HB-EGF consists of a membrane-bound form (proHB-EGF), a soluble form (sHB-EGF), and a C-terminal fragment (CTF), and each form has a unique biological activity. Among these 3 forms, the function of sHB-EGF has been widely evaluated using recombinant proteins. To date, the biological activity of proHB-EGF has been suggested on the basis of the effect of transfected proHB-EGF on the proliferation and survival of EGFR-expressing cells 
or on the basis of the effect of proHB-EGF overexpression on autonomous cell proliferation 
. However, information on proHB-EGF function has been scanty and inconsistent, possibly because of the lack of a method for specifically activating or inhibiting proHB-EGF. In some other studies, uncleavable mutant proHB-EGF (uc-proHB-EGF), which has 2 amino acid replacements (Leu148 and Pro149) in the cleavage site, was used to evaluate proHB-EGF function 
. uc-proHB-EGF has been shown to possess juxtacrine activity in formalin-fixed EGFR-expressing cells 
. If uc-proHB-EGF fully represents wild type proHB-EGF functions even in live cells, this mutant might be useful in identifying proHB-EGF functions in spheroid culture. Recently, another version of uc-proHB-EGF, whose cleavage site was deleted, was used to study proHB-EGF functions. This uc-proHB-EGF prevented anoikis of Madin-Darby canine kidney cells 
. However, in comparison with these approaches used so far, there are some advantages in our approach using a 3D spheroid culture and a functional anti-HB-EGF antibody: the functions of endogenously expressed proHB-EGF can be detected, and formalin fixation, which may cause artifacts, is not required because there is no endogenous sHB-EGF function.
Accumulated reports showed that spheroid culture mimics the cancer environment better than a 2D culture in terms of cell-cell contacts, drug resistance, drug penetration, and nutrient efficiency 
; therefore, the functions of endogenously expressed proHB-EGF found in the 3D spheroid suggest that proHB-EGF may play important roles in tumor progression. In this study, we used 3 cancer cell lines endogenously expressing proHB-EGF. Y-142 inhibited the proliferation of NUGC-3 cells by 72%, 5637 cells by 55%, and BxPC-3 cells by 24% at the maximum concentration (). As shown in , the 3 cell lines have different levels of proHB-EGF expression (NUGC-3 cells>5637 cells>BxPC-3 cells), which may explain the relative effect of Y-142 on proliferation. The 3 cell lines are derived from stomach, bladder, and pancreatic cancer tissues, which are known to overexpress HB-EGF 
. HB-EGF has been reported to be upregulated in other cancer types such as breast and ovarian cancers and glioblastoma 
; therefore, proHB-EGF may exert its functions in a broad range of cancer types.
In the spheroid culture, treatment with Y-142 increased the number of cells that diffused out of the spheroid core (). The inhibition of proHB-EGF juxtacrine activity was detected even 3 d after the Y-142 treatment (), when cell diffusion was seen. We speculate that the proHB-EGF-mediated cell-cell contact is directly associated with its proHB-EGF juxtacrine activity. The results of a previous study using uc-proHB-EGF supported this speculation in that the results indicated a cytoprotective role of proHB-EGF by enhancing EGFR-mediated cell-cell contact and showed that caspase activation was inhibited by the proHB-EGF/EGFR/AKT pathway 
. We also detected caspase activation and inhibited EGFR and AKT phosphorylation by Y-142 treatment in the spheroid culture (, ). Our results may be derived from the inhibition of the cytoprotective role of proHB-EGF. We speculate that the anti-caspase function of proHB-EGF contributes to cancer cell survival by inhibiting apoptosis signals such as those activated in cells subjected to chemotherapeutic agents, radiotherapy, hypoxia, and immune cell-mediated cytotoxicity. A previous study reported that the anti-apoptotic protein BAG-1 mediates the proHB-EGF cell survival effect 
. The BAG-1-mediated cell survival effect of proHB-EGF was noted in CHO cells, which are devoid of EGFR, suggesting that the proHB-EGF/BAG-1 pathway uses EGFR-independent cell survival signaling. proHB-EGF may use both the EGFR-dependent and EGFR-independent pathways to exert its cell survival effects. In addition, we noted the inhibition of ERK1/2 phosphorylation by Y-142 (), suggesting that the cell proliferation signal from proHB-EGF is mediated through the ERK1/2 pathway. While measuring the juxtacrine activity of proHB-EGF in a 3D spheroid culture, we noted the inhibition of EGFR signaling by Y-142 (), suggesting that proHB-EGF exerts the anti-apoptotic and cell proliferation functions on neighboring cells through their EGFR pathway. In cases where cancer cells express both proHB-EGF and EGFR, the same cell may act as the donor and the acceptor of the cell survival and proliferation signals. Taken together, we predict that proHB-EGF efficiently induces tumor progression by directly promoting cancer cell proliferation and by inhibiting apoptosis. As shown in , Y-142 did not inhibit ectodomain shedding, leading to CTF production. Therefore, we concluded that the biological activity of Y-142 found here was not attributable to the inhibition of CTF production. Previous studies have indicated that both proHB-EGF and CTF translocate to the nucleus, thereby inducing cancer cell proliferation and invasion 
. If Y-142 possesses an inhibitory activity against proHB-EGF translocation, further analyses could identify the importance of the proHB-EGF translocation in cancer cell proliferation and invasion.
In this study, to investigate proHB-EGF functions in cancer cells, we used 2 anti-HB-EGF antibodies, Y-142 and Y-073, which had activities similar to those of sHB-EGF, but different specificities toward proHB-EGF ( and ). Our previous study showed that the neutralizing activity of Y-142 is attributable to its recognition of amino acids in the EGF-like domain of HB-EGF 
. The EGF-like domain is required for the binding of HB-EGF to EGFR. We therefore presume that the epitope of neutralizing antibody Y-073 should also be located in the EGF-like domain. Y-073, however, did not bind to proHB-EGF. These findings allow us to speculate that Y-073 may also recognize the newly exposed site in sHB-EGF after proHB-EGF ectodomain shedding. Consistent with our speculation, several antibodies to different molecules such as type II collagen, aggrecan, fibrin, and link protein have been reported to specifically recognize a newly exposed site resulting from a protein cleavage 
. Epitope mapping studies will provide detailed information about Y-073 functionality.
In summary, we have shown that the endogenously expressed proHB-EGF acts as a cell proliferation and cell survival factor in cancer cells and that it may play an important role in cell proliferation and survival in tumor tissues.