Epigenetic regulation of RNA polymerase III transcription in early breast tumorigenesis.

RNA polymerase III (Pol III) transcribes medium-sized non-coding RNAs (collectively termed Pol III genes). Emerging diverse roles of Pol III genes suggest that individual Pol III genes are exquisitely regulated by transcription and epigenetic factors. Here we report global Pol III expression/methylation profiles and molecular mechanisms of Pol III regulation that have not been as extensively studied, using nc886 as a representative Pol III gene. In a human mammary epithelial cell system that recapitulates early breast tumorigenesis, the fraction of actively transcribed Pol III genes increases reaching a plateau during immortalization. Hyper-methylation of Pol III genes inhibits Pol III binding to DNA via inducing repressed chromatin and is a determinant for the Pol III repertoire. When Pol III genes are hypo-methylated, MYC amplifies their transcription, regardless of its recognition DNA motif. Thus, Pol III expression during tumorigenesis is delineated by methylation and magnified by MYC.

Involvement of polypyrimidine tract-binding protein (PTBP1) in maintaining breast cancer cell growth and malignant properties.

We have investigated some roles of splicing factor polypyrimidine tract-binding protein (PTBP1) in human breast cancer. We found that PTBP1 was upregulated in progressively transformed human mammary epithelial cells (HMECs), as well as in breast tumor cell lines compared with HMECs with finite growth potential and found that the level of PTBP1 correlated with the transformation state of HMECs. Knockdown of PTBP1 expression substantially inhibited tumor cell growth, colony formation in soft agar and in vitro invasiveness of breast cancer cell lines, a result similar to what we have reported in ovarian cancer. However, ectopic expression of PTBP1 (as a PTBP1-EGFP fusion protein) did not enhance the proliferation of immortalized HMEC. Rather, PTBP1 expression promoted anchorage-independent growth of an immortalized HMEC as assessed by increased colony formation in soft agar. In addition, we found that knockdown of PTBP1 expression led to upregulation of the expression of the M1 isoform of pyruvate kinase (PKM1) and increase of the ratio of PKM1 vs PKM2. PKM1 has been reported to promote oxidative phosphorylation and reduce tumorigenesis. Correspondingly, we observed increased oxygen consumption in PTBP1-knockdown breast cancer cells. Together, these results suggest that PTBP1 is associated with breast tumorigenesis and appears to be required for tumor cell growth and maintenance of transformed properties. PTBP1 exerts these effects, in part, by regulating the splicing of pyruvate kinase, and consequently alters glucose metabolism and contributes to the Warburg effect.

Epigenetic changes accompanying human mammary epithelial cell immortalization.

Acquisition of immortality may be an early and crucial step in malignant progression. We hypothesize that acquisition of unlimited growth potential in individual human mammary epithelial cells (HMEC) requires inactivation of several distinct negative growth constraints as well as reactivation of a mechanism to maintain telomeres on chromosomes. Some of the heritable changes that occur during HMEC immortalization, i.e., loss of expression of cyclin dependent kinase inhibitors p16INK4a and p57KIP2, loss of TGFbeta-mediated growth inhibition, and derepression of telomerase, appear to occur without identifiable mutations in the genes and pathways involved. The absence of mutations, combined with the fact that the changes are often incremental over several cell generations even in clonal populations indicates that some changes associated with immortalization can be epigenetic. We have used the term "conversion" to describe the gradual epigenetic process in chemical carcinogen-immortalized HMEC that leads to activation of telomerase, stabilization of telomere length, and ability to grow uniformly well in the presence or absence of TGFbeta. Characterization of the epigenetic mechanisms involved in immortalization may uncover additional factors that drive tumor progression, and that may be responsive to novel forms of intervention.

Expression of the telomerase catalytic subunit, hTERT, induces resistance to transforming growth factor beta growth inhibition in p16INK4A(-) human mammary epithelial cells.

Failures to arrest growth in response to senescence or transforming growth factor beta (TGF-beta) are key derangements associated with carcinoma progression. We report that activation of telomerase activity may overcome both inhibitory pathways. Ectopic expression of the human telomerase catalytic subunit, hTERT, in cultured human mammary epithelial cells (HMEC) lacking both telomerase activity and p16(INK4A) resulted in gaining the ability to maintain indefinite growth in the absence and presence of TGF-beta. The ability to maintain growth in TGF-beta was independent of telomere length and required catalytically active telomerase capable of telomere maintenance in vivo. The capacity of ectopic hTERT to induce TGF-beta resistance may explain our previously described gain of TGF-beta resistance after reactivation of endogenous telomerase activity in rare carcinogen-treated HMEC. In those HMEC that overcame senescence, both telomerase activity and TGF-beta resistance were acquired gradually during a process we have termed conversion. This effect of hTERT may model a key change occurring during in vivo human breast carcinogenesis.

The ZNF217 gene amplified in breast cancers promotes immortalization of human mammary epithelial cells.

The functional consequences of overexpression of a candidate oncogene on chromosome 20q13.2, ZNF217, were examined by transducing the gene into finite life span human mammary epithelial cells (HMECs). In four independent experiments, ZNF217-transduced cultures gave rise to immortalized cells. HMECs that overcame senescence initially exhibited heterogeneous growth and continued telomere erosion, followed by increasing telomerase activity, stabilization of telomere length, and resistance to transforming growth factor beta growth inhibition. The incremental changes in telomerase activity and growth that occurred in ZNF217-transduced cultures after they overcame senescence were similar to the conversion pattern we have described previously in rare HMEC lines immortalized after exposure to a chemical carcinogen. Aberrant expression of ZNF217 may be selected for during breast cancer progression because it allows breast cells to overcome senescence and attain immortality.

Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes.

Senescence and genomic integrity are thought to be important barriers in the development of malignant lesions. Human fibroblasts undergo a limited number of cell divisions before entering an irreversible arrest, called senescence. Here we show that human mammary epithelial cells (HMECs) do not conform to this paradigm of senescence. In contrast to fibroblasts, HMECs exhibit an initial growth phase that is followed by a transient growth plateau (termed selection or M0; refs 3-5), from which proliferative cells emerge to undergo further population doublings (approximately 20-70), before entering a second growth plateau (previously termed senescence or M1; refs 4-6). We find that the first growth plateau exhibits characteristics of senescence but is not an insurmountable barrier to further growth. HMECs emerge from senescence, exhibit eroding telomeric sequences and ultimately enter telomere-based crisis to generate the types of chromosomal abnormalities seen in the earliest lesions of breast cancer. Growth past senescent barriers may be a pivotal event in the earliest steps of carcinogenesis, providing many genetic changes that predicate oncogenic evolution. The differences between epithelial cells and fibroblasts provide new insights into the mechanistic basis of neoplastic transformation.

Interindividual variation in CYP1A1 expression in breast tissue and the role of genetic polymorphism.

The cytochrome P4501A1 (CYP1A1) enzyme is regulated at the transcriptional level and its expression is influenced by genetic factors, polymorphisms in the structural and regulatory genes, and by environmental factors such as exposure to polycyclic aromatic hydrocarbons (PAHs). To investigate the role of CYP1A1 in breast cancer, we studied CYP1A1 expression in breast tissue, thereby taking all possible modifying factors into account. We measured CYP1A1 expression in 58 non-tumor breast tissue specimens from both breast cancer patients (n = 26) and cancer-free individuals (n = 32) using a newly developed reverse transcription-polymerase chain reaction assay. CYP1A1 expression varied between specimens approximately 400-fold and was independent of age. CYP1A1 expression was somewhat higher in tissue from breast cancer patients than in that from cancer-free individuals, but this difference was not statistically significant. Analysis for CYP1A1 genetic polymorphisms revealed eight variants, seven in the cancer-free group and one in the patient group. The variant genotype was not a good predictor of expression level. We conclude that high CYP1A1 expression could be a risk factor for breast cancer and that the known CYP1A1 polymorphisms are not good predictors of CYP1A1 expression.

High frequency of hypermethylation at the 14-3-3 sigma locus leads to gene silencing in breast cancer.

Expression of 14-3-3 final sigma (final sigma) is induced in response to DNA damage, and causes cells to arrest in G(2). By SAGE (serial analysis of gene expression) analysis, we identified final sigma as a gene whose expression is 7-fold lower in breast carcinoma cells than in normal breast epithelium. We verified this finding by Northern blot analysis. Remarkably, final sigma mRNA was undetectable in 45 of 48 primary breast carcinomas. Genetic alterations at final sigma such as loss of heterozygosity were rare (1/20 informative cases), and no mutations were detected (0/34). On the other hand, hypermethylation of CpG islands in the final sigma gene was detected in 91% (75/82) of breast tumors and was associated with lack of gene expression. Hypermethylation of final sigma is functionally important, because treatment of final sigma-non-expressing breast cancer cell lines with the drug 5-aza-2'-deoxycytidine resulted in demethylation of the gene and synthesis of final sigma mRNA. Breast cancer cells lacking final sigma expression showed increased number of chromosomal breaks and gaps when exposed to gamma-irradiation. Therefore, it is possible that loss of final sigma expression contributes to malignant transformation by impairing the G(2) cell cycle checkpoint function, thus allowing an accumulation of genetic defects. Hypermethylation and loss of final sigma expression are the most consistent molecular alterations in breast cancer identified so far.

Establishment and characterization of a breast cell strain containing a BRCA1 185delAG mutation.

OBJECTIVE: The aim of this study was to examine whether cells containing the heterozygous form of a BRCA1 185delAG mutation would exhibit abnormal growth or an altered response to DNA damage. METHODS: A primary culture of human mammary epithelial cells (90P) was obtained from the nontumor breast tissue of a 35-year-old patient who had undergone a mastectomy for removal of a breast tumor. These cells were immortalized (90PE6E7) following retroviral infection with HPV-16 viral E6/E7. genes. Both the 90P cell strain and the cell line were characterized for their ability to grow in culture, form colonies in soft agar, and produce tumors in athymic nude mice compared to normal breast epithelial cells containing wild-type BRCA1. 90P cells were also analyzed for cellular response to gamma radiation and H(2)O(2). RESULTS: These cells were confirmed to contain a frameshift mutation, 185delAG, of the BRCA1 gene. Despite being heterozygous for wild-type BRCA1, the 220-kDa full-size BRCA1 protein was abundantly expressed. 90P and 90PE6E7 cells grew at a similar rate and were anchorage dependent. 90PE6E7 also failed to form tumors in athymic nude mice. Finally, 90P cells exhibited a survival response similar to that of normal mammary epithelial cells to radiation damage and exposure to oxidative stress. CONCLUSION: To our knowledge the 90P cells and the 90PE6E7 cells are the first characterized, non-tumor-derived breast epithelial cells that are heterozygous for the BRCA1 germline mutation 185delAG. Our conclusion is that these BRCA1 mutant cells appear to have growth and stress response characteristics similar to those of normal human breast cells, which is consistent with the hypothesis that loss of heterozygosity must occur to impair putative BRCA1 function.

Culture models of human mammary epithelial cell transformation.

Human pre-malignant breast diseases, particularly ductal carcinoma in situ (DCIS) already display several of the aberrant phenotypes found in primary breast cancers, including chromosomal abnormalities, telomerase activity, inactivation of the p53 gene, and overexpression of some oncogenes. Efforts to model early breast carcinogenesis in human cell cultures have largely involved studies of in vitro transformation of normal finite lifespan human mammary epithelial cells (HMEC) to immortality and malignancy. We present a model of HMEC immortal transformation consistent with the known in vivo data. This model includes a recently described, presumably epigenetic process, termed conversion, which occurs in cells that have overcome stringent replicative senescence and are thus able to maintain proliferation with critically short telomeres. The conversion process involves reactivation of telomerase activity, and acquisition of good uniform growth in the absence and presence of TGFbeta. We propose that overcoming the proliferative constraints set by senescence, and undergoing conversion, represent key rate-limiting steps in human breast carcinogenesis, and occur during early stage breast cancer progression.

p57KIP2 expression and loss of heterozygosity during immortal conversion of cultured human mammary epithelial cells.

We have uncovered a novel role for the cyclin-dependent kinase inhibitor, p57KIP2, during the immortalization of cultured human mammary epithelial cells (HMECs). HMECs immortalized after chemical carcinogen exposure initially expressed little or no telomerase activity, and their telomeres continued to shorten with passage. Cell populations whose mean terminal restriction fragment (TRF) length declined to < or = 3 kb exhibited slow heterogeneous growth and contained many nonproliferative cells. These conditionally immortal HMEC cultures accumulated large quantities of p57 protein. With continued passage, the conditionally immortal cell populations very gradually converted to a fully immortal phenotype of good uniform growth, expression of high levels of telomerase activity, and stabilization of telomere length. The fully immortal HMECs that grew well did not accumulate p57 in G0 or during the cell cycle. DNA and RNA analysis of mass populations and individual subclones of conditionally immortal HMEC line 184A1 showed that continued growth of conditionally immortal cells with critically short telomeres was repeatedly accompanied by loss of the expressed p57 allele and transient expression of the allele imprinted previously. Conditionally immortal 184A1 with mean TRF > 3 kb, infected with retroviruses containing the p57 gene, exhibited premature slow heterogeneous growth. Conversely, exogenous expression of human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, in 184A1 with mean TRF > 3 kb prevented both the slow heterogeneous growth phase and accumulation of p57 in cycling populations. These data indicate that in HMECs that have overcome replicative senescence, p57 may provide an additional barrier against indefinite proliferation. Overcoming p57-mediated growth inhibition in these cells may be crucial for acquisition of the unlimited growth potential thought to be critical for malignant progression.

Viral oncogenes accelerate conversion to immortality of cultured conditionally immortal human mammary epithelial cells.

Our recent studies on the process of immortalization of cultured human mammary epithelial cells (HMEC) have uncovered a previously undescribed, apparently epigenetic step, termed conversion. When first isolated, clonally derived HMEC lines of indefinite lifespan showed little or no telomerase activity or ability to maintain growth in the presence of TGFbeta. Cell populations whose mean terminal restriction fragment length had declined to <3 kb also exhibited slow heterogeneous growth, and contained many non-proliferative cells. With continued passage, these conditionally immortal cell populations very gradually converted to a fully immortal phenotype of good growth+/-TGFbeta, expression of high levels of telomerase activity, and stabilization of telomere length. We now show that exposure of the early passage conditionally immortal 184A1 HMEC line to the viral oncogenes human papillomavirus type 16 (HPV16)-E6, -E7, or SV40T, results in either immediate (E6) or rapid (E7; SV40T) conversion of these telomerase negative, TGFbeta sensitive conditionally immortal cells to the fully immortal phenotype. Unlike conditional immortal 184A1, the HPV16-E7 and SV40T exposed cells were able to maintain growth in TGFbeta prior to expression of high levels of telomerase activity. A mutated HPV16-E6 oncogene, unable to inactivate p53, was still capable of rapidly converting conditional immortal 184A1. Our studies provide further evidence that the transforming potential of these viral oncogenes may involve activities beyond their inactivation of p53 and pRB functions. These additional activities may greatly accelerate a step in HMEC immortal transformation, conversion, that would be rate-limiting in the absence of viral oncogene exposure.

Increased p16 expression with first senescence arrest in human mammary epithelial cells and extended growth capacity with p16 inactivation.

Aberrations affecting the tumor suppressor gene p16INK4a have been described for a variety of tumors. In breast cancer, approximately 50% of tumors show low or lack p16 expression. While evidence provided by some studies has implicated a possible role for p16 in normal replicative senescence, other studies have suggested that the Rb, pathway through which p16 functions, may not be involved in senescence control. Previously we observed that all immortal lines derived from normal mammary epithelium which were analysed for p16 displayed inactivation of this gene through distinct mechanisms, supporting p16 inactivation as a possible necessary event in escape from senescence. To further clarify this issue, we have analysed p16 expression in a panel of normal finite lifespan human mammary epithelial cells (HMEC) from initial propagation through growth arrest, using media which confer different replicative capacity. Approximately 10-25-fold increase in p16 expression was observed for all normal HMEC with initial onset of a senescence phenotype following 15-25 population doublings in culture. These cells also displayed expression of the senescence associated beta-galactosidase. Interestingly, HMEC with additional long term replicative capacity (approximately 80 population doublings) arose from these growth arrested cultures, showing lack of p16 expression. This extended growth capacity appears to be associated with a methylation phenomenon since treatment of these cells with the methylation inhibitor 5-aza-2-deoxycytidine resulted in growth arrest concurrent with reacquisition of p16 expression and senescence associated beta-galactosidase. Analysis of p21waf1 expression revealed no change in expression during growth in vitro. These results support p16INK4a as the 9p senescence gene and suggest a role for p16 loss in the escape from initial onset of senescence and in acquisition of an extended life span of human mammary epithelial cells.

Gradual phenotypic conversion associated with immortalization of cultured human mammary epithelial cells.

Examination of the process of immortal transformation in early passages of two human mammary epithelial cell (HMEC) lines suggests the involvement of an epigenetic step. These lines, 184A1 and 184B5, arose after in vitro exposure of finite lifespan 184 HMEC to a chemical carcinogen, and both are clonally derived. Although early-passage mass cultures of 184A1 and 184B5 maintained continuous slow growth, most individual cells lost proliferative ability. Uniform good growth did not occur until 20-30 passages after the lines first appeared. Early-passage cultures expressed little or no telomerase activity and telomeres continued to shorten with increasing passage. Telomerase activity was first detected when the telomeres became critically short, and activity levels gradually increased thereafter. Early-passage cultures had little or no ability to maintain growth in transforming growth factor-beta (TGFbeta); however, both mass cultures and clonal isolates showed a very gradual increase in the number of cells displaying progressively increased ability to maintain growth in TGFbeta. A strong correlation between capacity to maintain growth in the presence of TGFbeta and expression of telomerase activity was observed. We have used the term "conversion" to describe this process of gradual acquisition of increased growth capacity in the absence or presence of TGFbeta and reactivation of telomerase. We speculate that the development of extremely short telomeres may result in gradual, epigenetic-based changes in gene expression. Understanding the underlying mechanisms of HMEC conversion in vitro may provide new insight into the process of carcinogenic progression in vivo and offer novel modes for therapeutic intervention.

Breast cancer cells have lower activating protein 1 transcription factor activity than normal mammary epithelial cells.

To determine whether normal breast cells have different levels of activating protein 1 (AP-1) expression and activation relative to breast cancer cells, we have compared the level of c-Jun and c-Fos expression and AP-1 activity in human mammary epithelial cells (HMECs) at different stages of transformation (normal proliferating HMECs, immortal HMECs, oncogene-transformed HMECs, and breast cancer cell lines). These studies demonstrated that normal and immortal HMECs have a high basal level of expression of cJun and cFos and higher AP-1 DNA-binding and transcriptional activating activities than do oncogene-transformed HMECs or human breast cancer cells, with a gradual decrease in AP-1 transactivating activity as cells progress through the carcinogenesis pathway (normal > immortal > oncogene-transformed > cancer cell lines). The AP-1 activity in normal or immortal cells was not modulated by growth factor supplementation or oncogene overexpression, as it is in breast cancer cells. However, the addition of suramin, a nonspecific growth factor antagonist, did inhibit AP-1 in these HMECs, suggesting that this high level of AP-1 present in normal HMECs may be due to autocrine stimulation of growth factor pathways. The differences in AP-1 activity in normal and malignant breast cells may indicate that normal cells are more dependent on AP-1-mediated signals for their growth than are breast cancer cells.

Transforming growth factor beta stabilizes p15INK4B protein, increases p15INK4B-cdk4 complexes, and inhibits cyclin D1-cdk4 association in human mammary epithelial cells.

The effects of transforming growth factor beta (TGF-beta) were studied in closely related human mammary epithelial cells (HMEC), both finite-life-span 184 cells and immortal derivatives, 184A1S, and 184A1L5R, which differ in their cell cycle responses to TGF-beta but express type I and type II TGF-beta receptors and retain TGF-beta induction of extracellular matrix. The arrest-resistant phenotype was not due to loss of cyclin-dependent kinase (cdk) inhibitors. TGF-beta was shown to regulate p15INK4B expression at at least two levels: mRNA accumulation and protein stability. In TGF-beta-arrested HMEC, there was not only an increase in p15 mRNA but also a major increase in p5INK4B protein stability. As cdk4- and cdk6-associated p15INK4B increased during TGF-beta arrest of sensitive cells, there was a loss of cyclin D1, p21Cip1, and p27Kip1 from these kinase complexes, and cyclin E-cdk2-associated p27Kip1 increased. In HMEC, p15INK4B complexes did not contain detectable cyclin. p15INK4B from both sensitive and resistant cells could displace in vitro cyclin D1, p21Cip1, and p27Kip1 from cdk4 isolated from sensitive cells. Cyclin D1 could not be displaced from cdk4 in the resistant 184A1L5R cell lysates. Thus, in TGF-beta arrest, p15INK4B may displace already associated cyclin D1 from cdks and prevent new cyclin D1-cdk complexes from forming. Furthermore, p27Kip1 binding shifts from cdk4 to cyclin E-cdk2 during TGF-beta-mediated arrest. The importance of posttranslational regulation of p15INK4B by TGF-beta is underlined by the observation that in TGF-beta-resistant 184A1L5R, although the p15 transcript increased, p15INK4B protein was not stabilized and did not accumulate, and cyclin D1-cdk association and kinase activation were not inhibited.

Lactoferrin expression in mammary epithelial cells is mediated by changes in cell shape and actin cytoskeleton.

Lactoferrin is a secreted iron binding protein which is expressed during normal functional development of mammary epithelium. Murine mammary epithelial cell lines competent for milk protein expression were used to identify microenvironmental factors that regulate lactoferrin expression. While lactoferrin was not expressed in adherent monolayer cultures under standard subconfluent conditions on plastic, lactoferrin mRNA and protein steadily accumulated when the cells aggregated to form spheroids on a reconstituted basement membrane gel. However, unlike other milk proteins such as beta-casein, lactoferrin expression was also induced at high cell density in the absence of exogenously added basement membrane or prolactin. These results led us to examine whether changes in cell growth, cell-cell interactions and/or cell shape were responsible for regulation of lactoferrin gene expression. Rounded, non-proliferating cells in suspension in serum-free medium expressed lactoferrin even as single cells. Conversely, lactoferrin expression could be inhibited in non-proliferative cells in serum-free medium by maintaining them in contact with an air-dried extracellular matrix which caused the cells to retain flat, spread morphologies. These findings indicated that cessation of cell growth was not sufficient, that cell-cell interactions were not required, and that cell culture conditions which minimize cell spreading may be important in maintaining lactoferrin expression. Additional data supporting this latter concept were generated by treating spread cells with cytochalasin D. The resulting disruption of microfilament assembly induced both cell rounding and lactoferrin expression. Shape-dependent regulation of lactoferrin mRNA was both transcriptional and post-transcriptional. Surprisingly, treatment of rounded cells with a transcription inhibitor, actinomycin D, produced a stabilization of lactoferrin mRNA, suggesting that transcription of an unstable factor is required for degradation of lactoferrin mRNA. Importantly, lactoferrin mRNA expression was regulated similarly in early passage normal human mammary epithelial cells. In vivo, the changing extracellular matrix components of the mammary gland during different stages of normal and abnormal growth and differentiation may provide different physical constraints on the configurations of cell surface molecules. These physical constraints may be communicated to the cell interior through mechanical changes in the cytoskeleton. Unlike beta-casein whose expression is upregulated by specific integrin-mediated signals, lactoferrin may be representative of a class of proteins synthesized in the mammary gland using basal transcriptional and translational machinery. The suppression of lactoferrin expression that is observed in monolayer culture and in malignant tissues may reflect inappropriate cell shapes and cytoskeletal structures that are manifested under these conditions.

Selective cell culture of primary breast carcinoma.

We have used culture conditions which simulate the microenvironment of breast tumors for the isolation and propagation of primary breast tumor cells in vitro. In this monolayer setup, the mixture of cells dissociated from primary breast tumors is subjected to self-created gradients of oxygen and nutrients as well as metabolic waste and extracellular pH. The tumor populations isolated under these novel conditions have displayed phenotypic properties characteristic of breast carcinomas, including homogeneous expression of cytokeratin 19, and increased mitochondrial retention of the cationic dye rhodamine 123. Nonmalignant cultures from reduction mammoplasty were unable to survive these conditions. One tumor population which reached passage 10 was aneuploid for chromosomes 15 and 17, and displayed a p53 mutation in exon 8. These studies strongly suggest that the culture conditions described here can suppress the growth of normal breast cells, thereby allowing selective isolation of some populations of slow-growing primary tumor cells in vitro.

Insulin receptor overexpression in 184B5 human mammary epithelial cells induces a ligand-dependent transformed phenotype.

To determine the role of the insulin receptor overexpression in breast epithelial cell transformation, the 184B5 human breast epithelial cell line was transfected with human insulin receptor cDNA. In two cell lines transfected with and overexpressing human insulin receptors (IR) (223.8 and 184.5 ng IR/10(6) cells), but not in untransfected cells, insulin binding and tyrosine kinase activity were elevated, and insulin induced a dose-dependent increase in colony formation in soft agar.

Mouse mammary tumors express elevated levels of RNA encoding the murine homology of SKY, a putative receptor tyrosine kinase.

To gain insight into the signal transduction pathways utilized by the Wnt-1-responsive mammary epithelial cell line C57MG, we screened for non-src family member tyrosine kinases expressed in these cells using a polymerase chain reaction-based technique. We identified five cDNA clones encoding receptor tyrosine kinases for which the ligand is known (fibroblast growth factor receptor, platelet-derived growth factor receptor, epithelial growth factor receptor, insulin receptor, and insulin-like growth factor receptor), two putative receptor tyrosine kinases for which the ligand remains to be identified (the products of ryk and the mouse klg homolog), and a novel tyrosine kinase. We cloned cDNAs encoding both the murine and human homologs of this kinase, the sequences of which were subsequently published under the names sky (Ohashi, K., Mizuno, K., Kuma, K., Miyata, T., and Nakamura, T. (1994) Oncogene 9, 699-705) and rse (Mark, M. R., Scadden, D. T., Wang, Z., Gu, Q., Goddard, A., and Godowski, P. J. (1994) J. Biol. Chem. 269, 10720-10728). Mouse sky RNA levels are abundant in mammary tumors derived from transgenic mice that express wnt-1, fgf-3, or both oncogenes in their mammary glands. However, little or no expression of sky is detected in mammary glands from virgin animals or in preneoplastic mammary glands from wnt-1 transgenic mice. Moreover, we find that the human homolog of sky is expressed at elevated levels when normal human mammary epithelial cells are rendered tumorigenic by the introduction of two viral oncogenes. Transient transfection of the human SKY cDNA into the quail fibrosarcoma cell line QT6 reveals that SKY is an active tyrosine kinase that augments the level of cellular phosphotyrosine. Introduction of murine Sky into RatB1a fibroblasts by retrovirus-mediated gene transfer results in morphological transformation, growth in soft agar, and the formation of tumors in nude mice. These data raise the possibility that the Sky tyrosine kinase is involved in the development and/or progression of mammary tumors.