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Nuclear export of intron-containing human immunodeficiency virus type 1 RNA is mediated by the viral Rev protein. Rev is a nucleocytoplasmic transport protein that directly binds to its cis-acting Rev-responsive element RNA. Rev function depends on its ability to multimerize. The in vivo dynamics and the subcellular dependence of this process are still largely unexplored. To visualize and quantitatively analyze the mechanism of Rev multimeric assembly in live cells, we used high resolution in vivo fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching. By using two different dynamic FRET approaches (acceptor photobleaching and donor bleaching time measurements), we observed a strong Rev-Rev interaction in the nucleoli of living cells. Most interestingly, we could also detect Rev multimerization in the cytoplasm; however, FRET efficiency in the cytoplasm was significantly lower than in the nucleolus. By using fluorescence recovery after photobleaching, we investigated the mobility of Rev within the nucleolus. Mathematical modeling of the fluorescence recovery after photobleaching recoveries enabled us to extract relative association and dissociation constants and the diffusion coefficient of Rev in the nucleolus. Our results show that Rev multimerizes in the nucleolus of living cells, suggesting an important role of the nucleolus in nucleocytoplasmic transport. ispartof: Journal of Biological Chemistry vol:279 issue:48 pages:50167-50175 ispartof: location:United States status: published

The antimicrobial and proinflammatory neutrophil granule protein cathepsin G (CaG) has been reported as a chemoattractant for human phagocytic leukocytes by using a putative G protein coupled receptor. In an effort to identify potential CaG receptor(s), we found that CaG-induced phagocyte migration was specifically attenuated by the bacterial chemotactic peptide fMLP, suggesting these two chemoattractants might share a receptor. In fact, CaG chemoattracts rat basophilic leukemia cells (RBL cells) expressing the high affinity human fMLP receptor FPR, but not parental RBL cells or cells transfected with other chemoattractant receptors. In addition, a specific FPR Ab and a defined FPR antagonist, cyclosporin H, abolished the chemotactic response of phagocytes and FPR-transfected cells to CaG. Furthermore, CaG down-regulated the cell surface expression of FPR in association with receptor internalization. Unlike fMLP, CaG did not induce potent Ca2+ flux and was a relatively weaker activator of MAPKs through FPR. Yet CaG activated an atypical protein kinase C isozyme, protein kinase Cζ, which was essential for FPR to mediate the chemotactic activity of CaG. Thus, our studies identify CaG as a novel, host-derived chemotactic agonist for FPR and expand the functional scope of this receptor in inflammatory and immune responses.

The triggering receptors expressed on myeloid cells (TREMs) have drawn considerable attention due to their ability to activate multiple cell types within the innate immune system, including neutrophils, monocyte/macrophages, and dendritic cells, via their association with DAP12. TLT-1 (TREM-like transcript-1) lies within the TREM gene cluster and contains the characteristic single V-set immunoglobulin (Ig) domain of the family, but its longer cytoplasmic tail is composed of both a proline-rich region and an immune receptor tyrosine-based inhibitory motif, the latter known to be used for interactions with protein tyrosine phosphatases. Here we report that TLT-1 is expressed exclusively in platelets and megakaryocytes (MKs) and that TLT-1 expression is up-regulated dramatically upon platelet activation. Consistent with this observation, confocal microscopy demonstrates that TLT-1 is prepackaged, along with CD62P, into both MK and platelet α-granules. Differences in thrombin-induced redistribution of CD62P and TLT-1 indicate that TLT-1 is not simply cargo of α-granules but may instead regulate granule construction or dispersal. Together these data show that that TLT-1 does not function to inhibit members of the TREM family but instead may play a role in maintaining vascular hemostasis and regulating coagulation and inflammation at sites of injury.

Murine natural killer cells selectively express members of the Ly49 family of class I MHC receptors; however, the molecular mechanism controlling probabilistic expression of Ly49 proteins has not been defined. A pair of overlapping, divergent promoters discovered in the Ly49g gene functions as a molecular switch that can produce a forward transcript containing the coding region of the gene (on position) or a noncoding transcript in the opposite direction (off position), and this element maintains transcription in the chosen direction. Competition of C/EBP and TBP transcription factors for overlapping binding sites determines the relative strength of the competing promoters and the probability of transcription in a given direction. Similar elements precede all Ly49 family members, and the relative strength of the forward promoter in each inhibitory Ly49 gene correlates with the percentage of natural killer cells that express a given receptor, supporting a promoter competition model of selective gene activation.

A mathematical model was developed to predict the bi- directional transport rate of fluorescent proteins across the nuclear membrane during a fluorescence recovery after photobleaching (FRAP) experiment. The model assumes that the total amount of fluorescent protein remains the same in the cell (i.e. no production, loss or exchange with the outside of the cell) and that the cell is in a state of equilibrium; i.e. proteins are leaving and entering the nucleus at an equal rate. The latter assumption has the advantage of not needing to take into account the method of protein transport (e.g. active or passive). The model includes correction for the photobleaching that happens during image acquisition following the deliberate photobleach. In this study, the green fluorescent protein (GFP) was transfected into cells in order to study its free behavior. In the FRAP experiments, either the entire nucleus and part of the cytoplasm or only part of the cytoplasm was photobleached followed by time-series imaging of the fluorescence redistribution. The model was fitted to the curves of intensity loss or recovery after photobleaching using numerical, non-linear methods. In addition, the mobile fractions of free GFP in the cytoplasm and the nucleus could be determined.

OBJECTIVE: The nitric oxide synthase inhibitor L-N(G)-methylarginine hydrochloride (L-NMMA HC1 546C88) causes reductions in cardiac output (CO), a potential limitation to clinical application. This drop in CO exceeds that from phenylephrine at matched systemic arterial pressure. We tested the hypothesis that the greater fall in CO attributable to L-NMMA primarily reflects a difference in venoconstriction between agents, such that phenylephrine produces larger increases in preload (an independent determinant of CO). DESIGN: Random infusion of phenylephrine or L-NMMA. SETTING: An animal research laboratory. SUBJECTS: Eight healthy, conscious, male dogs. INTERVENTIONS: L-N(G)-methylarginine hydrochloride (20 mg/kg for 1 hr) and phenylephrine (0.5 to 3 microg/kg/min) were administered into eight dogs chronically instrumented to measure left ventricular pressure and dimension. Data were measured at a constant heart rate (140 beats/min) to render CO proportional to stroke dimension. MEASUREMENTS AND MAIN RESULTS: At a matched increase in afterload (effective arterial elastance), L-NMMA increased preload (end-diastolic dimension) to a lesser degree (3.8%+/-1.5%, p < .05) than phenylephrine (9.6%+/-1.6%, p < .05 vs. L-NMMA). Neither L-NMMA nor phenylephrine affected the slope of the end-systolic pressure dimension relationship, although L-NMMA shifted the relationship rightward (1.7+/-0.7 mm, p < .05), consistent with a mild negative inotropic effect. L-NMMA decreased the stroke dimension to a greater extent than phenylephrine (-24.1%+/-6.8% and -10.6%+/-3.4%, respectively, p < .05). CONCLUSIONS: Differential CO responses to phenylephrine and L-NMMA were primarily attributable to changes in preload. Variable venular vs. arteriolar constrictor effects must be considered when evaluating the integrated cardiovascular response to a vasoactive agent. (Less)

Background: The role of cleaved caspase-3 as an integral player in the apoptotic process of mammalian cells has prompted the investigation of an assay to detect cleaved caspase-3 in circulating tumor cells (CTCs) as one way to assess CTC viability. Methods: Using a previously developed non-enrichment based assay, nucleated blood cells and CTCs are adhered to a single slide. An antibody for cleaved caspase-3 was incorporated into the assay in addition to the existing antibodies for cytokeratin (to identify tumor cells) and CD45 (to identify white blood cells), as well as a nuclear stain (DAPI). The modified assay was initially tested on cultured MCF 10a cells spiked into normal donor blood. Before spiking, apoptosis was induced in these cells using staurosporine. Following validation of the assay in cell lines, the assay was performed using blood samples from 9 different breast cancer patients, as well as 3 different normal donor samples. Results: CTCs were detected in the patient samples as single cells, as well as clusters. The results of the assay showed that only 1 out of 896 detected CTCs was positive for cleaved caspase-3 (0.11%). Of these CTCs, 134 of them were found to be in clusters, and all 134 were negative for cleaved caspase-3, indicating that whether in clusters or as single cells, the vast majority of CTC's detected with this assay are not undergoing irreversible apoptotic processes via cleaved caspase-3. The results also showed that 0.40% of white blood cells in healthy donors expressed cleaved caspase-3, compared to 0.51% of the white cells in cancer patients, indicating that healthy donors and cancer patients express cleaved caspase-3 in their white blood cells at nearly the same rate. Conclusions: The morphologic characterization used in this platform to select healthy-appearing cells identifies a population of circulating tumor cells with virtually no evidence of caspase-related ongoing apoptosis, a finding that supports the morphologic impression of viability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3619. doi:1538-7445.AM2012-3619

Background: We have established a fluid phase biopsy approach that identifies CTCs which preserves cytologic features in high-definition (HD) for diagnostic pathology without using immune or surface receptor-based enrichment. HD-CTCs identified with this approach can be used for enumeration and molecular characterization. Methods: Blood was collected from metastatic prostate cancer patients and normal donors in Cyto-Chex® tubes (Streck, Omaha, NE) as part of IRB approved protocols at each site. Following erythrocyte lysis, 3 million nucleated cells were deposited on a glass slide. Samples were incubated with a pan-cytokeratin (CK), CD45, and androgen receptor (AR) antibodies and counter-stained with DAPI. LNCaP cells were spiked into normal blood. Images were obtained with a fluorescent scanning microscope and analyzed with a computer algorithm. Candidate HD-CTCs were subsequently verified by expert readers. Slides were re-imaged for quantitative analysis using at a fixed exposure and gain. Results: A total of 227 CTCs from ten patients were compared to 20 LNCaP cells. The median (range) HD-CTCs in this cohort was: 9 (1-62) cells/ml. The mean ± standard deviation measurements in HD-CTCs were observed: CK intensity 60.4±154; total cell area 89.0 ± 53.8 μm2; nuclear area 61.1 ± 36.0 μm2. LNCaP cells spiked into normal blood gave the following values: CK intensity 1166+/−306; total cell area 143 ± 48.1 μm2; nuclear area 63.1 ± 18.6 μm2. CTCs were additionally classified as either AR positive (AR+) or AR negative (AR-). 37 of the 227 (16.3%) HD-CTCs were AR+. The average CK intensity was significantly higher in AR+ versus AR- cells at 174.23 and 39.86, respectively (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3630. doi:1538-7445.AM2012-3630

194 Background: We have established a fluid phase biopsy approach that identifies CTCs which preserves cytologic features in high-definition (HD) for diagnostic pathology without using immune or surface receptor-based enrichment. HD-CTCs identified with this approach can be used for enumeration and molecular characterization. Methods: Blood was collected from metastatic prostate cancer patients and normal donors in Cyto-Chex tubes (Streck, Omaha, NE) as part of IRB approved protocols at each site. Following erythrocyte lysis, 3 million nucleated cells were deposited on a glass slide. Samples were incubated with a pan-cytokeratin (CK), CD45, and androgen receptor (AR) antibodies and counter-stained with DAPI. LNCaP cells were spiked into normal blood. Images were obtained with a fluorescent scanning microscope and analyzed with a computer algorithm. Candidate HD-CTCs were subsequently verified by expert readers. Slides were re-imaged for quantitative analysis using at a fixed exposure and gain. Results: A total of 227 CTCs from ten patients were compared to 20 LNCaP cells. The median (range) HD-CTCs in this cohort was: 9 (1-62) cells/ml. The mean ± standard deviation measurements in HD-CTCs were observed: CK intensity 60.4±154; total cell area 89.0 ± 53.8 µm2; nuclear area 61.1 ± 36.0 µm2. LNCaP cells spiked into normal blood gave the following values: CK intensity 1166+/−306; total cell area 143 ± 48.1 µm2; nuclear area 63.1 ± 18.6 µm2. CTCs were additionally classified as either AR positive (AR+) or AR negative (AR−). 37 of the 227 (16.3%) HD-CTCs were AR+. The average CK intensity was significantly higher in AR+ versus AR− cells at 174.23 and 39.86, respectively (p Conclusions: We find a positive association between AR and CK expression on a per cell basis. Further, we find AR is expressed at comparable levels in CTCs from patients and human prostate cancer cells in culture. The HD-CTC based approach may be used for enumeration and molecular interrogation of CTCs in patients with prostate cancer.

Circulating tumor cells (CTCs) have been implicated as a population of cells that may seed metastasis and venous thromboembolism (VTE), two major causes of mortality in cancer patients. Thus far, existing CTC detection technologies have been unable to reproducibly detect CTC aggregates in order to address what contribution CTC aggregates may have on metastasis or VTE. We report here an enrichment-free immunofluorescence detection method that can reproducibly detect and enumerate homotypic CTC aggregates in patient samples. We identified CTC aggregates in 43% of 86 patient samples. The fraction of CTC aggregation was investigated in blood draws from 24 breast, 14 non-small cell lung (NSCLC), 18 pancreatic, 15 prostate stage IV cancer patients, and 15 normal blood donors (NBD). Both single CTCs and CTC aggregates were measured to determine whether differences exist in the physical characteristics of these two populations. Cells contained in CTC aggregates had less area and length, on average, than single CTCs. Nuclear to cytoplasmic (N/C) ratio between single CTCs and CTC aggregates were similar. This detection method may assist future studies in determining which population of cells is more physically likely to contribute to metastasis and VTE.

Circulating tumor cell (CTC) counts are an established prognostic marker in metastatic prostate, breast and colorectal cancer, and recent data suggest a similar role in late stage non-small cell lung cancer (NSCLC). However, due to sensitivity constraints in current enrichment-based CTC detection technologies, there are few published data about CTC prevalence rates and morphologic heterogeneity in early-stage NSCLC, or the correlation of CTCs with disease progression and their usability for clinical staging. We investigated CTC counts, morphology and aggregation in early stage, locally advanced and metastatic NSCLC patients by using a fluid-phase biopsy approach that identifies CTCs without relying on surface-receptor-based enrichment and presents them in sufficiently high definition (HD) to satisfy diagnostic pathology image quality requirements. HD-CTCs were analyzed in blood samples from 78 chemotherapy-naive NSCLC patients. 73% of the total population had a positive HD-CTC count (>0 CTC in 1 mL of blood) with a median of 4.4 HD-CTCs mL⁻¹ (range 0-515.6) and a mean of 44.7 (±95.2) HD-CTCs mL⁻¹. No significant difference in the medians of HD-CTC counts was detected between stage IV (n = 31, range 0-178.2), stage III (n = 34, range 0-515.6) and stages I/II (n = 13, range 0-442.3). Furthermore, HD-CTCs exhibited a uniformity in terms of molecular and physical characteristics such as fluorescent cytokeratin intensity, nuclear size, frequency of apoptosis and aggregate formation across the spectrum of staging. Our results demonstrate that despite stringent morphologic inclusion criteria for the definition of HD-CTCs, the HD-CTC assay shows high sensitivity in the detection and characterization of both early- and late-stage lung cancer CTCs. Extensive studies are warranted to investigate the prognostic value of CTC profiling in early-stage lung cancer. This finding has implications for the design of extensive studies examining screening, therapy and surveillance in lung cancer patients.

Circulating tumor cell (CTC) counts are an established prognostic marker in metastatic prostate, breast, and colorectal cancer. However, there is little knowledge about CTC incidence rates and morphologic heterogeneity in non-small cell lung cancer (NSCLC) and their correlation to disease progression and usability for clinical staging. Here, we present a fluid phase biopsy approach that identifies CTCs without surface receptor-based enrichment, and presents them in sufficiently high definition (HD) to satisfy diagnostic pathology image quality requirements. The HD-CTC assay will be used to monitor HD-CTC counts and morphology in early stage, locally advanced, and metastatic NSCLC patient over time. 150 newly diagnosed NSCLC patients are being enrolled for this project. At the time of diagnosis 50 of the patients will have early stage cancers (stage I-II); 50 patients will have locally advanced (stage III) cancers, and 50 will be patients presenting with metastatic (stage IV) disease. Blood samples from each patient will be obtained at the time of diagnosis and 6 months later. We present here an interim data set that shows an incidence rate of 78% at baseline with a median CTC count of 19.5/ml of blood (range 0-441.6). Our preliminary data demonstrates that, despite stringent morphologic inclusion criteria for the definition of HD-CTCs, our HD-CTC assay shows high sensitivity in the detection of lung cancer CTCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4151. doi:10.1158/1538-7445.AM2011-4151

Circulating tumor cells (CTCs) have been implicated as a potential population of cells that may seed metastasis in cancer patients. In order to characterize and understand this population of cells, an enrichment-free immunofluorescence detection method was developed by our lab. This method take whole patient blood and classifies cells based on cytokeratin (CTCs) and CD45 (WBCs) antibody staining. Using this CTC detection method, a population of cells residing in tumor cell aggregates was observed in blood draws from Stage IV breast cancer patients. It was previously observed in ex vivo mouse experiments that homotypic aggregate formation of tumor cells may increase metastatic efficiency (Glinsky, et al., Cancer Research, 2003). In this work, we seek to determine if a significant proportion of CTCs reside in homotypic aggregates in patient blood by enumerating CTC aggregates from the blood draws of 23 Stage IV breast cancer patients and 16 healthy donor patients. Preliminary analysis measuring associations of CTCs with other nucleated cells found in patient blood draws revealed that ∼43% of cytokeratin-positive (CK+) cells found in breast cancer patient samples localize with either other CK+ or white blood cells (WBCs) versus 14% in healthy donor patient samples. Furthermore, ∼19% of WBCs were found to be associated with either CK+ or other WBCs in breast cancer patient samples versus ∼7% in healthy donor patient samples. Pathological review confirming CK+ cells as CTCs revealed that of the CTCs detected in patient blood after pathological review, ∼2% of CTCs were found as homotypic aggregates versus ∼0.07% in healthy donor samples (p=0.0005). Four breast cancer patients were found to have no CTC aggregates. Two breast cancer patients had significantly higher than average CTC aggregate percentages (∼6% and ∼7%). Patients will continue to be monitored to measure any differences in disease outcome of patients with high incidence of CTC aggregates versus patients with low incidence of CTC aggregates and healthy donors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5225. doi:10.1158/1538-7445.AM2011-5225

Epithelial-to-mesenchymal transition (EMT) is associated with increased breast tumor metastasis, although the specific mechanism by which EMT increases metastasis remains somewhat unclear. We have previously demonstrated that detached human breast tumor cells generate unique microtentacle (McTN) extensions of their plasma membrane that are supported by coordinated vimentin intermediate filaments and detyrosinated microtubules. Microtentacles are specifically induced by EMT and promote the reattachment of tumor cells to each other and endothelial cell layers. We report here that immunohistochemical staining of tumor sections from patients with ductal carcinoma in situ and invasive ductal carcinoma indicates that detyrosination of α-tubulin occurs at invasive fronts where tumor cells are moving through the stromal microenvironment. Costaining with the EMT marker, Twist, demonstrates that tubulin detyrosination occurs specifically within cells that are undergoing EMT. Exogenous expression of either Twist or Snail in immortalized human mammary epithelial cells leads to a downregulation of the tubulin tyrosine ligase (TTL) enzyme that catalyzes the readdition of tyrosine to α-tubulin. This provides a novel molecular mechanism for the increase in detyrosinated microtubules observed in cells undergoing EMT. Addition of the sesquiterpene lactone, Parthenolide, to breast tumor cells reversed this EMT-induced increase in detyrosinated tubulin. Treatment of either breast tumor cells or nontumorigenic MCF10A mammary epithelial cells with 10μM Parthenolide also led to reductions in microtentacles, without affecting cell viability. Given the role of microtentacles in adhesion to endothelial layers, whole-animal bioluminescence imaging was used to track circulating tumor cells following tail vein injection. Pretreating metastatic MDA-MB-231 cells with Parthenolide prior to tail vein injection caused up to a 75% decrease in the lung retention of circulating tumor cells. These data support a model where the tubulin detyrosination that supports microtentacles promotes the ability of circulating tumor cells to reattach in distant capillary beds. Furthermore, the tubulin detyrosination that occurs in cells undergoing EMT at invasive tumor fronts could prime these cells for metastatic success once entering the circulation. These findings provide a possible explanation for why increased levels of detyrosinated tubulin are associated with poor patient survival in breast cancer and a new potential mechanism for how EMT could promote tumor metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1464.

During metastasis, invading cells produce various actin-based membrane protrusions that promote directional migration and proteolysis of extracellular matrix (ECM). Our recent discovery of a filamentous actin (F-actin) component within thin tubulin-based microtentacle (McTN) protrusions in suspended MDA-MB-231 tumor cells prompted an investigation of whether these extensions are structural or functional analogs of invadopodia. We show here that MDA-MB-231s are capable of producing invadopodia and McTNs, both of which contain F-actin. Invadopodium formation was enhanced by expression of a constitutively-active c-Src kinase, and repressed by expression of dominant negative, catalytically inactive form of c-Src. In contrast expression of inactive c-Src significantly increased the McTN formation. Additionally, direct inhibition of c-Src with the SU6656 inhibitor compound significantly enhanced McTN formation, suppressed the appearance of F-actin cores and phospho-cortactin foci, and completely blocked focal degradation of extracellular matrix. These results identify McTNs and invadopodia as separate structures that provide unique and sequential contributions to metastatic progression, namely endothelial attachment and invasion, respectively. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5133.

Breast cancer is the most frequent malignancy in women, and although many breast cancers are curable via surgery, approximately one quarter maintain a latent and insidious characteristic of slow growth with early metastasis. The loss of the tumor suppressor PTEN has been associated with breast cancer stage, lymph node status, and disease-related death, and the high rate of loss in primary tumors suggests a potential role in initiation and/or progression of the disease. However, specific cellular alterations in human breast epithelium controlled by PTEN inactivation, which lead to an increased metastatic phenotype, remain poorly defined. We have determined that heterozygous and homozygous loss of PTEN in non-tumorigenic mammary epithelial cells (MECs) was insufficient to promote anchorage-independent growth. However, MECs with PTEN loss maintained elevated activation of the PI3K/Akt and MAPK pathways and apoptotic resistance to cell rounding and matrix detatchment. We have also recently determined that PTEN expression loss leads to the production of long, dynamic, tubulin-based membrane protrusions upon detachment, which are increased in frequency, number and length per cell as compared to their isogenic, PTEN-expressing parental counterparts. These novel structures, termed microtentacles (McTNs), are structurally distinct from classical actin-based extensions of adherent cells, persist for days in breast tumor lines that are resistant to anoikis, and aid in the reattachment to matrix or cell monolayers. Therefore, the combination of apoptotic resistance and enhanced McTN formation due to PTEN loss may have important consequences for facilitating tumor cell extravasation and efficient adherence in metastatic sites. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1123.

Abstract #55 Detached human breast tumor cell lines produce microtentacles composed of a unique kinesin-dependent coordination of vimentin filaments and detyrosinated microtubules. Compelling in vivo studies show that a mechanism consistent with microtentacles is responsible for the attachment of circulating tumor cells to blood vessel walls. However, the molecular regulators of microtentacle extension have only begun to be defined. Increased expression of the microtubule-binding protein, Tau, is associated with poor patient outcome in breast cancer, but the mechanism by which Tau protein affects prognosis remains unclear. We report here that expression of Tau directly regulates the formation of metastasis-associated microtentacles in detached and circulating breast tumor cells. First, increased expression of Tau correlates with increased microtentacle frequency in a panel of human breast tumor cell lines. Endogenous Tau protein colocalizes with tubulin microtentacles in cells detached from extracellular matrix. Exogenous expression of Tau significantly increases microtentacle frequency in weakly invasive breast tumor cell lines. Tau-induced microtentacles are longer, thicker and more rigid than those in cell lines without Tau expression. Although there is increasing interest in targeting the actin cytoskeleton to reduce tumor cell motility and division, our results indicate that breast tumor cells with high Tau expression have particularly dramatic increases in microtentacles when treated with inhibitors of actin polymerization. Likewise, tubulin-stabilizing compounds, like paclitaxel, increase microtentacles and may be ill-advised in patients with high Tau expression. Indeed, clinical studies show that high Tau expression increases the likelihood of recurrence following paclitaxel treatment. We observed an enrichment of Tau in metastatic tumors compared to matched primary tumors. So while stabilizing microtubules and disrupting actin filaments can each decrease tumor cell growth, it is important to consider the effects of these treatments on Tau-expressing cells to avoid accidentally enhancing the metastatic potential of circulating tumor cells while targeting cell division. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 55.