Mammary gland biology and breast cancer. Conference on Common Molecular Mechanisms of Mammary Gland Development and Breast Cancer Progression.
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Biomarkers, Tumor↗
Biomedical subjects
Bernd Groner
Publications and source records attributed to Bernd Groner.
Mammalian target of rapamycin regulates the growth of mammary epithelial cells through the inhibitor of deoxyribonucleic acid binding Id1 and their functional differentiation through Id2.
Organ development requires the integration of multiple extracellular signals to assure a proper balance between proliferation and differentiation and to achieve and maintain specialized functions. Considerable progress has been made in the study of hormones and growth factors and in the understanding of the regulated intracellular pathways and transcriptional events that contribute to mammogenesis. Cell culture experiments have pointed out crucial pathways and components, which were subsequently validated in vivo experiments. We found that the mammalian target of rapamycin (mTOR) pathway is essential for both growth and differentiation of mammary epithelial cells and that the action of mTOR is mediated through the induction of the helix-loop-helix transcriptional regulators Id1 and Id2. Pharmacological inhibition of mTOR activity in HC11 mammary epithelial cells reduced cellular proliferation and prevented the lactogenic hormone-induced expression of milk proteins. Treatment of female mice with rapamycin impaired mammary gland differentiation and milk protein synthesis. The effects of mTOR on proliferation and differentiation require the functions of the helix-loop-helix proteins Id1 and Id2. Rapamycin treatment of HC11 cells resulted in a suppression of Id1 expression and an inhibition of proliferation. This effect of rapamycin was reversed by the forced expression of Id1. Rapamycin also prevented the induction of Id2 by lactogenic hormones and milk protein gene expression. Expression of a Id2 transgene bypassed the requirement of mTOR activity for beta-casein induction. These data suggest that mTOR activity has distinguishable functions in the proliferative and the differentiated state of mammary epithelial cells: it is a prerequisite for proliferation through the induction of Id1 and for differentiation-specific gene expression through the induction of Id2. The relative strengths of these proliferation and differentiation signals reflected by the expression levels of the individual Id proteins are crucial to the functional life cycle of mammary epithelial cells and might be disturbed in tumorigenesis.
Animals↗
Expression of a carboxy terminally truncated Stat5 with no transactivation domain in the mammary glands of transgenic mice inhibits cell proliferation during pregnancy, delays onset of milk secretion, and induces apoptosis upon involution.
Signal transducer and activator of transcription (Stat5) is a transcription factor, which transduces extracellular cytokine and growth-factor signals to the nuclei of mammalian cells. As a major mediator of prolactin action, it is involved in the regulation of the development, function, and survival of mammary epithelial cells. The carboxyl terminal of Stat5 encodes a transactivation domain (TAD), which interacts with coactivators and is crucial for the transcriptional induction of Stat5 target genes. To study the role of the Stat5 TAD in mediating Stat5 functions, a carboxy terminally truncated Stat5 variant (Stat5Delta750) was directed for expression in the mammary glands of transgenic mice by regulatory sequences of the beta-lactoglobulin (BLG) gene. Expression of Stat5Delta750 in mammary tissue reduced the rates of cell proliferation at mid and late pregnancy. Subsequently, morphological signs of milk secretion upon parturition were delayed. In double-transgenic mice, expression of Stat5Delta750 drastically decreased BLG/luciferase activity during lactation, but did not affect the expression and secretion of the endogenous beta-casein or alpha-lactalbumin into the milk. Expression of Stat5Delta750 also caused an increase in the number of apoptotic cells during mammary involution by a factor of 3 relative to control glands. Our data established a role for the Stat5 TAD in mediating the effects of Stat5 on mammary development, regulation of milk protein gene activity, and cell survival. The full effects of Stat5Delta750 may be partially buffered by the expression of endogenous wild-type Stat5 and the formation of truncated and wild-type heterodimers.
Animals↗
The biological functions of the versatile transcription factors STAT3 and STAT5 and new strategies for their targeted inhibition.
Signal transducers and activators of transcription (STATs) comprise a unique family of transcription factors, which transmit the interactions of cytokines, hormones and growth factors with their cell surface receptors into transcriptional programs. The mechanism of STAT activation has been well-established and comprises tyrosine phosphorylation, dimerization, nuclear translocation, binding to specific DNA response elements, recruitment of co-activators or co-repressors and transcriptional induction or repression of target genes. Gene deletion, microarrays, proteomics and chromatin immunoprecipitation experiments have revealed target genes with a broad range of functions regulated by STAT3 and STAT5. In the mammary gland, STAT5-induced genes contribute mainly to the prolactin dependent lobulo-alveolar development, whereas STAT3 induced genes control apoptosis during involution. Crucial effects have also been observed in other tissues. The germ line deletion of STAT3 or STAT5 causes early embryonal or perinatal lethality in mice. STAT5 is also required for proliferation of T- and B-cells and hematopoietic stem cell self-renewal. Deregulated STAT activity is often found associated with tumorigenesis and activated STATs seem to be limiting components in tumor cells. This review summarizes the functions of STAT3 and STAT5 in different cell types and the strategies that are used to counteract their action in tumor cells.
Animals↗
Peptide aptamers with binding specificity for the intracellular domain of the ErbB2 receptor interfere with AKT signaling and sensitize breast cancer cells to Taxol.
The ErbB2 receptor tyrosine kinase is overexpressed in approximately 30% of breast tumor cases and its overexpression correlates with an unfavorable prognosis. A major contributor for this course of the disease is the insensitivity of these tumors toward chemotherapy. Monoclonal antibodies, inhibiting the ligand-induced activation of the receptor and tyrosine kinase inhibitors acting on the intrinsic enzymatic activity of the intracellular domain, have been developed as targeted drugs. Both have been shown to be beneficial for breast cancer patients. We targeted a third aspect of receptor function: its association with intracellular signaling components. For this purpose, we selected peptide aptamers, which specifically interact with defined domains of the intracellular part of the receptor. The peptide aptamers were selected from a random peptide library using a yeast two-hybrid system with the intracellular tyrosine kinase domain of ErbB2 as a bait construct. The peptide aptamer AII-7 interacts with high specificity with the ErbB2 receptor in vitro and in vivo. The aptamers colocalized with the intracellular domain of ErbB2 within cells. We investigated the functional consequences of the aptamer interaction with the ErbB2 receptor within tumor cells. The aptamer sequences were either expressed intracellularly or introduced into the cells as recombinant aptamer proteins. The phosphorylation of p42/44 mitogen-activated protein kinase was nearly unaffected and the activation of signal transducers and activators of transcription-3 was only modestly reduced. In contrast, they strongly inhibited the induction of AKT kinase in MCF7 breast cancer cells treated with heregulin, whereas AKT activation downstream of insulin-like growth factor I or epidermal growth factor receptor was not or only slightly affected. High AKT activity is responsible for the enhanced resistance of ErbB2-overexpressing cancer cells toward chemotherapeutic agents. Peptide aptamer interference with AKT activation resulted in the restoration of regular sensitivity of breast cancer cells toward Taxol.
Amino Acid Motifs↗
Current strategies for the development of peptide-based anti-cancer therapeutics.
The completion of the human genome sequence and the development of new techniques, which allow the visualisation of comprehensive gene expression patterns, has led to the identification of a large number of gene products differentially expressed in tumours and corresponding normal tissues. The task at hand is the sorting of these genes into correlative and causative ones. Correlative genes are merely changed as a consequence of transformation and have no decisive effects upon transformation. In contrast, causative genes play a direct role in the process of cellular transformation and the maintenance of the transformed state, which can be exploited for therapeutic purposes. Oncogenes and tumour suppressor genes are prime targets for the development of new inhibitors and gene therapeutic strategies. However, many target oncogene products do not exhibit enzymatic activity that can be inhibited by conventional small molecular weight compounds. They exert their functions through regulated protein-protein or protein-DNA interactions and might require other compounds for efficient interference with such functions. Peptides are emerging as a novel class of drugs for cancer therapy, which could fulfil these tasks. Peptide therapy aims at the specific inhibition of inappropriately activated oncogenes. This review will focus on the selection procedures, which can be employed to identify useful peptides for the treatment of cancer. Before peptide-based therapeutics can become useful, it will be necessary to increase their stability by modifications or the use of scaffolds. Additionally, various delivery methods including liposomes and particularly the use of protein transduction domains (PTDs) have to be explored. These strategies will yield highly specific and more effective peptides and improve the potential of peptide-based anti-cancer therapeutics.
Antineoplastic Agents↗
The IGF-1 receptor and its contributions to metastatic tumor growth-novel approaches to the inhibition of IGF-1R function.
Signals originating from the type 1 insulin-like growth factor receptor (IGF-1R) have pleiotropic effects on cell behavior. They regulate cell proliferation, survival, differentiation and transformation. IGF-1R also plays an essential role in the multistep process of cancer cell metastasis. Metastatic spread of tumor cells is the main reason for the high mortality rates associated with cancer. The development of strategies to inhibit this process promises important clinical benefits. Current attempts to block IGF-1R signaling has resulted in the reversion of the transformed phenotype, the induction of apoptosis, the sensitization to chemotherapeutic drugs and the reduction of the metastatic propensity of tumor cells. Since inhibition of IGF-1R has acceptable side effects on normal cells, the IGF-1R represents a favorable target for anticancer therapy. The well known structure and biochemical functions of the receptor suggest diverse strategies for interference. We will discuss strategies which have already been developed and suggest novel approaches based on peptide aptamers. These are peptides selected for specific binding to defined domains of the IGF-1R which offer subtle and specific possibilities to interfere with IGF-1R in the context of experimental tumor therapy.
Animals↗
Peptide aptamers: recent developments for cancer therapy.
During the past two decades, our understanding of oncogenesis has advanced considerably and many new signalling pathways have been identified. Differences in signalling events that distinguish normal cells from tumour cells provide new targets for the development of anticancer agents. Peptide aptamers are small peptide sequences that have been selected to recognise a predetermined target protein domain and are potentially able to interfere with its function. They represent useful molecules for manipulating protein function in vivo. The isolation and use of specific peptide aptamers as inhibitors of individual signalling components, essential in cancer development and progression, provides a new challenge for drug development. Although peptides make up only a small fraction of current therapeutics, their potential is being enhanced by new developments affecting their modification, stability, delivery and their successful application in preclinical settings. This review summarises the methods that can be used for the isolation and delivery of peptide aptamers, as well as the important achievements that have been made using such peptide aptamers in different systems. The applicability of peptide aptamers as novel cancer therapeutics will be discussed.
Amino Acid Sequence↗
Signal transducer and activator of transcription 5 (STAT5), a crucial regulator of immune and cancer cells.
STAT5 belongs to a small family of transcription factors with dual functions. The seven signal transducers and activators of transcription (STAT) act as signaling components between the plasma membrane and the nucleus, and as transcription factors with specific DNA binding ability in the nucleus. STAT5 regulates the expression of genes, which determine important cellular phenotypes. It can promote proliferation and inhibit apoptosis, but is also involved in the regulation of differentiation between specific gene expression. STAT5 can also contribute to the transformed phenotype. In many leukemias and some solid tumors, STAT5 is constitutively activated through receptors or receptor associated tyrosine kinases and contributes to the survival and the proliferation of malignant cells. STAT5 activity appears to be limiting for these phenotypes. Inhibition of STAT5 in these tumor cells results in growth arrest and apoptosis. Targeting of STATs and other downstream mediators of oncogenic tyrosine kinases provides a promising strategy for tumor therapy, which might be refractory to resistance mechanisms incapacitating tyrosine kinase inhibitors. The well-studied steps in the activation of STAT5 and its roles in different subcellular compartments suggest original interference strategies, which could be used to inhibit its function. The challenge for drug developers will be the exploitation of defined protein-protein or protein-DNA interactions as targets of inhibition.
Animals↗
Deregulation of Stat5 expression and activation causes mammary tumors in transgenic mice.
Members of the signal transducers and activators of transcription (Stat) family regulate essential cellular growth and survival functions in normal cells and have also been implicated in tumorigenesis. We have studied the potential role of Stat5 in mammary tumorigenesis by targeting Stat5 variants to the mammary gland of transgenic mice using regulatory sequences of the beta-lactoglobulin gene. Mammary-directed expression of the wild-type Stat5, constitutively activated Stat5 and carboxyl-terminally truncated dominant negative Stat5 forms resulted in mammary tumors with incidence rates of up to 22% and latency periods of 8-12 months. Undifferentiated carcinomas most frequently occurred in mice expressing the carboxyl-terminally truncated Stat5. The more differentiated papillary and micropapillary adenocarcinomas were primarily found in mice overexpressing the native and constitutively active transgenes. Higher levels of translation initiation factor 4E (eIF4E) and cyclin D1 expression but lower levels of activated Stat3 were found in tumors of mice expressing the constitutively active Stat5 when compared to mice expressing the wild-type or truncated forms. A higher expression of the estrogen receptor (ERalpha) was observed in carcinomas compared to other phenotypes. The ability of both forms of Stat5, the transactivating form and the dominant negative form, to participate in oncogenesis indicates that there is more than one mechanism by which Stat5 contributes to this process. The transactivation function of Stat5 is involved in the determination of tumors with a more differentiated phenotype.
Animals↗
The insulin like growth factor-1 receptor (IGF-1R) as a drug target: novel approaches to cancer therapy.
The insulin-like growth factor 1 receptor (IGF-1R) is an important signaling molecule in cancer cells and plays an essential role in the establishment and maintenance of the transformed phenotype. Inhibition of IGF-1R signaling thus appears to be a promising strategy to interfere with the growth and survival of cancer cells. Different classes of molecules, e.g., antisense RNA, monoclonal antibodies and dominant negative IGF-1R gene variants, have been employed towards this aim. These agents have been able to reverse the transformed phenotype in several rodent and human cancer cell lines. The application of peptide aptamers specifically binding to the IGF-1R represents a novel approach to target IGF-1R signaling. The integration of peptide aptamers into targeted protein degradation vehicles and their transduction into cells allows the temporary elimination of the receptor protein. This review summarizes recently published data about inhibition of IGF-1R signaling and provides a perspective on upcoming possibilities.
Animals↗
Therapeutic antibodies.
Monoclonal antibodies had the lure of drugs very much since their first description. The ability to bind to a predetermined chemical structure stimulated the imagination of drug discoverers and developers. Nevertheless it took many years before a drug was registered which started to make good on the promise. The complexity of the molecule, made up of four polypeptide chains, its large molecular weight, its multiple and versatile functional domains and its mouse origin initially were obstacles for the production and the utilisation. Also the selection of appropriate target structures on the surface of cells turned out be difficult. Many of these difficulties have been overcome. The replacement of most of the murine sequences with equivalent human sequences and the concomittant decrease in immunogenicity, and the identification of cell surface components which are causative and limiting in cellular transformation have made monoclonal antibodies valuable weapons in the fight against cancer. Multiple mechanisms of monoclonal antibody action are being exploited for this purpose. Antibodies can sequester growth factors and prevent the activation of crucial growth factor receptors. A monoclonal antibody directed against the vascular endothelial growth factor (VEGF) has been shown to be a potent neo-vascularisation inhibitor (bevacizumab). An antibody against the extracellular domain of the EGF receptor prevents the binding of the ligand to the receptor and thereby its activation (cetuximab). EGFR activity, however, is absolutely required for the survival and proliferation of certain human tumour cells. An antibody which interferes with the dimerisation of the ErbB2 and the ErbB3 members of the EGF receptor family prevents the association of a most potent signaling module (pertuxumab). The signals emenating from this dimer determine many phenotypic properties of e.g. human breast cancer cells. A monoclonal antibody also directed against ErbB2 has been most successful, clinically and commercially (trastuzumab). This antibody interferes with signals generated by the receptor and causes the arrest of the cell cycle in tumour cells. In addition, it recruits immune effector cells as cytotoxic agents. Finally, monoclonal antibody derivatives, single chain Fv fragments, have been used as a basis for the construction of recombinant tumour toxins. These molecules harness the exquisite binding specificity of the antibodies and combine them with the toxic principles of bacteria.
Animals↗
The interaction of specific peptide aptamers with the DNA binding domain and the dimerization domain of the transcription factor Stat3 inhibits transactivation and induces apoptosis in tumor cells.
The transcription factor signal transducer and activator of transcription (Stat) 3 is activated through the interleukin-6 family of cytokines and by binding of growth factors to the epidermal growth factor (EGF) receptor. It plays an essential role in embryonic development and assumes specialized tasks in many differentiated tissues. Constitutively activated Stat3 has been found in tumor cell lines and primary tumors and plays a crucial role in tumor cell survival and proliferation. To inhibit the oncogenic action of Stat3 in tumor cells, we have selected short peptides, so-called peptide aptamers, which specifically interact with defined functional domains of this transcription factor. The peptide aptamers were selected from a peptide library of high complexity by an adaptation of the yeast two-hybrid procedure. Peptide aptamers specifically interacting with the Stat3 dimerization domain caused inhibition of DNA binding activity and suppression of transactivation by Stat3 in EGF-responsive cells. Similarly, a peptide aptamer selected for its ability to recognize the Stat3 DNA binding domain inhibited DNA binding and transactivation by Stat3 following EGF stimulation of cells. Peptide aptamers were expressed in bacteria as fusion proteins with a protein transduction domain and introduced into human myeloma cells. This resulted in dose-dependent growth inhibition, down-regulation of Bcl-x(L) expression, and induction of apoptosis. The inhibition of Stat3 functions through the interaction with peptide aptamers counteracts the transformed phenotype and could become useful in targeted tumor therapy.
Animals↗
The coactivator of transcription CREB-binding protein interacts preferentially with the glycosylated form of Stat5.
The signal transducer and activator of transcription (Stat) gene family comprises seven members with similarities in their domain structure and a common mode of activation. Members of this gene family mediate interferon induction of gene transcription and the response to a large number of growth factors and hormones. Extracellular ligand binding to transmembrane receptors causes the intracellular activation of associated tyrosine kinases, phosphorylation of Stat molecules, dimerization, and translocation to the nucleus. Prolactin-induced phosphorylation of Stat5 is a key event in the development and differentiation of mammary epithelial cells. In addition to the crucial phosphorylation at tyrosine 694, we have identified an O-linked N-acetylglucosamine (O-GlcNAc) as another secondary modification essential for the transcriptional induction by Stat5. This modification was only found on nuclear Stat5 after cytokine activation. Similar observations were made with Stat1, Stat3, and Stat6. Glycosylation of Stat5, however, does not seem to be a prerequisite for nuclear translocation. Mass spectrometric analysis revealed a glycosylated peptide in the N-terminal region of Stat5. Replacement of threonine 92 by an alanine residue (Stat5a-T92A) strongly reduced the prolactin induction of Stat5a glycosylation and abolished transactivation of a target gene promoter. Only the glycosylated form of Stat5 was able to bind the coactivator of transcription CBP, an essential interaction for Stat5-mediated gene transcription.
Acetylglucosamine↗
Bifunctional recombinant proteins in cancer therapy: cell penetrating peptide aptamers as inhibitors of growth factor signaling.
The major aim of molecular cancer research is the development of new therapeutic strategies and compounds that target directly the genetic and biochemical causes of malignant transformation. Therapeutic genes, antibodies and their derivatives, but also small molecular weight compounds, have been used for this purpose. Small peptides might be able to complement these agents because of their ability to recognize specific protein domains and thus to interfere with enzymatic functions or protein-protein interactions. A variation of the yeast-two-hybrid procedure allows to select specifically binding peptides, so called peptide aptamers, from a peptide library of high complexity. This selection procedure can be adapted to any protein or protein fragment as a bait construct and selects for the intracellular interaction between the bait of choice and the peptide aptamer prey. Peptide aptamers thus selected can be cloned, provided with a protein transduction domain, expressed in bacteria and introduced into cancer cells. There they might disrupt protein-protein interactions crucial for cancer cell growth or survival. We introduce an example in which the Stat3 arm of EGF receptor signaling is selectively inhibited by a peptide aptamer and causes the growth arrest of EGF receptor-dependent tumor cells. The aptamer constructs can be supplemented with additional functional domains to enhance their inhibitory effects.
Amino Acid Sequence↗
Comparative proteomic analysis of proliferating and functionally differentiated mammary epithelial cells.
Proliferation and differentiation of mammary epithelial cells are governed by hormonal stimuli, cell-cell, and cell-matrix interactions. Terminal differentiation of mammary epithelial cells depends upon the action of the lactogenic hormones, insulin, glucocorticoids, and prolactin that enable them to synthesize and secrete milk proteins. These differentiated cells are polarized and carry out vectorial transport of milk constituents across the apical plasma membrane. To gain additional insights into the mechanisms governing differentiation of mammary epithelial cells, we identified proteins whose expression distinguishes proliferating from differentiated mammary epithelial cells. For this purpose we made use of the HC11 mammary epithelial line, which is capable of differentiation in response to lactogenic hormones. Using two-dimensional gel electrophoresis and mass spectrometry, we found about 60 proteins whose expression levels changed in between these two differentiation states. Bioinformatic analysis revealed differential expression of cytoskeletal components, molecular chaperones and regulators of protein folding and stability, calcium-binding proteins, and components of RNA-processing pathways. The actin cytoskeleton is asymmetrically distributed in differentiated epithelial cells, and the identification of proteins involved in mRNA binding and localization suggests that asymmetry might in part be achieved by controlling cellular localization of mRNAs. The proteins identified provide insights into the differentiation of mammary epithelial cells and the regulation of this process.
Animals↗
Sequence-specific peptide aptamers, interacting with the intracellular domain of the epidermal growth factor receptor, interfere with Stat3 activation and inhibit the growth of tumor cells.
Receptor tyrosine kinases of the epidermal growth factor (EGF) receptor family regulate essential cellular functions such as proliferation, survival, migration, and differentiation but also play central roles in the etiology and progression of tumors. We have identified short peptide sequences from a random peptide library integrated into the thioredoxin scaffold protein, which specifically bind to the intracellular domain of the EGF receptor (EGFR). These molecules have the potential to selectively inhibit specific aspects of EGF receptor signaling and might become valuable as anticancer agents. Intracellular expression of the aptamer encoding gene construct KDI1 or introduction of bacterially expressed KDI1 via a protein transduction domain into EGFR-expressing cells results in KDI1.EGF receptor complex formation, a slower proliferation, and reduced soft agar colony formation. Aptamer KDI1 did not summarily block the EGF receptor tyrosine kinase activity but selectively interfered with the EGF-induced phosphorylation of the tyrosine residues 845, 1068, and 1148 as well as the phosphorylation of tyrosine 317 of p46 Shc. EGF-induced phosphorylation of Stat3 at tyrosine 705 and Stat3-dependent transactivation were also impaired. Transduction of a short synthetic peptide aptamer sequence not embedded into the scaffold protein resulted in the same impairment of EGF-induced Stat3 activation.
3T3 Cells↗
The histone deacetylase inhibitor valproic acid selectively induces proteasomal degradation of HDAC2.
Histone-modifying enzymes play essential roles in physiological and aberrant gene regulation. Since histone deacetylases (HDACs) are promising targets of cancer therapy, it is important to understand the mechanisms of HDAC regulation. Selective modulators of HDAC isoenzymes could serve as efficient and well-tolerated drugs. We show that HDAC2 undergoes basal turnover by the ubiquitin-proteasome pathway. Valproic acid (VPA), in addition to selectively inhibiting the catalytic activity of class I HDACs, induces proteasomal degradation of HDAC2, in contrast to other inhibitors such as trichostatin A (TSA). Basal and VPA-induced HDAC2 turnover critically depend on the E2 ubiquitin conjugase Ubc8 and the E3 ubiquitin ligase RLIM. Ubc8 gene expression is induced by both VPA and TSA, whereas only TSA simultaneously reduces RLIM protein levels and therefore fails to induce HDAC2 degradation. Thus, poly-ubiquitination and proteasomal degradation provide an isoenzyme-selective mechanism for downregulation of HDAC2.
Animals↗