore, it is noteworthy that PFL enhanced the susceptibility of cancer cells to anti-cancer drugs such as gefitinib. Serum levels of surfactant protein D (SP-D), a C-type lectin with innate immune functions, also reportedly are associated with gefitinib efficacy [31]. Therefore, future work will be directed toward evaluating the in vivo adjuvant effect of PFL with various anti-cancer drugs to increase therapeutic efficacy and reduce side effects.