Debajit K. Biswas

The nuclear factor kappa B (NF-κB): A potential therapeutic target for estrogen receptor negative breast cancers

The effect of a kinase inhibitor Go6796 on growth of epidermal growth factor (EGF)-stimulated estrogen receptor negative (ER−) breast cancer cells in vivo and role of nuclear factor kappa B (NF-κB) on tumorogenesis have been investigated. This was studied in an animal model by implanting ER− mouse mammary epithelial tumor cells (CSMLO) in syngeneic A-J mice. (i) Local administration of Go6976 an inhibitor of protein kinases C alpha and beta inhibited growth of tumors and caused extensive necrotic degeneration and regression of the tumors without causing any microscopically detectable damage to the vital organs liver and lung. (ii) Stable expression of dominant-negative mutants of the beta subunit (dnIkkβ) of the inhibitory kappa B (IκB) kinase (dnIkk) that selectively blocked activation of NF-κB caused loss of tumorigenic potential of CSMLO cells. Stable expression of dnIkkβ also blocked phorbol 12-myristate 13-acetate (PMA)-induced activation of NF-κB and overexpression of cyclin D1, concomitantly with the loss or reduced tumorigenic potential of these cells. Thus, results from in vivo and in vitro experiments strongly suggest the involvement of NF-κB in ER− mammary epithelial cell-mediated tumorigenesis. We propose that blocking NF-κB activation not only inhibits cell proliferation, but also antagonizes the antiapoptotic role of this transcription factor in ER− breast cancer cells. Thus, NF-κB is a potential target for therapy of EGFR family receptor-overexpressing ER− breast cancers.

Linkage between EGFR family receptors and nuclear factor kappaB (NF‐κB) signaling in breast cancer

In the United States there are 225,000 new cases of invasive breast cancer annually, and at least 50,000 women are diagnosed with ductal carcinoma in situ (DCIS). Breast cancer is a collection of disorders of mammary epithelial cells with distinct pathological characteristics and diverse clinical manifestations. Breast cancers are divided broadly into four classes by the level of the biomarkers HER2 (erbB2/neu) and the estrogen receptor (ER). Histologic grade is also an important modifier of breast cancer taxonomy and behavior. Broadly speaking, breast cancer can be divided into those that are HER2‐positive, containing cancers that are both ER‐positive and negative, cancers that are ER‐positive and divided into high‐grade and low‐grade tumors, and the remaining but important class of cancers that are both ER‐negative and HER2‐negative. These last cancers are called basal‐like and were first recognized as a distinct group by gene expression arrays. Nuclear factor kappaB (NF‐κB) is family of multifunctional transcription factors that when activated generate pleotrophic changes in target cells. Elevated levels of active NF‐κB are detected in many human diseases including breast cancers. High‐level active NF‐κB is detected in specific subclasses of breast cancers briefly described above, predominantly in ER‐negative and epidermal growth factor family receptor (EGFR) overexpressing breast cancers (predominantly HER2 amplified cancers). This article is focused on the role of NF‐κB activation initiated by the EGFR family receptors in subclasses of breast cancer. The combined influence of EGFR family receptors and NF‐κB signaling on the transformation of ER‐negative human mammary epithelial cell is illustrated. J. Cell. Physiol. 209: 645–652, 2006.

Crossroads of estrogen receptor and NF-kappaB signaling.

Cellular homeostasis in higher organisms is maintained by balancing cell growth, differentiation, and death. Two important systems that transmit extracellular signals into the machinery of the cell nucleus are the signaling pathways that activate nuclear factor κB (NF-κΒ) and estrogen receptor (ER). These two transcription factors induce expression of genes that control cell fates, including proliferation and cell death (apoptosis). However, ER has anti-inflammatory effects, whereas activated NF-κB initiates and maintains cellular inflammatory responses. Recent investigations elucidated a nonclassical and nongenomic effect of ER: inhibition of NF-κB activation and the inflammatory response. In breast cancer, antiestrogen therapy might cause reactivation of NF-κB, potentially rerouting a proliferative signal to breast cancer cells and contributing to hormone resistance. Thus, ER ligands that selectively block NF-κB activation could provide specific potential therapy for hormone-resistant ER-positive breast cancers.

Nuclear factor-κB activation: A molecular therapeutic target for estrogen receptor-negative and epidermal growth factor receptor family receptor-positive human breast cancer

Nuclear factor-κB (NF-κB), a transcription factor with pleotropic effects, is a downstream mediator of growth signaling in estrogen receptor (ER)-negative and erbB family particularly erbB2 (HER-2/neu) receptor–positive cancer. We previously reported activation of NF-κB in ER-negative breast cancer cells and breast tumor specimens, but the consequence of inhibiting NF-κB activation in this subclass of breast cancer has not been shown. In this study, we investigated the role of NF-κB activation by studying the tumorigenic potential of cells expressing genetically manipulated, inducible, dominant-negative inhibitory κB kinase (IKK) β in xenograft tumor model. Conditional inhibition of NF-κB activation by the inducible expression of dominant-negative IKKβ simultaneously blocked cell proliferation, reinstated apoptosis, and dramatically blocked xenograft tumor formation. Secondly, the humanized anti-erbB2 antibody trastuzumab (Herceptin) and the specific IKK inhibitor NF-κB essential modifier–binding domain peptide both blocked NF-κB activation and cell proliferation and reinstated apoptosis in two ER-negative and erbB2-positive human breast cancer cell lines that are used as representative model systems. Combinations of these two target-specific inhibitors synergistically blocked cell proliferation at concentrations that were singly ineffective. Inhibition of NF-κB activation with two other low molecular weight compounds, PS1145 and PS341, which inhibited IKK activity and proteasome-mediated phosphorylated inhibitory κB protein degradation, respectively, blocked erbB2-mediated cell growth and reversed antiapoptotic machinery. These results implicate NF-κB activation in the tumorigenesis and progression of ER-negative breast cancer. It is postulated that this transcription factor and its activation cascade offer therapeutic targets for erbB2-positive and ER-negative breast cancer. [Mol Cancer Ther 2007;6(7):1973–82]

Expression of cyclins E1 and E2 during mouse development and in neoplasia

Cyclin E1 (formerly called cyclin E) and the recently described cyclin E2 belong to the family of E-type cyclins that operate during the G1/S phase progression in mammalian cells. The two E-cyclins share a catalytic partner, cyclin-dependent kinase 2 (CDK2), and activate their associated kinase activities at similar times during cell cycle progression. Despite these similarities, it is unknown whether the two proteins perform distinct functions, or, alternatively, they control S-phase entry of different cell types in a tissue-specific fashion. To start addressing in vivo functions of E-cyclins, we determined the expression pattern of cyclins E1 and E2 during normal mouse development. We found that the two E-cyclins showed very similar patterns of expression; both were expressed within the proliferating compartment during embryo development. Analyses of cells and tissues lacking members of the retinoblastoma (pRB) family of proteins revealed that the expression of both cyclins is controlled in a pRB-dependent, but p107- and p130-independent fashion, likely through the pRB-dependent E2F transcription factors. We also found that cyclins E1 and E2 are expressed at high levels in mouse breast tumors driven by the Myc oncogene. Last, we found that cyclin E2 is overexpressed in ≈24% of analyzed human mammary carcinomas. Collectively these findings suggest that the expression of cyclins E1 and E2 is governed by similar molecular circuitry.

Restriction, de-restriction and mistranslation in missense suppression. Ribosomal discrimination of transfer RNA's

Abstract The strA ribosomal mutations known to restrict the level of translational ambiguity and the efficiency of nonsense transfer RNA-suppressors, are shown also to restrict the efficiency of missense tRNA-suppressors; the efficiency of wildtype tRNA is not noticeably affected by these strA mutations and the extent by which the suppressor tRNA is restricted is shown to be dependent upon some structural aspect of the suppressor-tRNA molecule other than either the anticodon or the amino acid-accepting specificity. Ribosomal alteration mutants (ram), known to reverse strA restriction of translational ambiguity, are shown to reverse also strA restriction of the efficiency of nonsense and missense tRNA-suppressors. Furthermore, introduction of the ram mutation into missense suppressor strains is shown greatly to increase the amount of mistranslation (a property peculiar to missense suppressors), while it has no significant effect in su− strains. The strA mutation is shown to reverse this ram effect on mistranslation. Presence of streptomycin, known to act on the ribosome, is shown to reverse the effect of strA on restriction and mistranslation in a manner similar and additive to that of the ram mutation. These observations suggest that: (a) the ribosome provides a recognition screen for tRNAs prior to, or simultaneous with, their interaction with messenger RNA; and (b) this postulated ribosomal screen discriminates normal from mutated tRNA.

Induction of prolactin synthesis in rat pituitary tumor cells by 5-bromodeoxyuridine

Abstract The clonal strain of pituitary tumor cells GH 1 2C 1 does not produce detectable amounts of prolactin ( 1 2C 1 cells were grown in the presence of 5-bromodeoxyuridine (BrdU, 3 μg/ml), the cells did produce prolactin as determined by quantitative microcomplement fixation and incorporation of 3 H-leucine into 3 H-prolactin. BGH 1 2C 1 and F 1 BGH 1 2C 1 , two BrdU-resistant (r) substrains derived from GH 1 2C 1 which grow in the presence of 30 μg/ml BrdU, also synthesized prolactin (100–500 ng/mg cell protein per 24 hr). Growth of BrdU r strains was not dependent upon on the presence of the drug in the medium; however, the continued production of prolactin by F 1 BGH 1 2C 1 cells was dependent upon the presence of BrdU. Growth hormone production in both BrdU s and BrdU r strains was not affected by BrdU. Resistance of F 1 BGH 1 2C 1 cells to BrdU was not due to a defect in BrdU uptake. Thymidine inhibited the incorporation of 3 H-BrdU into DNA in both sensitive and resistant strains, and also reduced BrdU-induced prolactin synthesis in F 1 BGH 1 2C 1 . We postulate that induction of prolactin synthesis by BrdU in GH 1 2C 1 and F 1 BGH 1 2C 1 cells is mediated by the incorporation of the drug into cellular DNA. Furthermore, the lack of measurable prolactin synthesis by the parent strain GH 1 2C 1 is not due to deletion of the gene for prolactin, but is probably the result of regulatory mechanisms which do not permit expression of this gene.

Classification of breast cancer cells on the basis of a functional assay for estrogen receptor

BackgroundThe receptor (ER) for estrogen (E2) is routinely assayed as a marker to determine the feasibility of anti-hormone therapy against breast cancer because ER-positive (ER+) tumors are much more likely to respond to anti-hormone therapy than are ER-negative (ER−). However 40% of ER+ breast cancer patients do not respond to anti-hormone therapy. We suggest that this unpredictability of therapeutic responses lies in the current ER assays, which measure only an initial component of the E2-responsive pathway, and that the difference depends upon altered downstream processes. We propose a functional criterion that subclassifies breast cancers on the basis of specific binding of ER to its cognate DNA sequence, the estrogen response element (ERE).Materials and MethodsER was identified in breast cancer cell lines by immunofluorescence assay, Western blot analysis, identification of ER-specific mRNA, and by interaction of the ER-ERE complex with three different ER-specific antibodies. ER-ERE complex formation was measured by electrophoretic mobility shift assay (EMSA). Transactivation of the E2-responsive gene was studied by transfection of cells with fusion gene construct with the promoter-containing ERE sequence and assay of reporter gene activity in the cell extracts.ResultsThe growth of ER+ T47D cells was sensitive to tamoxifen, ICI-182,780, and ethynyl estradiol (EE2), whereas another ER+ breast cancer cell line, 21PT, was resistant to these compounds. The estrogen receptor (ER) in the nuclear extracts of MCF-7 and T47D demonstrated hormone-dependent interaction with the response element (ERE) and also downstream transactivation of the E2-responsive PS2 promoter. But in the 21PT cell line that was designated as ER− on the basis of ligand-binding assay and was found to be ER+ by all the other ER assays, ER-ERE interaction and PS2 promoter transactivation were independent of hormone.ConclusionsOn the basis of the downstream functional assay of ER interaction with ERE, ER+ breast tumor cells can be subclassified into two categories. The first is E2-dependent (ERd+) and these cells should respond to anti-hormone therapy. The second type of ER interacts with ERE independent of E2 (ERi+) and constitutively transactivates responsive genes. It is predicted that the latter type of breast cancers will not respond to antihormone therapy.

NF-κB activation-induced anti-apoptosis renders HER2-positive cells drug resistant and accelerates tumor growth

Breast cancers with HER2 overexpression are sensitive to drugs targeting the receptor or its kinase activity. HER2-targeting drugs are initially effective against HER2-positive breast cancer, but resistance inevitably occurs. We previously found that NF-κB is hyperactivated in a subset of HER2-positive breast cancer cells and tissue specimens. In this study, we report that constitutively active NF-κB rendered HER2-positive cancer cells resistant to anti-HER2 drugs and cells selected for lapatinib resistance upregulated NF-κB. In both circumstances, cells were antiapoptotic and grew rapidly as xenografts. Lapatinib-resistant cells were refractory to HER2 and NF-κB inhibitors alone but were sensitive to their combination, suggesting a novel therapeutic strategy. A subset of NF-κB–responsive genes was overexpressed in HER2-positive and triple-negative breast cancers, and patients with this NF-κB signature had poor clinical outcome. Anti-HER2 drug resistance may be a consequence of NF-κB activation, and selection for resistance results in NF-κB activation, suggesting that this transcription factor is central to oncogenesis and drug resistance. Clinically, the combined targeting of HER2 and NF-κB suggests a potential treatment paradigm for patients who relapse after anti-HER2 therapy. Patients with these cancers may be treated by simultaneously suppressing HER2 signaling and NF-κB activation. Implications: The combination of an inhibitor of IκB kinase (IKK) inhibitor and anti-HER2 drugs may be a novel treatment strategy for drug-resistant human breast cancers. Mol Cancer Res; 12(3); 408–20. ©2013 AACR.

Increased level of prolactin gene sequences in bromodeoxyuridine treated GH cells

The 5-bromodeoxyuridine-resistant (BrdUrdr) derivative (F1BGH12C1) of prolactin nonproducing (PRL-) rat pituitary tumor cell-subclone GH12C1, synthesize prolactin (PRL) in the presence of the drug. Analysis of nuclear RNA isolated from BrdUrd treated F1BHG12C1 cells demonstrated several high molecular weight RNA PRL sequences, similar to those observed in the nuclear RNA fraction of PRL producing (PRL+) GH3 cells. No such RNAPRL sequences could be detected in nuclear RNA fraction of untreated F1 BGH12C1 cells. PRL sequences in the genome of GH3 (PRL+), GH12C1 (PRL-) and F1BGH12C1 (PRL-, BrdUrdr) GH cells could be identified by blot analysis in 4.8-5.2kb fragment of restriction endonuclease, Hind III digested DNA. Both PRL+ and PRL- cells seem to have approximately the same level of PRL gene sequences in total cell DNA. However Hind III digested DNA of BrdUrd treated F1BGH12C cells revealed the presence of significantly higher levels of PRL gene sequences, in comparison, to that observed in total DNA of untreated cells. The increased level of PRL gene sequences was dependent on the period of drug treatment and a parallel increase in the cytoplasmic RNAPRL sequences was also observed.