Dedifferentiation of adenocarcinomas by activation of phosphatidylinositol 3-kinase

Abstract

Signet ring cell carcinoma is a malignant type of poorly differentiated adenocarcinomas in stomach, which is characterized by the occasional presence of signet ring-like cancer cells. We found that expression of constitutively active phosphatidylinositol 3-kinase (PI 3-kinase) in well differentiated adenocarcinoma cell lines induced the loss of cell-cell contact and some of the cells changed their shapes to signet ring cell-like, characterized by appearance of mucus droplets in the cytoplasm with well developed endplasmic reticulum and Golgi complexes. The active PI 3-kinase-expressing cells formed poorly differentiated tumors in nude mice, which were clearly different from those of the original cell lines. The PI 3-kinase activities detected in anti-phosphotyrosine immunoprecipitates were higher in several signet ring cell carcinoma-derived cell lines than in other adenocarcinoma cell lines. In addition, PI 3-kinase was found to be associated with a 200-kDa protein phosphorylated in tyrosine in 4 of 6 signet ring cells but not in other cell lines, suggesting that PI 3-kinase is possibly activated in these cells by binding to the 200-kDa protein. The 200-kDa protein-PI 3-kinase complex was exclusively fractionated in the membrane fractions. The specific activity of the PI 3-kinase immunoprecipitated with anti-phosphotyrosine antibody was approximately 3-fold higher than that with anti-PI 3-kinase antibody. These results suggest that PI 3-kinase in signet ring cell carcinoma is recruited to the membrane and activated by the binding to the 200-kDa protein.

Figure 1

Formation of signet ring-like cells after expression of the constitutively active PI 3-kinase. (a) Structure of the mutant PI 3-kinases used in establishment the stable transformants. The active PI 3-kinase, BD110, has the binding site for p110 in p85 at the amino-terminal end of p110 as well as the myc tag. The truncated versions of BD110, BD110X, and BD110E lack the kinase domain at the carboxyl-terminal portion of BD110 and almost all of the p110 sequence except the p85 binding domain, respectively. (b) Scheme of induction of the PI 3-kinases by the Cre–loxP system. A recombinase, Cre, cleaves out the sequence between the two loxP sequences. (c) Expression of PI 3-kinases after infection of the cells with an adenovirus bearing the Cre recombinase. The HCC2998 and MKN45 cells were infected with AxCANCre or AxCANLacZ. Expression of the PI 3-kinases was analyzed by Western blotting with anti-myc monoclonal antibody, 9E10. The numbers show the incubation time after infection with the viruses. (d) Elevation of levels of 3′-phosphorylated polyphosphoinositides after induction of pBD110. HCC2998/BD110 cells were infected with AxCANCre. After indicated periods of incubation, the cells were labeled with [³²P]orthophosphate for 4 hr, and the lipid was extracted. After deacylation reaction, the resulting water-soluble components were analyzed by a strong anion column chromatography. The radioactivities in the PI 3,4-diphosphate and PI 3,4,5-triphosphate fractions were plotted on the graph. The numbers were normalized by the total radioactivity in the lipid fraction. A representative of two experiments with two cell lines is shown in the figure. (e) Morphological change of HCC2998 and MKN45 cells expressing the PI 3-kinase mutants. Cells bearing PI 3-kinase genes, HCC2998/BD110, HCC2998/BD110X, HCC2998/BD110E, and MKN45/BD110 cells were infected with AxCANCre (Cre) or with AxCANLacZ (LacZ) and photographed at the indicated time. The cells expressing BD110 changed their shapes, becoming round and separated. Large signet ring-like cells appeared among the round cells after 2–3 days (indicated by arrows). No such changes were observed in the control cells. (f) Phosphorylation of p38 MAP kinase and PKB after expression of pBD110. HCC2998/BD110 and MKN45/BD110 cells were incubated for 3 days after infection with AxCANCre (Cre) or AxCANLacZ (LacZ). The cells were harvested, and Western blotting was performed with anti-phospho-p38 antibody (phospho-p38) and anti-phospho-PKB antibody (phospho-PKB), respectively. Total p38 MAP kinase and PKB were detected by using anti-p38 antibody (p38) and anti-PKB antibody (PKB). Expression of the myc-pBD110 was detected by anti-myc antibody (Bottom). (−), mock-infected cells.

Figure 2

Characterization of the signet ring-like cells formed by the expression of pBD110 in HCC2998/BD110 cells. (a and b) PAS staining of mucinous substance in the pBD110-expressing cells (b) or control cells (a). HCC2998/BD110 cells were cultured on a coverslip for 3 days after infection with AxCANCre, fixed with 4% paraformaldehyde, and PAS stained. Eccentric nuclei and large droplets containing diastase-resistant PAS-positive substance can be seen in the signet ring-like cells (indicated by an arrowheads). (c) Intracellular localization of a secretory glycoprotein antigen, CA15–3. Cells prepared as above were embedded in paraffin and the section was stained with anti-CA15–3 antibody. CA15–3 antigen was detected in various sized vacuoles in cytoplasm as well as the entire cell surface. Various cross sections of the cells were observed. (d) Electron micrograph of a signet ring-like cell found in the BD110-expressing cells. HCC2998/BD110 cells cultured for 3 days after AxCANCre infection were used to prepare the section. A typical signet ring-like cell was photographed. Dilation of the Golgi apparatus was observed from perinuclear to the submembranous regions (indicated by arrowheads). Irregular elongation of microvilli was localized above the dilated Golgi apparatus (indicated by arrows). (Original magnification, ×4000.)

Figure 3

Conversion of HCC2998 and MKN45 cells to a more malignant, invasive type after expression of constitutively active PI 3-kinase. (a) Elevation of colony-forming efficiency in soft agar after expression of constitutively active PI 3-kinase. Three hundred cells of HCC2998/BD110 and MKN45/BD110 before and after induction of BD110 were embedded in a medium containing 0.4% agarose. After two weeks, the numbers of colonies over 0.5 mm in diameter were scored in two independent experiments each with duplicate dishes. (b) Representative colonies formed by active PI 3-kinase-expressing cells or their parental cells. (c) Constitutively active PI 3-kinase-expressing cells formed malignant, poorly differentiated carcinomas showing high invasiveness in nude mice. HCC2998/BD110 cells or MKN45/BD110 cells were transplanted into subcutaneous tissue of nude mice before or 40 days after the infection with AxCANCre. Cells without pBD110 expression preserved their intestinal cell nature and polarity as shown by the presence of papillary or tubular structures (arrowheads) with lumen (L, Upper), in which the presence of mucus was detected by the PAS reaction (data not shown). In contrast, the BD110-expressing cells (shown by arrows) in the tumors lost their polarity and proliferated diffusely in solid or trabecular patterns. Stromal fibroblasts proliferated among the cancer cells. The histological features of the xenotransplants are summarized in Table 1.

Figure 4

Activation of PI 3-kinase in the native signet ring cell lines. (a) Signet ring cells exhibited relatively high PI 3-kinase activity in the anti-phosphotyrosine immunoprecipitates among gastric cancer cell lines. Phosphotyrosine-containing proteins were immunoprecipitated with anti-phosphotyrosine antibody, PY20, from various gastric tumor cell lines (the morphologies of the cell lines are shown on the abscissa). PI 3-kinase activity in the immunoprecipitates was analyzed with PI as a substrate. The radioactivity in the PI 3-P spots on TLC was quantified with BAS2000 imaging analyzer (Fuji). (b) Association of a 200-kDa protein phosphorylated on tyrosine with PI 3-kinase in signet ring cell lines. PI 3-kinase was immunoprecipitated with AB6 from various gastric tumor cell lines. The blots of precipitated proteins were probed for phosphotyrosine-containing proteins (PY20, Upper) or p85 α (AB6, Lower). (c)Membrane localization of PI 3-kinase bound to the 200-kDa protein in the signet ring cells. Cells were fractionated into cytosol (C) and membranes (M). The levels of the 200-kDa protein bound to PI 3-kinase (Upper) in anti-p85 immunoprecipitates and of total PI 3-kinase (Lower) in total lysates of each fraction were analyzed by using Western blotting probed for phosphotyrosine and p85, respectively. (d) Elevation of specific activities of PI 3-kinase in anti-phosphotyrosine immunoprecipitates from signet ring cell lines. PI 3-kinase was immunoprecipitated with PY20 or CA3, which was shown to precipitate both p85α and p85β regardless of binding to the 200-kDa protein from the serial dilutions of the total lysates, and the PI 3-kinase activities in the immunoprecipitates were analyzed (Lower). The levels of p85 were analyzed by using Western blotting with CA3 (Upper). The numbers under each panel show the relative intensity of the p85 bands (Upper) and radioactivities of PI 3-P spots (Lower). The numbers under the bottom line show the relative specific activities of PI 3-kinase. (e) Activation of p38–MAP kinase in native signet ring cell lines. Phosphorylation of p38–MAP kinase in indicated cell lines was detected by using Western blotting for anti-phospho-p38 antibody (Upper). Total p38 was detected by anti-p38 antibody (Lower).