Correlation of hormone receptor status between circulating tumor cells, primary tumor, and metastasis in breast cancer patients.

PURPOSE: Estrogen receptor (ER) and progesterone receptor (PR) status is prognostic and predictive in breast cancer. Because metastatic breast tumor biopsies are not routinely feasible, circulating tumor cells (CTCs) offer an alternative source of determining ER/PR tumor status. METHODS/PATIENTS: Peripheral blood was collected prospectively from 36 patients with metastatic breast cancer. CTCs were isolated using the microfluidic OncoCEE™ platform. Detection was accomplished with an expanded anti-cytokeratin (CK) cocktail mixture and anti-CD45. ER/PR protein expression was assessed by immunocytochemistry (ICC) on the CK+ cells and compared to the primary and/or metastatic tumor by immunohistochemistry (IHC). RESULTS: Among the 24 CK + CTC cases, a concordance of 68 % (15/22) in ER/PR status between primary breast tumor and CTCs and 83 % (10/12) between metastatic tumor and CTCs was observed. An overall concordance of 79 % (19/24) was achieved when assessing CTC and metastatic tumor (primary tumor substituted if metastatic breast biopsy not available). A test sensitivity of 72 % and specificity of 100 % was identified when comparing CTCs to tumor tissue. Of the 7 discordant cases between CTCs and primary tumor tissue, 2 were concordant with the metastatic biopsy. CONCLUSIONS: CTC ER/PR status using the OncoCEE™ platform is feasible, with high concordance in ER/PR status between tumor tissue (IHC) and CTCs (ICC). The prognostic and predictive significance of CTC ER/PR protein expression needs further evaluation in larger trials.

An optimized system for studies of EPO-dependent murine pro-erythroblast development.

OBJECTIVE: Objectives were to develop new means to isolate useful numbers of primary progenitor cells and to quantitatively assay the stepwise maturation of erythroblasts. METHODS: Approaches involved dosing mice with thiamphenicol (TAP) to yield staged cohorts of pro-erythroid cells; optimizing conditions for their EPO-dependent in vitro growth and survival; developing assays for CFU-E maturation; analyzing stage-specific transcript expression; and expressing a heterologous, erythroid-specific tag (EE372) in transgenic mice. RESULTS: Per TAP-treated mouse, 3 x 10(7) highly EPO-responsive erythroid progenitor cells were generated that represented up to 30% of total splenocytes and showed strict dependence on EPO for survival, growth, and immediate response gene expression. In this developing cohort, a tightly programmed sequence of gene expression was observed, and maximal expression of c-kit, EPO receptor, and beta-globin transcripts occurred at 72, 96, and 120 hours post-TAP withdrawal, respectively. Also, the newly discovered erythroid-specific dual-specificity kinase, DYRK3, was revealed to be expressed at a late CFU-E stage. In vitro, these progenitor cells matured stepwise from high FALS Ter119- cells (24-hour culture) to high FALS Ter119+ cells (24-36 hours) to low FALS Ter119+ maturing erythroblasts (40-48 hours) and sharp differences in their morphologies were observed. Finally, a MACS-based procedure for the purification of erythroid progenitor cells from TAP-treated EE372 transgenic mice also was developed. CONCLUSIONS: A comprehensive new system for isolating large numbers of primary murine erythroid progenitor cells and quantitatively monitoring their development is established that should serve well in investigations of endogenous and pharmacological regulators of red blood cell development.

Integrative signaling by minimal erythropoietin receptor forms and c-Kit.

Erythroid homeostasis depends critically upon erythropoietin (Epo) and stem cell factor cosignaling in late progenitor cells. Epo bioresponses are relayed efficiently by minimal receptor forms that retain a single Tyr-343 site for STAT5 binding, while forms that lack all cytoplasmic Tyr(P) sites activate JAK2 and the transcription of c-Myc plus presumed additional target genes. In FDCER cell lines, which express endogenous c-Kit, the signaling capacities of such minimal Epo receptor forms (ER-HY343 and ER-HY343F) have been dissected to reveal: 1) that Epo-dependent mitogenesis, survival, and bcl-x gene expression via ER-HY343 depend upon the intactness of the Tyr-343 STAT5 binding site; 2) that ER-HY343-dependent bcl-x(L) gene transcription is enhanced markedly via c-Kit; 3) that socs-3, plfap, dpp-1, and cacy-bp gene transcription is induced via ER-HY343, whereas dpp-1 and cacy-bp gene expression is also supported by ER-HY343F; 4) that ectopically expressed SOCS-3 suppresses proliferative signaling by not only ER-HY343 but also c-Kit; and 5) that in FDCER and primary erythroid cells, c-Kit appears to provide the primary route to MAPK activation. Thus, integration circuits exist in only select downstream pathways within Epo and stem call factor receptor signaling.

Pim-1 kinase protects hematopoietic FDC cells from genotoxin-induced death.

The hematopoietic cell S/T kinase Pim-1 was originally discovered as a target of murine leukemia provirus integration, and when expressed at increased levels is predisposing to lymphomagenesis. Recently, Pim-1 has been shown to enhance the activities of p100, c-Myb and cdc25a, and in part this might explain reported effects on mitogenesis. In the context of cytokine withdrawal, Pim-1 also can attenuate programmed cell death (PCD). Cytokine withdrawal, however, alters signaling pathways and can complicate the dissection of mitogenic vs apoptotic responses. To better study possible effects of Pim-1 on PCD, a hematopoietic cell model was developed in which proliferation was supported efficiently by SCF plus EPO in the absence of endogenous Pim-1 gene expression. This was provided by factor-dependent FDCW2 cells that express endogenous and functional c-Kit, and were transfected stably with truncated Epo receptor form mutated at a Y343 STAT5 binding site. In proliferating cells, exogenously expressed Pim-1 was observed to efficiently inhibit PCD as induced by either Co60 or adriamycin, and the dose-dependent nature of this effect was established in several independent clones. By comparison, effects of exogenous Pim-1 on mitogenesis were nominal. In addition, in cell fractionation studies an estimated 25% of Mr 34000 Pim-1 (but not Mr 44000 Pim-1) was present in nuclear extracts. Thus, Pim-1 efficiently buffers hematopoietic progenitor cells against death as induced by several clinically important apoptotic agents, and may directly target nuclear effectors.

Signal transduction in the erythropoietin receptor system.

Events relayed via the single transmembrane receptor for erythropoietin (Epo) are essential for the development of committed erythroid progenitor cells beyond the colony-forming unit-erythroid stage, and this clearly involves Epo's inhibition of programmed cell death (PCD). Less well resolved, however, are issues regarding the precise nature of Epo-dependent antiapoptotic mechanisms, the extent to which Epo might also promote mitogenesis and/or terminal erythroid differentiation, and the essential vs modulatory nature of certain Epo receptor cytoplasmic subdomains, signal transducing factors, and downstream pathways. Accordingly, this review focuses on the following aspects of Epo signal transduction: (1) Epo receptor/Jak2 activation mechanisms; (2) the critical vs dispensable nature of (P)Y sites and SH2 domain-encoding effectors in survival, growth, and differentiation responses; (3) primary mechanisms by which Epo inhibits PCD; (4) the integration of signals relayed by coexpressed and possibly directly interacting cytokine receptors; and (5) predictions regarding effector function which are provided by the association of certain primary and familial polycythemias with mutated human Epo receptor forms.

Extracellular signal-regulated kinase (ERK) interacts with signal transducer and activator of transcription (STAT) 5a.

Serine phosphorylation of signal transducers and activators of transcription (STAT) 1 and 3 modulates their DNA-binding capacity and/or transcriptional activity. Earlier we suggested that STAT5a functional capacity could be influenced by the mitogen-activated protein kinase (MAPK) pathway. In the present study, we have analyzed the interactions between STAT5a and the MAPKs, extracellular signal-regulated kinases ERK1 and ERK2. GH treatment of Chinese hamster ovary cells stably transfected with the GH receptor (CHOA cells) led to rapid and transient activation of both STAT5a and ERK1 and ERK2. Pretreatment of cells with colchicine, which inhibits tubulin polymerization, did not inhibit STAT5a translocation to the nucleus and ERK1/2 activation. In vitro precipitation with a glutathione-S-transferase-fusion protein containing the C-terminal transactivation domain of STAT5a showed GH-regulated association of ERK1/2 with the fusion protein, while this was not seen when serine 780 in STAT5a was changed to alanine. In vitro phosphorylation of the glutathione-S-transferase-fusion proteins using active ERK only worked when the fusion protein contained wild-type STAT5a sequence. The same experiment, performed with full-length wild-type STAT5a and the corresponding S780A mutant, showed that serine 780 is the only substrate in full-length STAT5a for active ERK. In coimmunoprecipitation experiments, larger amounts of STAT5a-ERK1/2 complexes were detected in cytosol from untreated CHOA cells than in cytosol from GH-treated cells, suggesting the presence of preformed STAT5a-ERK1/2 complexes in unstimulated cells. Transfection experiments with COS cells showed that kinase-inactive ERK1 decreased GH stimulation of STAT5-regulated reporter gene expression. These observations show, for the first time, direct physical interaction between ERK and STAT5a and also clearly identify serine 780 as a target for ERK. Furthermore, it is also established that serine phosphorylation of STAT5a transactivation domain, via the MAPK pathway, is a means of modifying GH-induced transcriptional activation.

Growth hormone promotion of tubulin polymerization stabilizes the microtubule network and protects against colchicine-induced apoptosis.

We have investigated the effect of GH on microtubular physiology in Chinese hamster ovary (CHO) cells stably transfected with the complementary DNA for the rat GH receptor (CHO-GHR(1-638)). We show here that after 30 min of human GH (hGH) treatment of CHO-GHR(1-638) cells, there was a significant increase in the level of polymerization of all four tubulin isoforms (alpha-, beta-, gamma-, and tyrosinated alpha-tubulin) compared with the serum-deprived state. However, this transient increase in the levels of polymerized tubulin after hGH treatment was particularly pronounced for beta- and tyr alpha-tubulin. For alpha- and gamma-tubulin, the hGH-induced increase in polymerization state lasted to approximately 3 h and then declined by 7 h, whereas for beta- and tyr alpha-tubulin there was a decrease in the polymerization state at 1-2 h after hGH treatment compared with the level at 30 min (but still greater than the serum-deprived state) followed by a second but lesser wave of increased polymerization lasting to 7 h. The changes in the polymerization state of the tubulins were not accompanied by comparative changes in the level of total cellular tubulin. The proline rich box 1 region of the GH receptor was required for hGH to stimulate tubulin polymerization indicative that this event is JAK dependent. Increased tubulin polymerization still occurred in response to hGH in a receptor truncation lacking the carboxyl terminal half of the intracellular domain of the GH receptor indicative that hGH induced changes in intracellular calcium concentration is not required for tubulin polymerization. Prior treatment of CHO-GHR(1-638) cells with hGH retarded colchicine induced microtubule depolymerization and also prevented colchicine induced apoptotic cell death. The integrity of the microtubule network was not required for GH-induced STAT5 mediated transcription as treatment of cells with colchicine, vincristine, or vinblastine did not alter the fold stimulation of the STAT5 mediated transcriptional response to GH. Thus one consequence of cellular treatment with GH is alteration in microtubule physiology.

In vitro interaction between STAT 5 and JAK 2; dependence upon phosphorylation status of STAT 5 and JAK 2.

A working model for haematopoietic cytokine signal transduction has been hypothesised as follows. Binding of cytokines to specific receptor molecules leads to phosphorylation and activation of receptor associated members of the Janus kinase family. This is followed by tyrosine phosphorylation of the associated receptor and members of the STAT (signal transducer and activator of transcription) family of DNA-binding transcription factors. Phosphorylation is accompanied by STAT dimerisation, nuclear transport and activation of gene transcription. Activation of gene transcription is mediated by the binding of STAT dimers to palindromic STAT response elements. A number of areas of confusion remain; not least the mechanism by which multiple cytokines signal via a limited number of STATs. A role has been suggested for phosphorylated receptor tyrosine residues as STAT docking sites on activated receptor-JAK complexes. According to this model the amino acid sequence context of key tyrosine residues confers receptor specificity upon STAT activation. There is some controversy as to whether this model applies to STAT 5. The heterologous expression of STAT 5 in Sf 9 insect cells using the baculovirus expression system is described here. Protein of the correct molecular weight was expressed and found to be phosphorylated on tyrosine residues and to bind to a STAT response DNA element. This binding was dependent upon the phosphorylation status of the STAT protein. DNA binding could be abolished in vitro by treatment with a phosphotyrosine phosphatase and restored in vitro by treatment with activated recombinant JAK 2. The protein was purified to near homogeneity using a simple ion exchange/gel filtration chromatography procedure. The interaction between purified recombinant STAT 5 and JAK 2, either expressed by baculovirus or endogenously expressed in Buffalo rat liver cells, was studied. In both cases STAT 5 in its non-phosphorylated form was found to form a stable complex with activated JAK 2. Non-activated JAK 2 and phosphorylated STAT 5 were unable to participate in complex formation. The results presented provide a mechanistic basis for the activation of STAT 5 by a wide range of cytokines capable of activating JAK 2.

Mitogen-activated protein kinase kinase inhibition decreases growth hormone stimulated transcription mediated by STAT5.

We have investigated the possible involvement of the MAPK pathway in the growth hormone(GH)-induced activation of one of the members of signal transducers and activators of transcription, STAT5, by using the MAPK kinase (MEK) inhibitor PD98059. PD98059 treatment of Chinese hamster ovarian cells, stably transfected with the GH receptor (CHOA cells), abolished the GH-induced MAPK activity. PD98059 decreased the amount of GH-induced STAT5 in nuclear extract with DNA-binding capacity. Furthermore, GH dependent transcription of a STAT5 regulated reporter gene was inhibited by PD98059. The MEK inhibitor did not reduce GH-stimulated nuclear translocation of STAT5. We also investigated if PD98059 differentially influences the activation of the two STAT5 homologs, STAT5a and STAT5b, which differ mainly at the C-terminal end, one of the differences being the presence of a possible MAPK phosphorylation site in STAT5a. Expression plasmids for these transcription factors were transfected into CHOA cells together with a reporter gene. GH-stimulated fold induction of transcription was reduced by PD98059 in STAT5a but not in STAT5b overexpressing cells. A MAPK phosphorylation site-mutated version of STAT5a was also transfected into CHOA cells. GH-stimulated fold induction of cotransfected reporter gene was not reduced by PD98059 in cells overexpressing mutant STAT5a. The above data show that the MAPK pathway is required for the full activation of one of the STAT5 isoforms (STAT5a).

Growth hormone-induced reorganization of the actin cytoskeleton is not required for STAT5 (signal transducer and activator of transcription-5)-mediated transcription.

We have investigated the effect of GH on the organization of the actin cytoskeleton within the cell. Human GH (hGH) treatment (50 nM) of Chinese hamster ovary (CHO) cells stably transfected with the complementary DNA for the rat GH receptor (CHO-GHR(1-638)) resulted in a reorganization of actin filaments in the cell that was not observed upon GH treatment of the untransfected parental CHO cell line. hGH initially induced depolymerization of actin stress fibers similar in magnitude to that induced by treatment of the cells with 100 nM human insulin-like growth factor I. This loss of stress fibers was observed as early as 30 sec after addition of hGH to the medium, and maximal depolymerization of stress fibers was observed between 1-4 min after addition of hGH. This was followed by a slow, but submaximal, repolymerization of the stress fibers and the formation of localized focal filamentous actin containing complexes. Similar cytoskeletal changes were observed after hGH treatment in Swiss 3T3 fibroblasts and BRL cells stably transfected with rat GH receptor complementary DNA (BRL-GHR(1-6381)). Pretreatment of CHO-GHR(1-638) cells with wortmannin (a phosphatidylinositol 3-kinase inhibitor) and verapamil (a calcium channel antagonist) both inhibited the hGH-induced actin reorganization. The integrity of the actin cytoskeleton was not required for GH-induced STAT5 (signal transducer and activator of transcription-5)-mediated transcription, as treatment of cells with cytochalasins B and D did not alter the fold stimulation of the STAT5-mediated transcriptional response to GH. We conclude that GH induces a rapid reorganization of the actin cytoskeleton by a process requiring phosphatidylinositol 3-kinase activation and calcium influx, but this cytoskeletal reorganization is not required for the STAT5-mediated transcriptional response to GH.

Mediation of growth hormone-dependent transcriptional activation by mammary gland factor/Stat 5.

Previous observations have shown that binding of growth hormone to its receptor leads to activation of transcription factors via a mechanism involving phosphorylation on tyrosine residues. In order to establish whether the prolactin-activated transcription factor Stat 5 (mammary gland factor) is also activated by growth hormone, nuclear extracts were prepared from COS-7 cells transiently expressing transfected Stat 5 and growth hormone receptor cDNA. Gel electrophoresis mobility shift analyses revealed the growth hormone-dependent presence of specific DNA-binding proteins in these extracts. The complexes formed could be supershifted by polyclonal anti-Stat 5 antiserum. In other experiments nuclear extracts from growth hormone-treated Chinese hamster ovary cells stably expressing transfected growth hormone receptor cDNA and liver from growth hormone-treated hypophysectomized rats were used for gel electrophoresis mobility shift analyses. These also revealed the presence of specific DNA-binding proteins sharing antigenic determinants with Stat 5. Stat 5 cDNA was shown to be capable of complementing the growth hormone-dependent activation of transcription of a reporter gene in the otherwise unresponsive COS-7 cell line. This complementation was dependent on the presence of Stat 5 tyrosine 694, suggesting a role for phosphorylation of this residue in growth hormone-dependent activation of DNA-binding and transcription.