Down-regulation of riα subunit of camp-dependent protein kinase induces growth inhibition of human mammary epithelial cells transformed by c-ha-ras and c-erbb-2 proto-oncogenes

MCF- 10A is a spontaneously immortalized, non-transformed human mammary epithelial cell line. We have recently obtained MCF- 10A clones (MCF- 1OA HE cells) that are transformed following over-expression of both a human point-mutated c-Ha-ras and the c-erbB-2 proto-oncogenes. Two isoforms of the cAMP-dependent protein kinase (cAK) have been described in mammalian cells. Enhanced levels of type-I cAK (cAKI) are generally found in tumor cells. To determine whether inhibition of cAKl expression may interfere with ras and erbB-2 oncogene-induced transformation of human mammary epithelial cells, we have tested the effects of 2 agents that specifically down-regulate cAKI, such as 8-chloro-cAMP and an anti-sense oligodeoxynucleotide targeted against the R1α regulatory subunit of cAKl on MCF-10A HE cells. Treatment of MCF-10A HE cells with 8-chloro-cAMP induces a dose-dependent growth inhibition under both monolayer and soft-agar growth conditions, that is correlated with an accumulation of MCF-10A HE cells in G₀/G, phases of the cell cycle and a reduction of the number of cells in S phase. In contrast, 8-chloro-cAMP has no effect on MCF-10A cell growth. Furthermore, 8-chloro-cAMP treatment of MCF-10A HE cells induces a 4- to 6-fold reduction in p185erbB-2 expression and brings p21 ras expression to levels comparable to those found in MCF-10A cells. Treatment of MCF-10A HE cells with an Rlα anti-sense oligodeoxynucleotide determines a comparable inhibition of both anchorage-dependent and anchorage-independent cell growth. Our results suggest that cAKl may act as a mediator of ras and erbB-2 oncogene action in human breast cells and that interference with cAKl action provides a potential tool for inhibiting the growth-promoting effects of these oncogenes.     

Inhibition of tumorigenicity in lung adenocarcinoma cells by c-erbB-2 antisense expression

The lung carcinoma cell line Calu3, which overexpresses the c-erbB-2 oncogene, was stably transfected with antisense (AS) cDNA constructs encompassing different regions of the c-erbB-2 gene. Transfected cells were analyzed for their tumorigenic properties in vitro and in nude mice. Two independent clones, AS F1 (low erbB-2 expressor) and AS B12 (high erbB-2 expressor), as well as the polyclonal Calu3/AS 5′, were selected for these analyses. In Calu3/AS 5′ transfected cells and in the AS F1 clone, c-erbB-2 RNA and protein levels were lower than those detected in the parental cell line and the AS B12 clone. Anchorage-independent growth and tumor take were also significantly reduced. Furthermore, cells derived from primary tumors of Calu3/AS 5′, AS F1 and AS B12 lost the AS c-erbB-2 DNA insert but retained the gene for G418 resistance. Our results suggest that a correlation between c-erbB-2 overexpression and tumorigenicity may exist in the Calu3 lung carcinoma cell line. Int. J. Cancer 72:631–636, 1997. © 1997 Wiley-Liss, Inc.     

A humanized Anti-c-erbB-2 monoclonal antibody for the treatment of breast cancer

The c-erbB-2 product is thought to be a unique and useful target for antibody therapy of cancers that overexpress the c-erbB-2 gene. Its overexpression is also speculated to be correlated with chemoresistance to doxorubicin. The in vitro and in vivo anti-tumor effects of a humanized antibody directed against the extracellular domain of the c-erbB-2 gene product, rhu4D5, were examined. Rhu4D5 had direct antiproliferative activity against the SK-BR-3 cell line which overexpresses c-erbB-2. The in vivo treatment, using rhu4D5, of SCID mice carrying xenografts of 4-1ST human gastric carcinoma, which overexpresses c-erbB-2, revealed that the recombinant protein had potent anti-tumor activity. Furthermore, the cytotoxic action of human peripheral blood mononuclear cells against the SK-BR-3 cell line was significantly augmented with the administration of rhu4D5, but not with mu4D5. These results indicate that rhu4D5 might be a more efficacious treatment than previously predicted by preclinical studies.     

Insulin-like growth factor and epidermal growth factor independence in human mammary carcinoma cells with c-erbB-2 gene amplification and progressively elevated levels of tyrosine-phosphorylated p185erbB-2

Growth factor-independent proliferation is an essential aspect of the transformation process. To study the influence of c-erbB-2 overexpression on the autonomous growth of human mammary cancer cells, we used a series of non-neoplastic and neoplastic human mammary epithelial cell lines isolated from a patient with intraductal and invasive ductal carcinoma of the breast. The non-neoplastic cell line, H16N-2, which expresses a normal level (single gene copy) of c-erbB-2, was used for comparison with the neoplastic cell lines. Both the metastatic tumor cell lines, 21MT-1 and 21MT-2, showed equivalent amplification of the c-erbB-2 gene; however, 21MT-1 cells showed a higher level of c-erbB-2 overexpression. Therefore, the H16N-2, 21MT-2, and 21MT-1 cell series forms a distinct gradient of progressively increasing c-erbB-2 gene expression. Furthermore, the overexpression of c-erbB-2 in the 21MT cell lines was concordant with increases in the constitutive tyrosine kinase activity of p185^erbB-2 measured in the absence of exogenous growth factors in culture. Normal mammary epithelial cells require both insulin-like growth factor (IGF)-1 (or supraphysiological concentrations of insulin) and epidermal growth factor (EGF) to proliferate under serum-free conditions in culture. By contrast, 21MT-2 cells showed a reduced requirement for IGF but still required EGF to proliferate. 21MT-1 cells did not require either insulin or EGF to proliferate. Therefore, the progressive increases in constitutive p185^erbB-2 tyrosine kinase activity in the 21MT-2 and 21MT-1 cell lines was directly correlated with IGF independence and combined IGF and EGF independence under defined conditions in culture. Experiments using conditioned media and anti-IGF-1 receptor and anti-EGF receptor neutralizing antibodies showed that the growth-factor independence of the tumor cells did not involve detectable IGF- or EGF-like autocrine activity expressed by the 21MT cells. Furthermore, neu differentiation factor/heregulin, a ligand that indirectly activates p185^erbB-2 by direct binding to erbB-3 receptors, potently stimulated the proliferation of the growth factor-dependent H16N-2 cells (which expressed cerbB-2 and c-erbB-3 but not c-erbB-4) in the absence of both IGF and EGF. Thus, HRG-induced mitogenesis mimicked the autonomous growth seen in the 21MT cells that have the highest level of constitutive p185^erbB-2 activation. These data support the hypothesis that the constitutive activation of p185^erbB-2 in human mammary carcinoma cells causes growth-factor independence by directly activating multiple signal-transduction pathways that substitute for both IGF and EGF during proliferation. © 1996 Wiley-Liss, Inc.     

Growth suppression of transformed BALB/c 3T3 cells by transforming growth factor {beta}1 occurs only in the presence of their normal counterparts

Transforming growth factor ß1 (TGF-ß1) enhancesthe yield of transformed foci of BALB/c 3T3 cells, but the continuouspresence of TGF-ß1 after foci formation inhibits thegrowth of transformed foci. The focus-forming ability of Ha-ras-,v-src-and PyMT-transformed cells growing on a monolayer of non-transformedcells was completely suppressed by TGF ß1, whereasgrowth of the transformed cells was little inhibited by TGF-ß1in the absence of their normal counterparts. The inhibitionby TGF-ß1 of focus formation by transformed BALB/c3T3 cells on a normal cell monolayer remained when TGF-ß1was removed from the culture medium after 2 weeks. However,the transformed cells were not killed, since they grew in cultureconditions under which only transformed cells are able to grow(soft agar). These results suggest that TGF-ß1 suppressesgrowth of transformed cells in the presence of normal cells.Furthermore, when non-transformed cells were treated with TGF-ß1before co-culture with Ha-ras-transformed cells, formation oftransformed foci was inhibited. When normal and transformedcells were cultured in the same dish but separated physically,focus formation was still Inhibited. On the other hand, TGF-ß1enhanced the growth and changed the morphology of non-transformedcells only in the presence of transformed counterparts. Thegrowth inhibitory effect of TGF-ß1 on transformedcells and its growth stlmulatory effect on non-transformed cellsin co-culture conditions suggest the induction of reciprocalparacrine growth regulatory factors. As TGF-ß1 inhibitsthe growth of transformed BALB/c 3T3 cells only in the presenceof their normal counterparts, a paracrine negative growth controlmechanism appears to be operating.     

Episomally mediated overexpression of wild-type erbB-2 transforms MCF-10A breast epithelial cells

MCF-10A are human non-transformed, EGF and insulin dependent breast epithelial cells. The cells were transfected with an episomal pCEP4 vector library containing cDNA from SKBR3 breast carcinoma cells, and selected in media without EGF. After two cycles of expression cloning, morphologically transformed cells appeared. Extracted episomes contained a high proportion of erbB-2 cDNA with wild-type transmembrane domains. Transfection of MCF-10A with individual erbB-2 containing episomes induced significant foci formation in low serum (0.1%) without EGF. MCF-10A sublines expanded from these foci contained a high number of erbB-2 gene copies, highly expressed erbB-2, and lost E-cadherin expression. These results show that if wild-type erbB-2 is sufficiently overexpressed, erbB-2 alone can cause EGF independent transformation of these nonmalignant breast cells. This expression system may be useful for expression cloning in MCF-10A cells, and simulating the effects of high erbB-2 gene amplification in breast epithelial cells.     

Analysis of CerbB2 protein content of human glioma cells and tumor tissue

Summary This study was designed to determine whether or not overexpression of the cerbB2 protein plays a role in the etiology of human gliomas. The cerbB2 gene codes for a 185 kDa cell membrane glycoprotein (gpl85^cerbB2), which is similar to the receptor for epidermal growth factor. In initial studies, four human glioma cell lines (A-172, U118MG, U138MG and SW608) were used to develop techniques for detecting and quantifying gpl85^cerbB2, using immunofluorescence microscopy, immunoblot analysis and flow cytometry. A-172 cells were found to have the highest content of gpl85^cerbB2. More detailed studies utilizing A-172 cells indicated that cellular gpl85^cerbB2 content changed little in response to conditions affecting cellular proliferative status, including serum deprivation, growth in low glucose medium and treatment with dimethyl sulfoxide. Ten human glioma specimens were then analyzed for cellular gpl85^cerbB2 fluorescence and DNA content, using A-172 cells as a biological standard. Results indicated that gpl85^cerbB2 was expressed at levels comparable to that of A-172 cells in many specimens, and at a very high level in one specimen. These data reiterate the problem of themolecular heterogeneity of human gliomas and indicate that gpl85^cerbB2 may have a role in at least asubset of malignant glial tumors.     

Epidermal growth factor receptor and c-erbB-2 expression in normal breast tissue during the menstrual cycle

Summary EGF receptor (EGF-R) and c-erbB-2 are homologous tyrosine kinase transmembrane receptors. They are involved in controlling proliferation, and probably differentiation, of normal breast epithelial cells, and their expression has been linked to the prognosis of breast cancer. Their physiological roles in normal breast tissue remain to be elucidated, as most studies to date have involved breast cancer cell lines. We studied the location of EGF-R and c-erbB-2 in 100 samples of normal breast with standard immunohistochemical methods and double-labelling techniques. EGF-R was mainly expressed on the stroma and myoepithelial cells, whereas c-erbB-2 expression was exclusively epithelial. An image analyser was used to quantitate variations in their expression during the mentrual cycle. EGF-R and c-erbB-2 expression on epithelial cells was stronger during the luteal phase than the follicular phase (p < 0.01 for EGF-R). The pattern of expression was also compared with that in 28 breast cancers and 7 fibroadenomas.     

Adipogenic, osteogenic and myofibrogenic differentiations of a rat malignant fibrous histiocytoma (MFH)-derived cell line, and a relationship of MFH cells with embryonal mesenchymal, perivascular and bone marrow stem cells

Malignant fibrous histiocytoma (MFH) is regarded as an undifferentiated pleomorphic sarcoma with unproven histogenesis. We investigated pathobiological characteristics of a rat MFH cell line (MT-9). Immunocytochemically, MT-9 cells and MT-9-induced tumours reacted to vimentin, A3 (rat MFH cell-specific antibody), macrophage markers and α-SMA (myofibroblastic marker), indicating that MT-9 showed both histiocytic and (myo)fibroblastic features. Adipogenic supplement-added MT-9 showed increased accumulation of lipid droplets. Addition of BMP-2 or osteogenic supplement to MT-9 enhanced osteoblastic markers (ALP activity, osteocalcin mRNA expression and calcification). TGF-β1-treated MT-9 revealed increased numbers of α-SMA-immunopositive cells, and enhanced protein levels of α-SMA and fibronectin, indicating myofibrogenesis. In rat tissues, A3 labelled with immature mesenchymal and perivascular cells in foetuses and neonates, and with marrow stem cells in adults. c-kit mRNA expression was seen in bone marrows and MT-9. Collectively, progenitors of MFH should be sought in lineage of marrow stem cells capable of differentiating into mesenchymal cells.     

Immunohistochemical evaluation of erbB-2 and p53 protein expression in benign and atypical human meningiomas

Meningiomas arise from the arachnoidal cells surrounding the brain and are one of the most common tumors of the central nervous system. These tumors are known to be hormonally modulated and may occur in association with breast carcinoma. Overexpression of the erbB-2 oncogene product and mutation of the tumor suppressor p53 gene are considered causal driving forces in the pathogenesis of adenocarcinomas of the breast. To determine whether abnormal expression of these genes also plays a role in the pathogenesis of meningiomas, we analyzed the expression of the erbB-2 and p53 proteins in 17 atypical and 35 typical meningioma tissue specimens by immunohistochemistry. The staining intensity was assigned a relative value of 0 to 5+, where 5+ denoted confluent immunoreactivity, 4+ to 1+ denoted varying degrees of focal positivity, and 0 denoted no evidence of staining. Levels of p53 and erbB-2 immunohistochemical staining were then correlated with tumor histology. For p53 immunoreactivity, typical meningiomas had a median staining score of 1.0, compared to 4.0 for atypical meningiomas (P < 0.0001, Mann-Whitney U test). For erbB-2 immunoreactivity, typical meningiomas had a median staining score of 5.0 compared to 1.0 for atypical meningiomas (P < 0.0001, Mann-Whitney U test). The inverse relationship between levels of erbB-2 and p53 immunoreactivity was found to be statistically significant (P < 0.0001, ANOVA). Expression of the erbB-2 protein was not associated with gene amplification or the presence of activating mutations in the transmembrane region of the protein. These findings may improve our understanding of the molecular events that occur in the neoplastic transformation of meningothelial cells. The patterns of erbB-2 and p53 immunoreactivity may prove to be useful markers with which to identify potentially more malignant meningiomas.     

Involvement of the TGF-beta and mTOR/p70S6Kinase pathways in the transformation process induced by v-ErbA

v-ErbA is the oncogenic form of TRα/c-ErbA which transforms chicken erythrocytic progenitors by blocking differentiation. The oncogenic property of v-ErbA has been correlated with its ability to antagonize ligand-dependent gene activation by TRα/c-ErbA and retinoic acid receptors. Nevertheless, its cytoplasmic retention suggests that v-ErbA could interfere with intracellular signaling pathways. We demonstrate that only the transforming form of v-ErbA confers to chicken erythroid progenitors a TGF-β independency and induces an activation of the mTOR/p70S6K pathway. In these cells, TGF-β and mTOR/p70S6K pathways regulate the expression of a known target gene of v-ErbA, band3. This is the first demonstration that v-ErbA is able to modulate specifically some signaling pathways leading to changes in the expression level of a gene involved in transformation.