**2. Functions of LAPTM4B-35 involved in signaling network**

Current evidence indicates that the interaction between cancer cells with their microenviron‐ ment plays key roles in oncogenesis and progression. Cancer microenvironment is composed of variant signal molecules, including solvable signal molecules (growth factors, cytokines, etc.), insolvable extracellular matrix (ECM), and variant cells nearby. Cancer cells and their microenvironment are reciprocally affected. Cancer cell proliferation, survival, and migratio‐ nare all motivated and dependent on not only solvable signal molecules but also ECM. Cancer cells accept positive or negative regulations of signal molecules from solvable factors, ECM, and other cells in their microenvironment through signal transduction pathways, which are organized as a very complicated network. In other words, cancer may be known as a disease of signaling network. Disturbances of signaling pathways and the converging network initiate at the early stage and go through the whole process of cancer development. In addition, the disturbance of signaling pathways results from oncogenic alternation in genetics and epige‐ netics and contributes to the molecular and cellular malignant phenotypes of cancer cells, which include disregulations of proliferation, survival/apoptosis, differentiation and metab‐ olism, as well as enhancement of migration/invasion and multidrug resistance. Therefore, signaling pathways and the network are of importance from a therapeutic perspective because targeting them may help reverse, delay, or prevent oncogenesis. Notably, since cirrhosis is associated with hepatic regeneration after tissue damages, which are caused by hepatitis infection, toxins (for example, alcohol oraflatoxin) or metabolic influences, and is often the prerequisite of hepato-oncogenesis, it is noticed that the ECM and the ECM-related signaling pathways, that are commonly alternated in cirrhosis and HCC, are of very importance. Our preliminary study has indicated that LAPTM4B-35 is most likely an assembly platform or organizer for a number of signaling molecules which are integrated in the cell membranes or soluble in the cytoplasm. Overexpression of LAPTM4B-35 would therefore be expected to lead to disturbance of a wide range of signaling pathways and their networks. We found LAPTM4B-35 can interact or co-localize with a number of these signal molecules, including membrane-integrated receptors and cytoplasmic signal molecules. These membrane-integrat‐ ed receptors involve the growth factor receptors of the RTK (receptor tyrosine kinase) family, such as EGFR [9-11] and IGF-1R (Figure 1a), and ECM receptors of the integrin family, such as α6β1 [11] and α5β1 (Figures 1d and 2). The cytoplasmic signaling molecules that can interact with LAPTM4B-35 include FAK (Figure 2c) and PI3K p85α (Figure 3a). Given that LAPTM4B-35 is a tetra-transmembrane protein and localizes in plasma membrane and endomembranes (including lysosomes and endosomes). The interaction of LAPTM4B-35 with both RTK under the stimulation of growth factors, and integrin under ECM stimulation would be expected to integrate related signal transduction pathways triggered by growth factors and ECM components at the cell surface. It is well known that based on binding of growth factors (ligand) to their corresponding RTK receptor, Ras and ERK1/2 (MAPK family)downstream is subsequently activated [12]. At the same time, based on binding of ECM components (ligand), such as fibronectin (FN) or laminin (LN), to their corresponding integrin receptor (α5β1 or α6β1, respectively), FAK397 is phosphorylated and activated, and may subsequently activate downstream Ras/ERK and PI3K/AKT signaling pathways [13,14]. As has been previously recognized, the RTK/Ras/ERK signaling pathway and the ECM/Integrin/FAK signaling pathway converge at Ras and/or FAK. However, we found that over expression of LAPTM4B-35 can not only dramatically activate Ras (Figure 1b) and the downstream ERK1/2 (MAPK) under the stimulation of growth factors (Figures 1c) or FN (Figure 1f), respectively, but also activates FAK. This was originally suggested by knock down experiments. When LAPTM4B-35 is knocked down by RNAi in HCC cells, binding of integrin α5 with LAPTM4B-35 is dramatically decreased under stimulation with FN, as shown in Figure 1d. Knockdown of LAPTM4B-35 also coincidently significantly reduces phosphorylation and activation of FAK397 (Figure 1e) under stimulation by FN or LN. These experiments further provide evidence for the involvement of LAPTM4B-35 in the ECM/integrin/FAK signaling pathway. In addition, inhibition of FAK by PP2 (FAK inhibitor) can attenuate phosphorylation/ activation of ERK1/2 in both LAPTM4B35-up-regulated HCC cells (AE) and in wild-type HCC control cells (Mock) as shown in Figure 1f. AE and Mock cells are both LAPTM4B-35 overex‐ pressed, but to different extents. These results suggest that in LAPTM4B-35 overexpressed HCC cells, activation of ERK results from both the upstream growth factor/Ras and FN/ Integrin/FAK signaling cascades. Taken together, it is reasonable to propose that overex‐ pressed LAPTM4B-35 as a linker at the cell surface (plasma membrane) simultaneously over activates both the growth factor (EGF or IGF-1R)/RTK/Ras/ERK and the ECM (FN or LN)/ Integrin/FAK/ERK signaling pathways by interacting with growth factor receptor (RTK) and ECM receptor (integrin) under the stimulation of growth factor and ECM components (FN, LN), respectively. In other words, the growth factor/RTK/Ras/ERK and ECM/ integrin/FAK/ERK signaling pathways initiallyconverge at the plasma membrane level through overexpression of membrane-integrated LAPTM4B-35 in HCC cells, instead of at Ras and FAK in the cytoplasm in normal hepatocytes whichexpress LAPTM4B-35 and FAK at rather low level. Moreover, simultaneous overactivation of these two signaling pathways caused by LAPTM4B-35 overexpression would result in enhancement of proliferation, survival, migration and invasion of cancer cells.

disturbance of signaling pathways results from oncogenic alternation in genetics and epige‐ netics and contributes to the molecular and cellular malignant phenotypes of cancer cells, which include disregulations of proliferation, survival/apoptosis, differentiation and metab‐ olism, as well as enhancement of migration/invasion and multidrug resistance. Therefore, signaling pathways and the network are of importance from a therapeutic perspective because targeting them may help reverse, delay, or prevent oncogenesis. Notably, since cirrhosis is associated with hepatic regeneration after tissue damages, which are caused by hepatitis infection, toxins (for example, alcohol oraflatoxin) or metabolic influences, and is often the prerequisite of hepato-oncogenesis, it is noticed that the ECM and the ECM-related signaling pathways, that are commonly alternated in cirrhosis and HCC, are of very importance. Our preliminary study has indicated that LAPTM4B-35 is most likely an assembly platform or organizer for a number of signaling molecules which are integrated in the cell membranes or soluble in the cytoplasm. Overexpression of LAPTM4B-35 would therefore be expected to lead to disturbance of a wide range of signaling pathways and their networks. We found LAPTM4B-35 can interact or co-localize with a number of these signal molecules, including membrane-integrated receptors and cytoplasmic signal molecules. These membrane-integrat‐ ed receptors involve the growth factor receptors of the RTK (receptor tyrosine kinase) family, such as EGFR [9-11] and IGF-1R (Figure 1a), and ECM receptors of the integrin family, such as α6β1 [11] and α5β1 (Figures 1d and 2). The cytoplasmic signaling molecules that can interact with LAPTM4B-35 include FAK (Figure 2c) and PI3K p85α (Figure 3a). Given that LAPTM4B-35 is a tetra-transmembrane protein and localizes in plasma membrane and endomembranes (including lysosomes and endosomes). The interaction of LAPTM4B-35 with both RTK under the stimulation of growth factors, and integrin under ECM stimulation would be expected to integrate related signal transduction pathways triggered by growth factors and ECM components at the cell surface. It is well known that based on binding of growth factors (ligand) to their corresponding RTK receptor, Ras and ERK1/2 (MAPK family)downstream is subsequently activated [12]. At the same time, based on binding of ECM components (ligand), such as fibronectin (FN) or laminin (LN), to their corresponding integrin receptor (α5β1 or α6β1, respectively), FAK397 is phosphorylated and activated, and may subsequently activate downstream Ras/ERK and PI3K/AKT signaling pathways [13,14]. As has been previously recognized, the RTK/Ras/ERK signaling pathway and the ECM/Integrin/FAK signaling pathway converge at Ras and/or FAK. However, we found that over expression of LAPTM4B-35 can not only dramatically activate Ras (Figure 1b) and the downstream ERK1/2 (MAPK) under the stimulation of growth factors (Figures 1c) or FN (Figure 1f), respectively, but also activates FAK. This was originally suggested by knock down experiments. When LAPTM4B-35 is knocked down by RNAi in HCC cells, binding of integrin α5 with LAPTM4B-35 is dramatically decreased under stimulation with FN, as shown in Figure 1d. Knockdown of LAPTM4B-35 also coincidently significantly reduces phosphorylation and activation of FAK397 (Figure 1e) under stimulation by FN or LN. These experiments further provide evidence for the involvement of LAPTM4B-35 in the ECM/integrin/FAK signaling pathway. In addition, inhibition of FAK by PP2 (FAK inhibitor) can attenuate phosphorylation/ activation of ERK1/2 in both LAPTM4B35-up-regulated HCC cells (AE) and in wild-type HCC control cells (Mock) as shown in Figure 1f. AE and Mock cells are both LAPTM4B-35 overex‐ pressed, but to different extents. These results suggest that in LAPTM4B-35 overexpressed HCC cells, activation of ERK results from both the upstream growth factor/Ras and FN/

150 Recent Advances in Liver Diseases and Surgery

**Figure 1.** Activation of Ras/ERK and FAK/ERK signaling pathways by LAPTM4B-35 overexpression*. (a)* Co-IP assay indicates the interaction between LAPTM4B-35 and IGF-1R, but not PDGFR. Lysate from BEL-7402 HCC cells was im‐ munoprecipitated by anti-LAPTM4B pAb, and the supernatant (S) and precipitant (P) were then subjected to Western blot with anti-PDGFR-mAb and anti-IGF-1R-mAb. *(b)* GST pull-down experiments with GST-RafRBD fusion protein to show Ras activation under stimulation of 20% fetal calf serum. The left panel indicates that activated Rasis increased inthe LAPTM4B-35 overexpressed BEL-7402 HCC cells (AE) as compared to the control cells (MOCK). The right pane‐ lindicates that activated Rasis decreased in the BEL-7402 HCC cells (RNAi)in which theLAPTM4B-35 has been knocked down via transient transfection by LAPTM4B-shRNA as compared with its control cells (MOCK1). It is obvi‐ ous that activation of Ras in HCC cells is associated with overexpression of LAPTM4B-35. *(c)* Western blot analysis indicates that phosphorylated ERK1 and ERK 2 are increased in LAPTM4B-35 upregulated BEL-7402 HCC cells (AE) as compared with its control (MOCK) under stimulation of 20% fetal calf serum.*(d)* Co-IP assay indicates that the interac‐ tion between LAPTM4B-35 and integrin α5 and its dependent on the overexpression of LAPTM4B-35. The lysate of BEL-7402 HCC cells was immunoprecipitated with anti-LAPTM4B pAb, the supernatant (S) and precipitant (P) were then separately subjected to Western blot analysis with anti-integrin α5-mAb. In the Western blot profiles, Lanes 1 and 2 show the integrin α5 from the HCC MOCK1 cells (as a control) in the supernatant and immunoprecipitant, respec‐

tively; Lanes 3 and 4 show the integrin α5 from LAPTM4B-35 knocked down (RNAi) HCC cells in the supernatant and immunoprecipitant, respectively. It is obvious, that integrin α5 in Lane 4 from LAPTM4B-immunoprecipitant of RNAi HCC cells Is dramatically reduced (disappear) as compared with Lane 2 from LAPTM4B-immunoprecipitant of wildtype HCC control cells that over express LAPTM4B-35. *(e)* Western blot analysis indicates that the phosphorylation/ activation of FAK397 is reduced depending on knock down of LAPTM4B-35. The cells are stimulated by ECM compo‐ nent, either fibronectin (FN) or laminin (LN), for 15 min. The lysate of cells was then subjected to Western blot analy‐ sis. Anti- phosphorylated FAK397 mAb was used for blotting. The Western blot profiles indicate that based on stimulation of FN or LN, the phosphorylated FAK397 is reduced in cells which LAPTM4B-35 expression is knocked down as compared with the control cells.*(f)* Western blot analysis indicates that FAK inhibitor (PP2) inhibits the phos‐ phorylation of ERK1/2. After treatment of BEL-7402 HCC cells (AE) by 1 μM PP2, the phosphorylation of ERK1/2 was analyzed via Western blot for the LAPTM4B-35 up-regulated cells and the MOCK cells under the stimulation of lami‐ nin substrate. The Western blot profile shows that phosphorylation/activation of ERK1/2 is associated with FAK activi‐ ty.

**Figure 2.** Colocalization between LAPTM4B-35 and integrinα5 or FAK. Cells were attaching and spreading onto fibro‐ nectin for 6 h *(a, c)* or 24 h*(b)*. *(a)* and *(b) show* the colocalization of LAPTM4B-35 (red) and integrin α5 (green). *(c)* shows the colocalization of LAPTM4B-35 (red) and FAK(green).

We found that not only membrane-integrated receptors, but also some solvable signaling molecules in cytoplasm can interact with LAPTM4B-35, such as FAK (Figure 2c) and PI3K p85α [6]. It is known that PI3K is a kinase which catalyzes phosphorylation of proteins andlipids. An important phosphorylated product catalyzed byPI3K is membrane-integrated PIP3 which can recruit cytoplasmic PH domain-containing proteins, including Akt and the corresponding kinases (PDK1 and PDK2) to the plasma membrane where Akt is phosphory‐ lated by PDK1 and PDK2. Phosphorylated Akt is commonly known as a marker for PI3K/Akt signaling pathway activation. In view of the fact PI3K consists of two subunits: p110 catalytic subunit and p85α regulatory subunit. The kinase activity of p110 is normally inhibited by binding of p85α. The inhibitory effect of p85αcan be released by binding to an appropriate molecule [15]. We found that LAPTM4B-35 can interact with p85α, but not with p110 (Figure 3a). Moreover, using site-directed mutation experiments we found that binding of LAPTM4B-35 to PI3K p85α is mediated by two motifs. One is the proline-rich motif (PPRP) in the N-terminus of LAPTM4B-35, which may bind to the SH3 domain of PI3K p85αsubunit, and the other is phosphorylated Tyr285 in the C-terminus of LAPTM4B-35, which may bind to the SH2 domain of the PI3K p85α subunit (Figure 3b). To demonstrate this a series of HCC cell variants with highly expressed wild type and mutated LAPTM4B-35 were prepared by transfection with variant plasmids containing LAPTM4B-35 with mutation at PPRP or at Try (Y)285, or with deleted N-terminus. These plasmids containg a FLAG sequence as a tag are designated as pcDNA3-LAPTM4B-flag (AF) containing wild type LAPTM4B-35, pcDNA3- LAPTM4B-flag (PA) containing P12,13,15A mutated LAPTM4B-35, pcDNA3-LAPTM4B-flag (△N) containing LAPTM4B-35 with a deletion of N10-19 amino acid residues), pcDNA3- LAPTM4B-flag (YF) containing Y285F mutated LAPTM4B-35, or pcDNA3-LAPTM4B-flag (△N+YF). As shown in Figure 3b, the binding of p85α to LAPTM4B-35 in HCC AF cells (upregulated wild-type LAPTM4B-35) is dramatically increased under the stimulation of fetal calf serum, as compared with Mock cells (the control). In contrast, the binding of p85α to LAPTM4B-35 in the PA, △N, YF, and △N+YF-mutated HCC cell variants are all significantly attenuated under the same condition, as compared with AF cells. Therefore, the overexpression of LAPTM4B-35 in HCC cells would promote the interaction of both PPRP and Tyr-p motifs of LAPTM4B-35 with PI3K p85α and thus release the inhibitory effect of p85α regulatory subunit to the p110 catalytic subunit, and would cause the phosphorylation of the downstream AKT. Accordingly, Western blot analysis (Figure 3c) demonstrated that the phosphorylated Akt (Akt-p) is decreased in the mutated AF(PA) and AF(YF) cells as compared with the wildtype LAPTM4B-35 (AF), indicating that the proline-rich domain in the N-terminal and the Tyr285 in the C-terminal tails of LAPTM4B-35 are both required for Akt phosphorylation/ activation. We also found that in the serum-starved HCC cells, LAPTM4B-35 and Akt sepa‐ rately distributes (Figure 4a); conversely under the stimulation of fetal calf serum which provides growth factors, co-localization of activated Akt and LAPTM4B-35 appears in the AF cells (Figure 4b); however, there is no co-localization in the PA-mutated cells (Figure 4c), YFmutated cells (Figure 4d), and also in the cells in which PI3K is inhibited by its inhibitor LY294002 (Figure 4e). It is obvious that the co-localization of LAPTM4B-35 and Akt appears merely in cells wherein wild-type LAPTM4B-35 is up-regulated, but not in the cells transfected by the empty vector (Mock) nor in any of the cells with mutation of PA, ΔN, and YF of LAPTM4B-35. These results further provide evidence that the PI3K-dependent activation of Akt is associated with the up-regulation of LAPTM4B-35 expression via both proline-rich motif in the N-terminus and the Tyr-p in the C-terminus (Figure 5). It is therefore proposed that LAPTM4B-35 activates PI3K/Akt signaling pathway through binding PI3K p85α by a prolinerich domain at the N terminaus and a phosphorylated Tyr285 at the C terminus to release the inhibitory effect of p85α on PI3K p110 activity, and consequently result in phosphorylation and activation of Akt.

tively; Lanes 3 and 4 show the integrin α5 from LAPTM4B-35 knocked down (RNAi) HCC cells in the supernatant and immunoprecipitant, respectively. It is obvious, that integrin α5 in Lane 4 from LAPTM4B-immunoprecipitant of RNAi HCC cells Is dramatically reduced (disappear) as compared with Lane 2 from LAPTM4B-immunoprecipitant of wildtype HCC control cells that over express LAPTM4B-35. *(e)* Western blot analysis indicates that the phosphorylation/ activation of FAK397 is reduced depending on knock down of LAPTM4B-35. The cells are stimulated by ECM compo‐ nent, either fibronectin (FN) or laminin (LN), for 15 min. The lysate of cells was then subjected to Western blot analy‐ sis. Anti- phosphorylated FAK397 mAb was used for blotting. The Western blot profiles indicate that based on stimulation of FN or LN, the phosphorylated FAK397 is reduced in cells which LAPTM4B-35 expression is knocked down as compared with the control cells.*(f)* Western blot analysis indicates that FAK inhibitor (PP2) inhibits the phos‐ phorylation of ERK1/2. After treatment of BEL-7402 HCC cells (AE) by 1 μM PP2, the phosphorylation of ERK1/2 was analyzed via Western blot for the LAPTM4B-35 up-regulated cells and the MOCK cells under the stimulation of lami‐ nin substrate. The Western blot profile shows that phosphorylation/activation of ERK1/2 is associated with FAK activi‐

**Figure 2.** Colocalization between LAPTM4B-35 and integrinα5 or FAK. Cells were attaching and spreading onto fibro‐ nectin for 6 h *(a, c)* or 24 h*(b)*. *(a)* and *(b) show* the colocalization of LAPTM4B-35 (red) and integrin α5 (green). *(c)* shows

We found that not only membrane-integrated receptors, but also some solvable signaling molecules in cytoplasm can interact with LAPTM4B-35, such as FAK (Figure 2c) and PI3K p85α [6]. It is known that PI3K is a kinase which catalyzes phosphorylation of proteins andlipids. An important phosphorylated product catalyzed byPI3K is membrane-integrated PIP3 which can recruit cytoplasmic PH domain-containing proteins, including Akt and the corresponding kinases (PDK1 and PDK2) to the plasma membrane where Akt is phosphory‐ lated by PDK1 and PDK2. Phosphorylated Akt is commonly known as a marker for PI3K/Akt

the colocalization of LAPTM4B-35 (red) and FAK(green).

ty.

152 Recent Advances in Liver Diseases and Surgery

Moreover, we demponstrated that theTyr285 is the one single site for phosphorylation of Tyr residues in the LAPTM4B-35 molecule (Figure 3f). Notably, under stimulation of LN, Tyr285 phosphorylation rises quickly and peaks at 10 min. Thereafter phosphorylation decreases steadily out to 40 minutes (Figure 3g -1). It is of importance that LAPTM4B-35 Tyr285 phos‐ phorylation can be markedly inhibited by LAPTM4B-EC2-pAb (Figure 3g -2), indicating the EC2 domain is required for Tyr285 phosphorylation of LAPTM4B-35. Kazarow (2002) reported that CD151, a member of the tetra-transmembrane protein family, can interact with the integrin α subunit via an QRD motif in the EC2 domain. Similarly, a YRD motif exists in the LAPTM4B-35 EC2 domain. We found that in LAPTM4B-35 YRD233-235INF mutated HCC cells, AKT phosphorylation/activation is significantly inhibited (Figure 3h), suggesting interaction of LAPTM4B-35 EC2 YRD and integrin is involved in PI3K/AKT activation. In addition, LAPTM4B-EC2-pAb and integrin α6-mAb can both inhibit FAK phosphorylation under stimulation by LN (Figure 3i), indicating interaction of LAPTM4B-EC2 with integrin α6 (the specific receptor of LN) is involved in FAK phosphorylation/activation. Moreover, we found that the FAK inhibitor PP2 can simultaneously inhibit phosphorylation of LAPTM4B-35 and interaction of LAPTM4B-35 with PI3K p85α (Figure 3e). These result suggest that FAK is likely the kinase that catalyzes the Tyr phosphorylation of LAPTM4B-35, by which the binding site of LAPTM4B-35 to the PI3K p85α SH2 domain is created, thus releasing inhibition of PI3K p85α to p110 kinase activity, and consequently resulting in activation of downstream AKT (Figure 5).

It is known that FAK, as a functionally complicated signal molecule with Tyr kinase activity and nonkinase scaffolding function, is overexpressed in many cancers (including 60% of HCC) and involves in many aspects of tumor growth, invasion, and metastasis. Given that the phophorylation of FAK Tyr397 is critical for trigering its Tyr kinase activity and enhancing its nonkinase scaffolding function, and is induced by binding of integrin with FN or LN. We found that the PI3K/Akt signaling pathway in LAPTM4B-35 overexpressed HCC cells can be activated by stimulation of not only serum but also fibronectin or laminin substrate (Figure 3d); additionally the interaction of LAPTM4B-35 with PI3K p85α is inhibited by FAK inhibitor PP2 (Figure 3e). These results suggest that overexpression and interaction of LAPTM4B-35 and FAK in cancer cells would be expected tocreate an alternative signaling pathway, i.e. ECM/ integrin/FAK/LAPTM4B-35/PI3K/AKT signaling pathway. In which FAK phosphorylation/ activation results from interaction of the LAPTM4B-35 EC2 domain and integrin α6 subunit at the cell surface under the stimulation by LN or FN, and results in phosphorylation of LAPTM4B-35 Tyr285 by FAK kinase activity. This model (shown in Figure 5 on the upper right) illustrates a novel putative mechanism by which the PI3K/AKT signaling pathway is over activated through the involvement LAPTM4B-35 in cancer cells. In other words, our prelimi‐ nary results suggest there might be a novel LAPTM4B-35 dependent pathway which gives rise to overactivation of the PI3K/AKT signaling pathway in HCC cells. In this mechanism, overexpressed LAPTM4B-35 interacts initially with integrin at the cell surface under stimula‐ tion of an ECM component (FN or LN) via its EC2 YRD motif. This interaction of LAPTM4B-35 and integrin induces phosphorylation and activation of FAK397 through a currently not fully understood mechanism. Activated FAK may catalyze phosphorylation of LAPTM4B-35 Tyr285 to create a binding site for PI3K p85α. Consequently, downstream AKT is phosphory‐ lated and activated by PI3K p110, the kinase activity of which comes into play through binding of phosphorylated LAPTM4B-35 Tyr285 to PI3K p85α. This proposed molecular mechanism remains to be further studied in detail.

Moreover, we demponstrated that theTyr285 is the one single site for phosphorylation of Tyr residues in the LAPTM4B-35 molecule (Figure 3f). Notably, under stimulation of LN, Tyr285 phosphorylation rises quickly and peaks at 10 min. Thereafter phosphorylation decreases steadily out to 40 minutes (Figure 3g -1). It is of importance that LAPTM4B-35 Tyr285 phos‐ phorylation can be markedly inhibited by LAPTM4B-EC2-pAb (Figure 3g -2), indicating the EC2 domain is required for Tyr285 phosphorylation of LAPTM4B-35. Kazarow (2002) reported that CD151, a member of the tetra-transmembrane protein family, can interact with the integrin α subunit via an QRD motif in the EC2 domain. Similarly, a YRD motif exists in the LAPTM4B-35 EC2 domain. We found that in LAPTM4B-35 YRD233-235INF mutated HCC cells, AKT phosphorylation/activation is significantly inhibited (Figure 3h), suggesting interaction of LAPTM4B-35 EC2 YRD and integrin is involved in PI3K/AKT activation. In addition, LAPTM4B-EC2-pAb and integrin α6-mAb can both inhibit FAK phosphorylation under stimulation by LN (Figure 3i), indicating interaction of LAPTM4B-EC2 with integrin α6 (the specific receptor of LN) is involved in FAK phosphorylation/activation. Moreover, we found that the FAK inhibitor PP2 can simultaneously inhibit phosphorylation of LAPTM4B-35 and interaction of LAPTM4B-35 with PI3K p85α (Figure 3e). These result suggest that FAK is likely the kinase that catalyzes the Tyr phosphorylation of LAPTM4B-35, by which the binding site of LAPTM4B-35 to the PI3K p85α SH2 domain is created, thus releasing inhibition of PI3K p85α to p110 kinase activity, and consequently resulting in activation of downstream AKT

It is known that FAK, as a functionally complicated signal molecule with Tyr kinase activity and nonkinase scaffolding function, is overexpressed in many cancers (including 60% of HCC) and involves in many aspects of tumor growth, invasion, and metastasis. Given that the phophorylation of FAK Tyr397 is critical for trigering its Tyr kinase activity and enhancing its nonkinase scaffolding function, and is induced by binding of integrin with FN or LN. We found that the PI3K/Akt signaling pathway in LAPTM4B-35 overexpressed HCC cells can be activated by stimulation of not only serum but also fibronectin or laminin substrate (Figure 3d); additionally the interaction of LAPTM4B-35 with PI3K p85α is inhibited by FAK inhibitor PP2 (Figure 3e). These results suggest that overexpression and interaction of LAPTM4B-35 and FAK in cancer cells would be expected tocreate an alternative signaling pathway, i.e. ECM/ integrin/FAK/LAPTM4B-35/PI3K/AKT signaling pathway. In which FAK phosphorylation/ activation results from interaction of the LAPTM4B-35 EC2 domain and integrin α6 subunit at the cell surface under the stimulation by LN or FN, and results in phosphorylation of LAPTM4B-35 Tyr285 by FAK kinase activity. This model (shown in Figure 5 on the upper right) illustrates a novel putative mechanism by which the PI3K/AKT signaling pathway is over activated through the involvement LAPTM4B-35 in cancer cells. In other words, our prelimi‐ nary results suggest there might be a novel LAPTM4B-35 dependent pathway which gives rise to overactivation of the PI3K/AKT signaling pathway in HCC cells. In this mechanism, overexpressed LAPTM4B-35 interacts initially with integrin at the cell surface under stimula‐ tion of an ECM component (FN or LN) via its EC2 YRD motif. This interaction of LAPTM4B-35 and integrin induces phosphorylation and activation of FAK397 through a currently not fully understood mechanism. Activated FAK may catalyze phosphorylation of LAPTM4B-35 Tyr285 to create a binding site for PI3K p85α. Consequently, downstream AKT is phosphory‐

(Figure 5).

154 Recent Advances in Liver Diseases and Surgery

**Figure 3.** Mechanism for interaction of LAPTM4B-35 with of PI3K p85α and activation of Akt. *(a)* Co-IP analysis dem‐ onstrates interaction of LAPTM4B-35 with p85α regulatory subunit, but not PI3K p110 catalytic subunit. Anti-LAPTM4B35-pAb was used to precipitate the binding proteins, and a mixture of anti-PI3K p110-mAb and anti-PI3Kp85α-mAb was applied to blot the binding proteins. *(b)* Co-IP analysis demonstrates that the proline-rich domain in N-terminus and Tyr285 in C-terminus of LAPTM4B-35 are involved in the interaction of LAPTM4B-35 with PI3K p85α via a serious of mutants, including PA, △N, YF, and △N+YF mutants. PA mutant (P): Prolines in the PPRP motif in N-terminus of LAPTM4B-35 were mutated to alanines(P12,13,15A). △N mutant: The 10th-19th amino acid residues in the N-terminus of LAPTM4B-35 were deleted. YF mutant: The Tyr285 in the C-terminus of LAPTM4B-35 was mutat‐ ed to phenylalanine (Y285F). △N+YF mutant: △N mutant plus YF mutant. Anti-FLAG-mAb was used to immunopreci‐ pitate the binding proteins in lysates from variant BEL-7402 HCC cell lines, which were transfected separately by pcDNA3-Mock-flag (Mock), pcDNA3-LAPTM4B-flag (AF), pcDNA3-LAPTM4B-flag (PA), pcDNA3-LAPTM4B-flag (△N), pcDNA3-LAPTM4B-flag (YF), or pcDNA3-LAPTM4B-flag (△N+YF) plasmids. Then anti-PI3Kp85α-mAb was applied to blot the binding proteins. The interaction of LAPTM4B-35 and PI3K p85α was dramatically enhanced in LAPTM4B-35 up-regulated AF cells as compared with the Mock cells and was significantly attenuated in the variant LAPTM4B-mutated cells as compared with the AF cells. *(c)* Western blot profile demonstrates that Akt-p is decreased in the mutated AF(PA) and AF(YF) cells as compared with wild-type LAPTM4B-35 (AF), indicating that the prolinerich domain in N-terminal and the Tyr285 in C-terminal tails of LAPTM4B-35 are necessary for Akt phosphorylation. *(d)* Western blot demonstrates that ECM components, fibronectin (FN) or laminin (LN), can promote phosphorylation/ activation of Akt in cells in which LAPTM4B-35 expression is up-regulated, indicating association of phosphorylation/ activation of Akt with FN and LN in HCC cells. *(e)* Co-IP analysis demonstrates that FAK inhibitor PP2 can simultane‐ ously inhibit phosphorylation of Tyr285 and interaction of p85α with LAPTM4B-35. Anti-LAPTM4B-pAb was used to immunoprecipitate the binding proteins, then anti-phosphorylated Tyr mAb or anti-Akt mAb was used to blot the binding protein. *(f)* Co-IP and Western blot profile show that LAPTM4B-35 Tyr285 is the only phosphorylation site by mutation analysis. HepG2 cells were transfected by AF or AF(YF) mutant. The phosphorylation appeared merely in the wild type HepG2 cells, but not the Tyr 285 mutated YF cells. *(g)* Co-IP analysis indicates that LAPTM4B-35 Tyr can be phophorylated in a peaky manner under the stimulation of LN. HCC cells were placed on LN-coated vials for var‐ iant times, LAPTM4B-EC2-pAb was used to precipitate LAPTM4B protein in the HCC lysates. The immuno-precipi‐ tants were subjected to Western blot analysis. The anti-phosphorylated Tyr-mAb was used to blot the phosphorylated LAPTM4B-35. *(g-1)* shows the time course of LAPTM4B-35 phosphorylation with the highest phosphorylation at 10 min. *(g-2)* shows the inhibition of LAPTM4B-35 phosphorylation by LAPTM4B-EC2-pAb. *(h)* Western blot analysis in‐ dicates that mutation of YRD motif in EC2 domain of LAPTM4B-35 can inhibit AKT phosphorylation. BEL-7402 HCC cells were transfected by pcDNA3-AF(YRD233-235INF) mutated plasmids (INF) and the wild type pcDNA3-AF (AF) plasmids, respectively. The lysates were analyzed by Western blot with a anti-phosphorylated AKT-mAb. *(i)* Co-IP analysis indicates that both LAPTM4B-EC2-pAb and integrin α6 mAb can inhibit FAK phosphorylation. The BEL-7402 HCC cells were pre-incubated with non-immune IgG (as a control), LAPTM4B-EC2-pAb and integrin α6 mAb, respec‐ tively. The lysates were precipitated by FAK mAb. The immuno-precipitants were then subjected to Western blot anal‐ ysis, and phosphorylated FAK mAb (the upper panel) or FAK-mAb (the lower panel) was used as the bloting antibody.

**Figure 4.** Co-localization of activated Akt and overexpressed LAPTM4B-35 under the stimulation of serum in BEL-7402 HCC cells. *(a)* Nonactivated Akt (green) and LAPTM4B-35 (red) are separately distributed in the cells cotransfected with pEGFP-PH-Akt plasmids and pcDNA3-LAPTM4B-flag plasmids (AF) after serum-starvation for 16 h. *(b)* Colocali‐ zation (yellow) of activated Akt (green) and overexpressed LAPTM4B-35 (red) understimulation of serum in HCC cells, which is stimulated by 20% fetal calf serum for 15 min after serum-starvation for 16 h. (c) No colocalization ap‐ peared in PA mutant HCC cells under the same conditions as described in *(b)*. (d) No colocalization appeared in YF mutant HCC cells under the same conditions as described in *(b)*. (e) No colocalization appeared in the presence of PI3K inhibitor (LY294002) in AF HCC (wild-type) cells under the same conditions as described in *(b)*.

In summary, cancer-targeted therapy currently focuses primarily on targeting key signaling molecules in one or more signaling pathways which are overactivated in a given cancer. Tetratransmembrane LAPTM4B-35 is believed to function as an assembly platform or organizer for a number of signaling molecules, which may either be integrated in the cell membranes or

**Figure 5.** Signaling pathways activated by the overexpression of LAPTM4B-35 in HCC cells.

binding protein. *(f)* Co-IP and Western blot profile show that LAPTM4B-35 Tyr285 is the only phosphorylation site by mutation analysis. HepG2 cells were transfected by AF or AF(YF) mutant. The phosphorylation appeared merely in the wild type HepG2 cells, but not the Tyr 285 mutated YF cells. *(g)* Co-IP analysis indicates that LAPTM4B-35 Tyr can be phophorylated in a peaky manner under the stimulation of LN. HCC cells were placed on LN-coated vials for var‐ iant times, LAPTM4B-EC2-pAb was used to precipitate LAPTM4B protein in the HCC lysates. The immuno-precipi‐ tants were subjected to Western blot analysis. The anti-phosphorylated Tyr-mAb was used to blot the phosphorylated LAPTM4B-35. *(g-1)* shows the time course of LAPTM4B-35 phosphorylation with the highest phosphorylation at 10 min. *(g-2)* shows the inhibition of LAPTM4B-35 phosphorylation by LAPTM4B-EC2-pAb. *(h)* Western blot analysis in‐ dicates that mutation of YRD motif in EC2 domain of LAPTM4B-35 can inhibit AKT phosphorylation. BEL-7402 HCC cells were transfected by pcDNA3-AF(YRD233-235INF) mutated plasmids (INF) and the wild type pcDNA3-AF (AF) plasmids, respectively. The lysates were analyzed by Western blot with a anti-phosphorylated AKT-mAb. *(i)* Co-IP analysis indicates that both LAPTM4B-EC2-pAb and integrin α6 mAb can inhibit FAK phosphorylation. The BEL-7402 HCC cells were pre-incubated with non-immune IgG (as a control), LAPTM4B-EC2-pAb and integrin α6 mAb, respec‐ tively. The lysates were precipitated by FAK mAb. The immuno-precipitants were then subjected to Western blot anal‐ ysis, and phosphorylated FAK mAb (the upper panel) or FAK-mAb (the lower panel) was used as the bloting

**Figure 4.** Co-localization of activated Akt and overexpressed LAPTM4B-35 under the stimulation of serum in BEL-7402 HCC cells. *(a)* Nonactivated Akt (green) and LAPTM4B-35 (red) are separately distributed in the cells cotransfected with pEGFP-PH-Akt plasmids and pcDNA3-LAPTM4B-flag plasmids (AF) after serum-starvation for 16 h. *(b)* Colocali‐ zation (yellow) of activated Akt (green) and overexpressed LAPTM4B-35 (red) understimulation of serum in HCC cells, which is stimulated by 20% fetal calf serum for 15 min after serum-starvation for 16 h. (c) No colocalization ap‐ peared in PA mutant HCC cells under the same conditions as described in *(b)*. (d) No colocalization appeared in YF mutant HCC cells under the same conditions as described in *(b)*. (e) No colocalization appeared in the presence of PI3K

In summary, cancer-targeted therapy currently focuses primarily on targeting key signaling molecules in one or more signaling pathways which are overactivated in a given cancer. Tetratransmembrane LAPTM4B-35 is believed to function as an assembly platform or organizer for a number of signaling molecules, which may either be integrated in the cell membranes or

inhibitor (LY294002) in AF HCC (wild-type) cells under the same conditions as described in *(b)*.

antibody.

156 Recent Advances in Liver Diseases and Surgery

soluble in the cytoplasm. The LAPTM4B-35 overexpression, which occurs in more than 80% of HCC tissues, and the interactions with membrane-integrated receptors and cytoplasmic signal molecules are expected to act as an amplified assembly platform for upstream signal molecules of several signaling pathways, and leads to over activation of related signaling pathways (Figure 5), such as growth factor/ RTK/Ras/ERK, growth factor/RTK/Ras/PI3K/Akt, ECM/integrin/FAK/ERK, ECM/integrin/FAK/PI3K/Akt, and so on. Since these signaling pathways and their networks are closely associated with malignant molecular and cellular phenotypes, including cell proliferation/differentiation and survival/apoptosis as well as migration/invasion, it is believed that over activation of these signaling pathways is linked with hepatic carcinogenesis and progression [12-15]. Collectively, our data strongly suggest that LAPTM4B-35 would be an ideal target for HCC treatment, and that LAPTM4B-targeted therapy is a promising potential therapeutic strategy for HCC which will act in down regula‐ tion of the expression of LAPTM4B-35, or act by obstructing the interaction of LAPTM4B-35 with growth factors, integrins, FAK, PI3K p85α and other LAPTM4B-35 binding signal molecules.
