Interaction of papillomavirus E6 oncoproteins with a putative calcium-binding protein.

Human papillomaviruses (HPVs) are associated with the majority of cervical cancers and encode a transforming protein, E6, that interacts with the tumor suppressor protein p53. Because E6 has p53-independent transforming activity, the yeast two-hybrid system was used to search for other E6-binding proteins. One such protein, E6BP, interacted with cancer-associated HPV E6 and with bovine papillomavirus type 1 (BPV-1) E6. The transforming activity of BPV-1 E6 mutants correlated with their E6BP-binding ability. E6BP is identical to a putative calcium-binding protein, ERC-55, that appears to be localized in the endoplasmic reticulum.

DSS1 is required for the stability of BRCA2.

DSS1 is an evolutionarily conserved acidic protein that binds to BRCA2. However, study of the function of DSS1 in mammalian cells has been hampered because endogenous DSS1 has not been detectable by Western blotting. Here, we developed a modified Western blotting protocol that detects endogenous DSS1 protein, and used it to study the function of DSS1 and its interaction with BRCA2 in mammalian cells. We found that essentially all BRCA2 in human cell lines is associated with DSS1. Importantly, we found that RNAi knockdown of DSS1 in human cell lines led to dramatic loss of BRCA2 protein, mainly due to its increased degradation. Furthermore, the stability of BRCA2 mutant devoid of the DSS1-binding domain is unaffected by the depletion of DSS1. Most notably, like BRCA2 depletion, DSS1 depletion also led to hypersensitivity to DNA damage. These results demonstrated that the stability of BRCA2 protein in mammalian cells depends on the presence of DSS1. Deletion or mutation of DSS1 or suppression of its expression by other mechanisms are therefore potential causative mechanisms for human breast and ovarian cancer. Such mechanisms may be relevant to sporadic as well as familiar breast cancer where BRCA1 and BRCA2 mutations are not present.

The E6 oncoproteins of high-risk papillomaviruses bind to a novel putative GAP protein, E6TP1, and target it for degradation.

The high-risk human papillomaviruses (HPVs) are associated with carcinomas of the cervix and other genital tumors. Previous studies have identified two viral oncoproteins, E6 and E7, which are expressed in the majority of HPV-associated carcinomas. The ability of high-risk HPV E6 protein to immortalize human mammary epithelial cells (MECs) has provided a single-gene model to study the mechanisms of E6-induced oncogenic transformation. In this system, the E6 protein targets the p53 tumor suppressor protein for degradation, and mutational analyses have shown that E6-induced degradation of p53 protein is required for MEC immortalization. However, the inability of most dominant-negative p53 mutants to induce efficient immortalization of MECs suggests the existence of additional targets of the HPV E6 oncoprotein. Using the yeast two-hybrid system, we have isolated a novel E6-binding protein. This polypeptide, designated E6TP1 (E6-targeted protein 1), exhibits high homology to GTPase-activating proteins for Rap, including SPA-1, tuberin, and Rap1GAP. The mRNA for E6TP1 is widely expressed in tissues and in vitro-cultured cell lines. The gene for E6TP1 localizes to chromosome 14q23.2-14q24.3 within a locus that has been shown to undergo loss of heterozygosity in malignant meningiomas. Importantly, E6TP1 is targeted for degradation by the high-risk but not the low-risk HPV E6 proteins both in vitro and in vivo. Furthermore, the immortalization-competent but not the immortalization-incompetent HPV16 E6 mutants target the E6TP1 protein for degradation. Our results identify a novel target for the E6 oncoprotein and provide a potential link between HPV E6 oncogenesis and alteration of a small G protein signaling pathway.

Loss of p53 protein during radiation transformation of primary human mammary epithelial cells.

The causative factors leading to breast cancer are largely unknown. Increased incidence of breast cancer following diagnostic or therapeutic radiation suggests that radiation may contribute to mammary oncogenesis. This report describes the in vitro neoplastic transformation of a normal human mammary epithelial cell strain, 76N, by fractionated gamma-irradiation at a clinically used dose (30 Gy). The transformed cells (76R-30) were immortal, had reduced growth factor requirements, and produced tumors in nude mice. Remarkably, the 76R-30 cells completely lacked the p53 tumor suppressor protein. Loss of p53 was due to deletion of the gene on one allele and a 26-bp deletion within the third intron on the second allele which resulted in abnormal splicing out of either the third or fourth exon from the mRNA. PCR with a mutation-specific primer showed that intron 3 mutation was present in irradiated cells before selection for immortal phenotype. 76R-30 cells did not exhibit G1 arrest in response to radiation, indicating a loss of p53-mediated function. Expression of the wild-type p53 gene in 76R-30 cells led to their growth inhibition. Thus, loss of p53 protein appears to have contributed to neoplastic transformation of these cells. This unique model should facilitate analyses of molecular mechanisms of radiation-induced breast cancer and allow identification of p53-regulated cellular genes in breast cells.

Abrogation of wild-type p53-mediated transactivation is insufficient for mutant p53-induced immortalization of normal human mammary epithelial cells.

The p53 protein has become a subject of intense interest since the discovery that about 50% of human cancers carry p53 mutations. Mutations in the p53 gene are the most frequent genetic lesions in breast cancer, suggesting a critical role for p53 protein in normal mammary epithelial cell (MEC) growth control. We previously demonstrated that abrogation of the p53 function by a cancer-derived p53 mutant, del239, was sufficient to induce immortalization of normal MECs. To further extend these findings and to examine the mechanism of mutant p53-induced immortalization of MECs, we tested the immortalizing ability of four selected p53 mutants (R175H, R248W, R249S, and R273H), which involve residues that cluster close to N239 in the three-dimensional structure and which are critical for the DNA-binding function of p53. Interestingly, two of these mutants (R175H and R249S) reproducibly immortalized 76N normal MECs, whereas the other two mutants (R248W and R273H) induced an extension of life span but not immortalization. These results further substantiate that selective ablation of p53 function with dominant-negative mutants is sufficient for immortalization of MECs. To determine whether abrogation of the transactivation function of endogenous p53 was important for the differential immortalizing ability of p53 mutants, we measured the effects of mutant p53 on the endogenous wild-type p53-mediated transactivation of a chloramphenicol acetyltransferase reporter linked to a consensus p53 binding DNA sequence in transiently transfected 76N MECs. All of the mutants, regardless of their immortalizing phenotype, abrogated the endogenous wild-type p53-mediated transactivation to a similar extent. Thus, abrogation of transactivation function is not sufficient for mutant p53-induced immortalization of normal MECs. The p53-immortalized MECs showed substantial telomerase activity; however, induction of telomerase activity occurred at late passages and was undetectable in mutant p53-expressing cells prior to immortalization. We suggest that mechanisms other than abrogation of transactivation and induction of telomerase activity determine the differential MEC-immortalizing behavior of various p53 mutants.

Synchronization of tumor and normal cells from G1 to multiple cell cycles by lovastatin.

Synchronization of mammalian cells is essential for investigations involving cell proliferation. A simple method for obtaining synchrony in all types of cells, through several cycles and with minimal overall metabolic perturbations, has not yet been available. We describe a procedure for synchronizing normal as well as tumor cells reversibly in the G1 phase of the cell cycle using Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. This method of synchronization was successful with all cell lines tested, including normal and tumor cells of mouse, hamster, and human origins. For example, when MCF-7 human breast cancer cells were synchronized with Lovastatin and released by the addition of mevalonic acid (the product of the reaction catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A reductase), 3 phases of accelerated thymidine incorporation into DNA corresponding to 3 S phases of the cell cycle occurred during a 90-h period of cell replication. Thymidine incorporation was decreased to less than or equal to 4% during the initial lag of 18 h before the first S phase, and maximum incorporation was then achieved after only 6 h. The antibody Ki-67, which detects a nuclear antigen associated with proliferation, was present in cells arrested with Lovastatin. This fact, together with the lack of thymidine incorporation during the initial lag time, indicates that the cells were arrested in the G1 and not in the G0 phase of the cell cycle. Furthermore, in synchronized tumor-derived human breast epithelial cells, histone H4 RNA was low after Lovastatin release and increased with the onset of DNA synthesis. Concomitant synthesis of DNA and histone H4 RNA expression could be observed for 2 cycles. Minimal perturbations of general metabolic functions occurred since the rate of RNA, protein, and initial DNA synthesis were unaffected by Lovastatin, as evidenced by [3H]uridine, [3H]leucine, and initial [3H]thymidine incorporation. Finally, while the Lovastatin-induced synchronization was overcome by mevalonic acid, addition of squalene or cholesterol-ethanol had no such effect. Thus, Lovastatin appears to prevent formation of an early intermediate in the cholesterol pathway that is essential for progression of cells through early G1 phase.

E7 protein of human papilloma virus-16 induces degradation of retinoblastoma protein through the ubiquitin-proteasome pathway.

Rb protein is a critical regulator of entry into the cell cycle, and loss of Rb function by deletions, mutations, or interaction with DNA viral oncoproteins leads to oncogenic transformation. We have shown that the human papilloma virus (HPV)-16 E7 gene is sufficient to induce the immortalization of mammary epithelial cells (MECs). Surprisingly, the steady-state level of Rb protein in these immortal cells was drastically decreased. Here, we used pulse-chase analysis to show that the in vivo loss of Rb protein in E7-immortalized MECs is a consequence of enhanced degradation. Expression of HPV16 E7 in a cell line with a temperature-sensitive mutation in the E1 enzyme of the ubiquitin pathway demonstrated that degradation of Rb was ubiquitin dependent. Treatment of E7-immortalized MECs with aldehyde inhibitors of proteasome-associated proteases led to a marked stabilization of Rb protein, particularly the hypophosphorylated form. Taken together, our results provide evidence for HPV-16 E7-induced enhanced degradation of Rb protein via a ubiquitin-proteasome pathway and suggest a second mechanism of oncogenic transformation by E7, in addition to its previously identified ability to sequester Rb from E2F. Our analyses also show that normal Rb levels are regulated by the ubiquitin-proteasome degradation pathway.

Expression of growth factors and oncogenes in normal and tumor-derived human mammary epithelial cells.

The expression of genes which may be involved in the regulation of human mammary epithelial cell growth [transforming growth factors alpha and beta] and tumorigenesis [c-myc, erbB2, epidermal growth factor receptor (EGFR), Ha-ras, pS2] has been compared in similarly cultured normal cell strains and tumor cell lines. We have found that the normal breast cells produce high levels of EGFR mRNA, which are translated into nearly 10(5) low affinity epidermal growth factor-binding molecules/cell. In the estrogen receptor-negative lines examined, the EGFR gene was expressed at levels comparable to those in the normal cells. In contrast, EGFR and transforming growth factor alpha mRNAs were reduced in estrogen receptor-positive tumor lines compared to estrogen receptor-negative lines and normal cells. Steady state mRNA levels for transforming growth factor beta, erbB2, c-myc, and Ha-ras in the normal cells were greater than or comparable to those in all of the breast tumor lines. Furthermore, in the absence of gene amplification, only one of the genes examined (i.e., pS2) was overexpressed in a subset of the tumor cells compared to their normal counterparts. Several reports by other investigators have described overexpression of some of these genes in breast biopsies and in tumor lines in studies lacking normal controls. Thus, our results, in which the same genes were not overexpressed compared to normal cells unless amplified, underscore the importance of including appropriate normal controls in studies aimed at defining aberrant patterns of gene expression in tumor cells.

Identification of a novel serine protease-like gene, the expression of which is down-regulated during breast cancer progression.

In an effort to isolate genes with down-regulated expression at the mRNA level during oncogenic transformation of human mammary epithelial cells (MECs), we performed subtractive hybridization between normal MEC strain 76N and its radiation-transformed tumorigenic derivative 76R-30. Here, we report the isolation of cDNA clones corresponding to a 1.4-kb mRNA species that is abundantly expressed in 76N cells but is drastically reduced in 76R-30 cells. Based on its selective expression in MECs compared with fibroblasts, the corresponding gene is designated NES1 (normal epithelial cell-specific 1). Sequence analysis of the full-length NES1 cDNA clones revealed it to be a novel gene with a predicted polypeptide of 30.14 kilodaltons; in vitro transcription and translation confirmed this prediction. Database searches revealed a 50-63% similarity and 34-42% identity with several families of serine proteases, in particular the trypsin-like proteases, members of the glandular kallikrein family (including prostate-specific antigen, nerve growth factor gamma, and epidermal growth factor-binding protein) and the activators for the kringle family proteins (including the human tissue plasminogen activator and human hepatocyte growth factor activator). Importantly, all of the residues known to be crucial for substrate binding, specificity, and catalysis by the serine proteases are conserved in the predicted NES1 protein, suggesting that it may be a protease. An antipeptide antibody directed against a unique region of the NES1 protein (amino acids 120-137) detected a specific 30-kilodalton polypeptide almost exclusively in the supernatant of the mRNA-positive MECs, suggesting that NES1 is a secreted protein. The 1.4-kb NES1 mRNA was expressed in several organs (thymus, prostate, testis, ovary, small intestine, colon, heart, lung, and pancreas) with highest levels in the ovary; a 1.1-kb transcript was found in the pancreas. Although expression of the NES1 mRNA was observed in all normal and immortalized nontumorigenic MECs, the majority of human breast cancer cell lines showed a drastic reduction or a complete lack of its expression. The structural similarity of NES1 to polypeptides known to regulate growth factor activity and a negative correlation of NES1 expression with breast oncogenesis suggest a direct or indirect role for this novel protease-like gene product in the suppression of tumorigenesis.

Tumor progression in four mammary epithelial cell lines derived from the same patient.

Two primary and two metastatic cell lines with distinct phenotypes and genotypes have been established from a patient diagnosed as having infiltrating and intraductal mammary carcinoma (21T series). All four lines can be cultured in the same medium, DFCI-1, which also supports long-term growth of normal epithelial cells. Therefore, we have been able to compare normal and tumor cells at the cellular and molecular levels. The mammary origin of the 21T series was confirmed by using antibodies against the human milk fat globule antigen-2 epitope. The two primary tumor lines (21NT and 21PT) are both immortal and aneuploid, although only 21NT is tumorigenic in the nude mouse assay. The two populations derived from the metastatic pleural effusion (21MT-1 and 21MT-2) each exhibit distinct characteristics in morphology and growth factor requirements. The erbB2 gene is amplified and overexpressed in all of these cell lines compared to normal epithelial cell controls. These four tumor cell lines from a single patient represent a mammary tumor progression series that has been established in long-term cell culture.

Mutant p53-induced immortalization of primary human mammary epithelial cells.

Mutations of the p53 gene are the most frequent genetic lesions in breast cancer, suggesting a critical role for p53 protein in normal mammary cell growth control. Indeed, the p53-targeting human papillomavirus oncogene E6 induces efficient immortalization of normal human mammary epithelial cells (MECs). To assess whether selective loss of p53 is sufficient for MEC immortalization, we introduced seven missense mutants and one single-amino acid deletion mutant (del239) of p53 into the 76N normal MEC strain. Although the missense mutants failed to immortalize MECs, the del239 mutant reproducibly immortalized these cells. The immortal cells were anchorage dependent and nontumorigenic, indicating a preneoplastic transformation. Gamma-irradiation of these cells failed to induce G1 cell cycle arrest and did not lead to an increase in WAF1 and mdm-2 mRNA levels, demonstrating a loss of the endogenous p53 function. These results demonstrate that selective ablation of p53 function by a dominant-negative mutant is sufficient for immortalization of MECs. Availability of an immortalizing as well as several nonimmortalizing p53 mutants should help identify functions critical for cell growth control by p53 in mammary epithelial cells.

The role for NES1 serine protease as a novel tumor suppressor.

Previously (Liu et al, Cancer Res., 56: 3371-3379, 1996), we isolated a novel serine protease-like gene--Normal Epithelial Cell Specific-1 (NES1)--that is expressed in normal mammary epithelial cells but is down-regulated in most breast cancer cell lines. Here, we demonstrate that stable expression of NES1 in the NES1-negative MDA-MB-231 breast cancer cell line suppressed the oncogenicity as revealed by inhibition of the anchorage-independent growth and tumor formation in nude mice. Fluorescence in situ hybridization localized the NES1 gene to chromosome 19q13.3, a region that contains genes for related proteases (including the prostate-specific antigen) and is rearranged in human cancers. Similar to breast cancer cell lines, prostate cancer cell lines also lacked NES1 mRNA and protein expression. Together, these results strongly suggest a tumor-suppressor role for NES1 in breast and prostate cancer.

CpG methylation as a basis for breast tumor-specific loss of NES1/kallikrein 10 expression.

The normal epithelial cell-specific-1 (NES1)/kallikrein 10 gene is expressed in normal mammary epithelial cells, but its expression is dramatically decreased in breast cancer cell lines. Now, we have cloned and characterized the active promoter region of NES1. Using a luciferase reporter system, we demonstrate that most tumor cell lines are able to support full or partial transcription from the NES1 promoter, suggesting a role for promoter-independent cis-acting mechanisms of loss of NES1 expression. We show that hypermethylation of the NES1 gene represents one such mechanism. Using methylation-specific PCR and sequence analysis of sodium bisulfite-treated genomic DNA, we demonstrate a strong correlation between exon 3 hypermethylation and loss of NES1 mRNA expression in a panel of breast cancer cell lines and in primary tumors. Treatment of NES1-nonexpressing cells with a demethylating agent led to reexpression of NES1, suggesting an important role of hypermethylation in the loss of NES1 expression. We suggest that hypermethylation is responsible for tumor-specific loss of NES1 gene expression. Our results also suggest that hypermethylation of the NES1 gene may serve as a potential marker for breast cancer.

Effects of p53 mutants on wild-type p53-mediated transactivation are cell type dependent.

The spectrum of p53 mutations differs among human cancer types. We have hypothesized that the p53 mutational spectrum observed in particular tumor types reflects the functional ability of different p53 mutants to modulate wild-type (WT) p53-dependent gene transcription. Missense p53 mutants representing several mutational hotspot codons were cotransfected with WT p53 and analysed for their effects on p53-dependent transactivation of a reporter construct containing a specific p53 binding sequence (PG13-CAT) in human tumor cell lines lacking endogenous p53. Our results show that the ability of p53 mutants to inhibit WT p53-mediated transactivation is cell type dependent. In cell lines derived from a lung adenocarcinoma and a mesothelioma, the transactivation function of WT p53 was strongly inhibited by all p53 mutants examined. However, in cell lines derived from a prostate carcinoma and an osteosarcoma, the mutants examined generally had only minimal dominant negative effects. In cell lines derived from a hepatocellular carcinoma and an ovarian carcinoma, two mutants (248trp and 273his) enhanced WT p53-mediated transactivation of the reporter construct. Additional mutants retained the ability to inhibit WT p53-mediated transactivation in these cell lines. In addition, in a series of four breast tumor cell lines, the p53 mutants examined had similar effects on WT p53 transactivation ability including enhanced transactivation activity in the 273his cotransfectants. The p53 mutants were incapable of transactivating the PG13-CAT reporter in the absence of WT p53 expression. Therefore, the dominant negative effects of p53 mutants on WT p53 function may vary depending on the particular cell type. In addition, mutants with stronger inhibitory capabilities may confer a selective advantage during the tumorigenic process.

Analysis of normal epithelial cell specific-1 (NES1)/kallikrein 10 mRNA expression by in situ hybridization, a novel marker for breast cancer.

PURPOSE: Normal epithelial cell specific-1 (NES1)/kallikrein 10 gene is expressed in normal mammary and prostate epithelial cells, but the expression of NES1 mRNA and protein is markedly reduced in established breast and prostate cancer cell lines although the NES1 gene is intact. Here, we wished to assess whether NES1 expression is down-regulated in primary breast cancers. EXPERIMENTAL DESIGN: We developed and used an in situ hybridization technique with an antisense NES1 probe to detect NES1 mRNA in sections of normal breast specimens, typical and atypical ductal hyperplasia, ductal carcinoma in situ, and infiltrating ductal carcinoma. RESULTS: All of the 30 normal breast specimens showed high NES1 expression. Notably, 18 (75%) of 24 typical and atypical breast hyperplasia specimens showed high NES1 expression, with weak-to-moderate expression in 6 (25%). Significantly, 13 (46%) of 28 ductal carcinoma in situ specimens lacked NES1 expression, and the remaining 15 (54%) showed weak-to-moderate expression. Finally, 29 of 30 (97%) infiltrating ductal carcinoma grades I-III samples lacked NES1 mRNA, with weak expression in the remaining one sample. CONCLUSIONS: Our results demonstrate that NES1 mRNA is expressed in normal breast tissue and benign lesions, with loss of NES1 expression during tumor progression. We suggest that NES1 expression may serve as a molecular tool in the study of breast cancer progression. Studies with larger series of specimens should help assess whether NES1 expression can be a diagnostic and/or prognostic marker in breast and other cancers.

Alterations in growth phenotype and radiosensitivity after fractionated irradiation of breast carcinoma cells from a single patient.

PURPOSE: To investigate growth regulation and radiosensitivity in surviving clonogens after fractionated irradiation. METHODS AND MATERIALS: Four breast carcinoma cell lines isolated from the primary tumor (21NT, 21PT) and metastases (21MT-1, 21MT-2) of a single patient were exposed to cumulative radiation doses of 30 Gy yielding cell lines designated-IR with respect to their parent. The irradiated lines were then compared to their parent for serum-and growth factor-requirements under defined media conditions, ability to proliferate in soft agar, concentration of TGF-alpha in conditioned medium, and radiosensitivity. RESULTS: The irradiated lines showed no change in proliferative doubling times under serum- and growth factor-supplemented media conditions. A single line, 21MT-1-IR, acquired a limited ability to proliferate in serum-and growth factor-deplete medium with a day 2-4 doubling time of 44.5 hr. Three lines, 21MT-1-IR, 21MT-2-IR, and 21NT-IR, formed colonies in soft agar in contrast to none of the unirradiated parent lines. There were significant 6-8 fold increases in conditioned media TGF-alpha concentrations for 21MT-2-IR and 21NT-IR cells. The 21MT-1-IR and 21NT-IR cells were significantly less radiosensitive than their respective parent lines. This decrease in radiosensitivity appeared to be at least partially mediated by a released factor as the radiosensitivity of 21MT-1 cells was significantly decreased by pre-incubation with conditioned medium from 21MT-1-IR cells. CONCLUSION: Radiation-induced changes in growth phenotype vary with respect to clonal origin of the cell line and may influence the radiosensitivity of surviving clonogens after fractionated treatment.

The role of retinoblastoma and p53 tumor suppressor pathways in human mammary epithelial cell immortalization.

Breast cancer is the second leading cause of cancer-related deaths among women in the United States. Approximately 180,000 new cases of breast cancer are diagnosed each year and a quarter of these are fatal. Early detection is a key to survival of these patients. Unfortunately, no definitive markers are available to diagnose breast cancer at early stages. Identification of such early markers, therefore, is an important priority in breast cancer research. In order to identify early markers, we have focussed on understanding the molecular mechanisms that can lead to conversion of the normal mammary epithelial cells into precancerous immortal cells. Over last several years, we have developed in vitro models of human mammary epithelial cell immortalization which have allowed us to invoke the critical roles of the known tumor suppressor pathways in the maintenance of the untransformed state of mammary epithelial cells. These models are now being used to identify novel genes whose expression is important for normal mammary epithelial cell growth and whose altered expression contributes to breast cell transformation. Characterization of the molecular machinery whose alterations result in early preneoplastic transformation should help identify candidate genes for evaluation as potential early diagnostic markers.

Multiple genetic changes are required for efficient immortalization of different subtypes of normal human mammary epithelial cells.

Multiple Genetic Changes Are Required for Efficient Immortalization of Different Subtypes of Normal Human Mammary Epithelial Cells. Breast cancer is the second leading cause of cancer-related deaths of women in the U.S. About 180,000 new cases of breast cancer are diagnosed each year, a quarter of them fatal. Early detection is the key to the survival of these patients. However, there are no molecular markers to detect breast cancer at very early stages. A hurdle in understanding the early molecular changes in breast cancer has been the difficulty in establishing premalignant lesions and primary breast tumors as in vitro cell cultures. Normal epithelial cells grow for a finite life span and then senesce. Immortalization is defined by continuous growth of otherwise senescing cells and is believed to represent an early stage in tumor progression. To examine these early stages, we and others have developed in vitro models of mammary epithelial cell immortalization. These models have been extremely important in understanding the role of various tumor suppressor pathways that maintain the normal phenotypes of mammary epithelial cells. In this paper, we describe the establishment of these models and their relevance to understanding the molecular changes that occur in early breast cancer. These models have helped to identify molecular changes that occur in early breast cancers and appear to be well suited to identify novel markers for early diagnosis of breast cancer.

The role of cooperativity with Src in oncogenic transformation mediated by non-small cell lung cancer-associated EGF receptor mutants.

Non-small cell lung cancer (NSCLC)-associated epidermal growth factor receptor (EGFR) mutants are constitutively active and induce ligand-independent transformation in non-malignant cell lines. We investigated the possibility that the ability of mutant EGFRs to transform cells reflects a constitutive cooperativity with Src using a system in which the overexpression of mutant, but not wild-type, EGFR induced anchorage-independent cell growth. Src was constitutively activated and showed enhanced interaction with mutant EGFRs, suggesting that constitutive EGFR-Src cooperativity may contribute to mutant EGFR-mediated oncogenesis. Indeed, the mutant EGFR-mediated cell transformation was inhibited by Src- as well as EGFR-directed inhibitors. Importantly, a tyrosine to phenylalanine mutation of the major Src phosphorylation site on EGFR, Y845, reduced the constitutive phosphorylation of NSCLC-EGFR mutants, as well as that of STAT3, Akt, Erk and Src, and reduced the mutant EGFR-Src association as well as proliferation, migration and anchorage-independent growth. Reduced anchorage-independent growth and migration were also observed when dominant-negative-Src was expressed in mutant EGFR-expressing cells. Overall, our findings show that mutant EGFR-Src interaction and cooperativity play critical roles in constitutive engagement of the downstream signaling pathways that allow NSCLC-associated EGFR mutants to mediate oncogenesis, and support the rationale to target Src-dependent signaling pathways in mutant EGFR-mediated malignancies.

Preneoplastic transformation of human mammary epithelial cells.

Recent in-vitro models of mammary epithelial cell (MEC) immortalization have provided a practical approach to begin to dissect the molecular mechanisms of breast tumorigenesis. Introduction of a single oncogene, the human papilloma virus (HPV)-16 E6, induces efficient and reproducible preneoplastic transformation of normal MECs, by inducing degradation of the tumor suppressor protein p53. The role of p53 has also been demonstrated by analyses of a model of gamma-radiation-induced MEC transformation. Recently, efficient retroviral gene transfer has allowed identification of multiple mammary epithelial cell types that show distinct susceptibilities to HPV E6 and E7 oncogenes, indicating a cell-type-specific predominance of the tumor suppressor proteins p53 and Rb which are targeted by E6 and E7, respectively. Further analyses of these models are likely to elucidate the biochemical mechanisms of early mammary tumorigenesis.