PPAR-delta modulates membrane cholesterol and cytokine signaling in malignant B cells.

A deeper understanding of the mechanisms that underlie aberrant signal transduction in B-cell cancers such as chronic lymphocytic leukemia (CLL) may reveal new treatment strategies. The lipid-activated nuclear receptor peroxisome proliferator-activated receptor delta (PPARδ) accounts for a number of properties of aggressive cancers and was found to enhance Janus kinase (JAK)-mediated phosphorylation of signal transducer and activator of transcription (STAT) proteins in B lymphoma cell lines and primary CLL cells. Autocrine production of cytokines such as IL10 and interferon-beta was not increased by PPARδ but signaling responses to these cytokines were amplified and associated with increased cholesterol biosynthesis and plasma membrane levels. Plasmalemmal cholesterol and STAT phosphorylation from type 1 interferons (IFNs) were increased by PPARδ agonists, transgenes and exogenous cholesterol, and decreased by cyclodextrin, PPARD deletion and chemical PPARδ inhibitors. Functional consequences of PPARδ-mediated perturbation of IFN signaling included impaired upregulation of co-stimulatory molecules. These observations suggest PPARδ modulates signaling processes in malignant B cells in part by altering cholesterol metabolism and changes the outcomes of signaling from cytokines such as IFNs. PPARδ antagonists may have therapeutic activity as anti-leukemic signal transduction modulators.

PPAR-delta promotes survival of chronic lymphocytic leukemia cells in energetically unfavorable conditions.

Targeting the mechanisms that allow chronic lymphocytic leukemia (CLL) cells to survive in harsh cancer microenvironments should improve patient outcomes. The nuclear receptor peroxisome proliferator activated receptor delta (PPARδ) sustains other cancers, and in silico analysis showed higher PPARD expression in CLL cells than normal lymphocytes and other hematologic cancers. A direct association was found between PPARδ protein levels in CLL cells and clinical score. Transgenic expression of PPARδ increased the growth and survival of CD5+ Daudi cells and primary CLL cells in stressful conditions including exhausted tissue culture media, low extracellular glucose, hypoxia and exposure to cytotoxic drugs. Glucocorticoids and synthetic PPARδ agonists up-regulated PPARD expression and also protected Daudi and primary CLL cells from metabolic stressors. Survival in low glucose was related to increased antioxidant expression, substrate utilization and mitochondrial performance, and was reversed by genetic deletion and synthetic PPARδ antagonists. These findings suggest PPARδ conditions CLL cells to survive in harsh microenvironmental conditions by reducing oxidative stress and increasing metabolic efficiency. Targeting PPARδ may be beneficial in the treatment of CLL.

Self-reinforcing loop of amphiregulin and Y-box binding protein-1 contributes to poor outcomes in ovarian cancer.

The Y-box binding protein-1 (YB-1) transcription factor is associated with unfavorable clinical outcomes. However, the mechanisms underlying this association remain to be fully elucidated. We demonstrate that YB-1 phosphorylation, indicative of YB-1 activation, is a powerful marker of outcomes for ovarian cancer patients. In ovarian cancer, YB-1 phosphorylation is induced by activation of the lysophosphatidic acid (LPA) receptor (LPAR) via SRC-dependent transactivation of the epidermal growth factor receptor (EGFR) that is coupled to MAPK/p90 ribosomal S6 kinase (p90RSK), but not phosphatidylinositol 3-kinase (PI3K)/AKT signaling. Activation of the LPAR/SRC/EGFR/MAPK/p90RSK/YB-1 axis leads to production of the EGFR ligand amphiregulin (AREG). AREG induces ongoing YB-1 phosphorylation as well as YB-1-dependent AREG expression, thus constituting an AREG/YB-1 self-reinforcing loop. Disruption of transactivation of the EGFR and the downstream self-reinforcing loop decreases invasiveness of ovarian cancer cells in vitro and limits ovarian cancer growth in xenograft models. These findings established the regulation and significance of YB-1 phosphorylation, therefore further exploration of this signaling axis as a therapeutic avenue in ovarian cancer is warranted.

MKNK1 is a YB-1 target gene responsible for imparting trastuzumab resistance and can be blocked by RSK inhibition.

Trastuzumab (Herceptin) resistance is a major obstacle in the treatment of patients with HER2-positive breast cancers. We recently reported that the transcription factor Y-box binding protein-1 (YB-1) leads to acquisition of resistance to trastuzumab in a phosphorylation-dependent manner that relies on p90 ribosomal S6 kinase (RSK). To explore how this may occur we compared YB-1 target genes between trastuzumab-sensitive cells (BT474) and those with acquired resistance (HR5 and HR6) using genome-wide chromatin immunoprecipitation sequencing (ChIP-sequencing), which identified 1391 genes uniquely bound by YB-1 in the resistant cell lines. We then examined differences in protein expression and phosphorylation between these cell lines using the Kinexus Kinex antibody microarrays. Cross-referencing these two data sets identified the mitogen-activated protein kinase-interacting kinase (MNK) family as potentially being involved in acquired resistance downstream from YB-1. MNK1 and MNK2 were subsequently shown to be overexpressed in the resistant cell lines; however, only the former was a YB-1 target based on ChIP-PCR and small interfering RNA (siRNA) studies. Importantly, loss of MNK1 expression using siRNA enhanced sensitivity to trastuzumab. Further, MNK1 overexpression was sufficient to confer resistance to trastuzumab in cells that were previously sensitive. We then developed a de novo model of acquired resistance by exposing BT474 cells to trastuzumab for 60 days (BT474LT). Similar to the HR5/HR6 cells, the BT474LT cells had elevated MNK1 levels and were dependent on it for survival. In addition, we demonstrated that RSK phosphorylated MNK1, and that this phosphorylation was required for ability of MNK1 to mediate resistance to trastuzumab. Furthermore, inhibition of RSK with the small molecule BI-D1870 repressed the MNK1-mediated trastuzumab resistance. In conclusion, this unbiased integrated approach identified MNK1 as a player in mediating trastuzumab resistance as a consequence of YB-1 activation, and demonstrated RSK inhibition as a means to overcome recalcitrance to trastuzumab.

The first identified case of pandemic H1N1 influenza in pigs in Australia.

A 300-sow farrow-to-finish herd in New South Wales was infected with influenza pandemic (H1N1) 2009 (H1N1/09) virus in July 2009 and became the first recorded case of influenza in pigs in Australia. The outbreak resulted from human-to-pig transmission. Clinical signs in affected pigs were mild compared with overseas reports of 'classical' swine influenza virus and included coughing and decreased appetite in a small proportion of non-lactating breeding stock, weaners, growers and finishers. A diagnosis of H1N1/09 influenza virus infection was confirmed using a combination of serology (haemagglutination inhibition, blocking enzyme-linked immunosorbent assay) and real-time reverse transcription polymerase chain reaction. Attempts at virus isolation were unsuccessful. Results of a longitudinal study of pigs on this farm suggested that the virus continued to circulate for 9 weeks after the onset of infection, but was not present 6 months later. This report highlights the difficulties in preventing transmission of H1N1/09 influenza virus from infected humans to pigs during a human pandemic.

YB-1 evokes susceptibility to cancer through cytokinesis failure, mitotic dysfunction and HER2 amplification.

Y-box binding protein-1 (YB-1) expression in the mammary gland promotes breast carcinoma that demonstrates a high degree of genomic instability. In the present study, we developed a model of pre-malignancy to characterize the role of this gene during breast cancer initiation and early progression. Antibody microarray technology was used to ascertain global changes in signal transduction following the conditional expression of YB-1 in human mammary epithelial cells (HMEC). Cell cycle-associated proteins were frequently altered with the most dramatic being LIM kinase 1/2 (LIMK1/2). Consequently, the misexpression of LIMK1/2 was associated with cytokinesis failure that acted as a precursor to centrosome amplification. Detailed investigation revealed that YB-1 localized to the centrosome in a phosphorylation-dependent manner, where it complexed with pericentrin and γ-tubulin. This was found to be essential in maintaining the structural integrity and microtubule nucleation capacity of the organelle. Prolonged exposure to YB-1 led to rampant acceleration toward tumorigenesis, with the majority of cells acquiring numerical and structural chromosomal abnormalities. Slippage through the G(1)/S checkpoint due to overexpression of cyclin E promoted continued proliferation of these genomically compromised cells. As malignancy further progressed, we identified a subset of cells harboring HER2 amplification. Our results recognize YB-1 as a cancer susceptibility gene, with the capacity to prime cells for tumorigenesis.

Suppression of Her2/neu expression through ILK inhibition is regulated by a pathway involving TWIST and YB-1.

In a previous study it was found that the therapeutic effects of QLT0267, a small molecule inhibitor of integrin-linked kinase (ILK), were influenced by Her2/neu expression. To understand how inhibition or silencing of ILK influences Her2/neu expression, Her2/neu signaling was evaluated in six Her2/neu-positive breast cancer cell lines (LCC6(Her2), MCF7(Her2), SKBR3, BT474, JIMT-1 and KPL-4). Treatment with QLT0267 engendered suppression (32-87%) of total Her2/neu protein in these cells. Suppression of Her2/neu was also observed following small interfering RNA-mediated silencing of ILK expression. Time course studies suggest that ILK inhibition or silencing caused transient decreases in P-AKT(ser473), which were not temporally related to Her2/neu downregulation. Attenuation of ILK activity or expression was, however, associated with decreases in YB-1 (Y-box binding protein-1) protein and transcript levels. YB-1 is a known transcriptional regulator of Her2/neu expression, and in this study it is demonstrated that inhibition of ILK activity using QLT0267 decreased YB-1 promoter activity by 50.6%. ILK inhibition was associated with changes in YB-1 localization, as reflected by localization of cytoplasmic YB-1 into stress granules. ILK inhibition also suppressed TWIST (a regulator of YB-1 expression) protein expression. To confirm the role of ILK on YB-1 and TWIST, cells were engineered to overexpress ILK. This was associated with a fourfold increase in the level of YB-1 in the nucleus, and a 2- and 1.5-fold increase in TWIST and Her2/neu protein levels, respectively. Taken together, these data indicate that ILK regulates the expression of Her2/neu through TWIST and YB-1, lending support to the use of ILK inhibitors in the treatment of aggressive Her2/neu-positive tumors.

The expression of activated Y-box binding protein-1 serine 102 mediates trastuzumab resistance in breast cancer cells by increasing CD44+ cells.

The development of acquired resistance to trastuzumab remains a prevalent challenge in the treatment of patients whose tumors express human epidermal growth factor 2 (HER2). We previously reported that HER2 overexpressing breast cancers are dependent on Y-box binding protein-1 (YB-1) for growth and survival. As YB-1 is also linked to drug resistance in other types of cancer, we address its possible role in trastuzumab insensitivity. Employing an in vivo model of acquired resistance, we demonstrate that resistant cell lines have elevated levels of P-YB-1(S102) and its activating kinase P-RSK and these levels are sustained following trastuzumab treatment. Further, to demonstrate the importance of YB-1 in mediating drug resistance, the expression of the active mutant YB-1(S102D) rendered the BT474 cell line insensitive to trastuzumab. Questioning the role of tumor-initiating cells (TIC) and their ability to escape cancer therapies, we investigate YB-1's role in inducing the cancer stem cell marker CD44. Notably, the resistant cells express more CD44 mRNA and protein compared with BT474 cells, which correlated with increased mammosphere formation. Expression of YB-1(S102D) in the BT474 cells increase CD44 protein levels, resulting in enhanced mammosphere formation. Further, exposing BT474 cells to trastuzumab selected for a resistant sub-population enriched for CD44. Conversely, small intefering RNA inhibition of CD44 restored trastuzumab sensitivity in the resistant cell lines. Our findings provide insight on a novel mechanism employed by tumor cells to acquire the ability to escape the effects of trastuzumab and suggest that targeting YB-1 may overcome resistance by eliminating the unresponsive TIC population, rendering the cancer sensitive to therapy.

Anti-CD13 Abs in children with extensive chronic GVHD and their relation to soluble CD13 after allogeneic blood and marrow transplantation from a Children's Oncology Groups Study, ASCT0031.

Our group previously demonstrated a strong association between elevated plasma soluble CD13 enzyme activity and newly diagnosed extensive chronic GVHD (cGVHD) in children. As cytotoxic anti-CD13 Abs have been documented after blood and marrow transplant (BMT) in association with CMV infection and cGVHD, we hypothesized that soluble CD13 contributes to cGVHD pathogenesis by induction of CD13 reactive Abs and that anti-CD13 Abs could be additional biomarkers for newly diagnosed pediatric extensive cGVHD. Using prospectively collected plasma samples from pediatric allogeneic BMT (allo-BMT) subjects with cGVHD and controls without cGVHD enrolled in a large multi-institution Children's Oncology Group cGVHD therapeutic trial, we evaluated whether soluble CD13 correlates with induction of anti-CD13 Abs. We found that CD13 reactive Abs are present in a proportion of patients after allo-BMT, but did not seem to correlate with the presence of soluble CD13. Anti-CD13 Abs also did not meet our criteria as a diagnostic biomarker for cGVHD. These data do not confirm that induction of CD13 reactive Abs is a mechanism for cGVHD in children nor are part of the pathogenesis of cGVHD associated with elevated soluble CD13. The exact role of CD13 in cGVHD remains to be determined.

The transcriptional induction of PIK3CA in tumor cells is dependent on the oncoprotein Y-box binding protein-1.

PIK3CA, which codes for the p110alpha catalytic subunit of phosphatidylinositol-3-kinase (PI3K), is implicated as an oncogene. Despite importance of PIK3CA in cancer, little is known about what drives up its expression in tumor cells. We recently characterized the PIK3CA promoter and reported that it is transcriptionally silenced by the tumor suppressor protein p53. In the present study, we demonstrate that PIK3CA can be induced by the oncogenic transcription factor Y-box binding protein-1 (YB-1). Three YB-1-responsive elements were identified on the PIK3CA promoter using chromatin immunoprecipitation and electrophoretic mobility shift assays. Interestingly, silencing YB-1 with siRNA in models of basal-like breast cancer decreased p110alpha protein levels regardless of whether PIK3CA was wild type, amplified or mutated. This decrease in p110alpha led to a reduction in PI3K activity and the downstream signaling primarily through p90 ribosomal S6 kinase and S6 ribosomal protein. Disruption in PIK3CA-dependent signaling suppressed cellular invasion correlative with loss of urokinase plasminogen activator (uPA). Similarly, silencing YB-1 suppressed invasion and uPA production however this was reversible through the introduction of constitutively active PIK3CA. In conclusion, YB-1 is the first reported oncogene to induce the expression of PIK3CA through transcriptional control of its promoter.

Profiling YB-1 target genes uncovers a new mechanism for MET receptor regulation in normal and malignant human mammary cells.

Basal-like breast cancers (BLBCs) are aggressive tumors with high relapse rates and poor survival. We recently reported that >70% of primary BLBCs express the oncogenic transcription/translation factor Y-box binding protein-1 (YB-1) and silencing it with small interfering RNAs (siRNAs) attenuates the growth of BLBC cell lines. To understand the basis of these earlier findings, we profiled YB-1:DNA complexes by chromatin immunoprecipitation (ChIP)-on-chip. Several tumor growth-promoting genes such as MET, CD44, CD49f, WNT and NOTCH family members were identified. In addition, YB-1 and MET are coordinately expressed in BLBC cell lines, as well as in normal human mammary progenitor cells. MET was confirmed to be a YB-1 target through traditional ChIP and gel-shift assays. More specifically, YB-1 binds to -1018 bp on the MET promoter. Silencing YB-1 with siRNA decreased MET promoter activity, transcripts, as well as protein levels and signaling. Conversely, expressing wild-type YB-1 or a constitutively active mutant YB-1 (D102) increased MET expression. Finally, silencing YB-1 or MET attenuated anchorage-independent growth of BLBC cell lines. Together, these findings implicate MET as a target of YB-1 that work in concert to promote BLBC growth.

The phosphoinositide-dependent kinase-1 inhibitor 2-amino-N-[4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-acetamide (OSU-03012) prevents Y-box binding protein-1 from inducing epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) is integral to basal-like and human epidermal growth factor receptor-2 (Her-2)-overexpressing breast cancers. Such tumors are associated with poor prognosis, the majority of which express high levels of EGFR. We reported that EGFR expression is induced by the oncogenic transcription factor Y-box binding protein-1 (YB-1) that occurs in a manner dependent on phosphorylation by Akt. Herein, we questioned whether blocking Akt with 2-amino-N-[4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-acetamide (OSU-03012), a phosphoinositide-dependent protein kinase-1 (PDK-1) small-molecule inhibitor, could prevent YB-1 from binding to the EGFR promoter. MDA-MB-468 and SUM 149 are basal-like breast cancer (BLBC) cells that were used for our studies because they express high levels of activated PDK-1, YB-1, and EGFR compared with the immortalized breast epithelial cell line 184htrt. In these cell lines, YB-1 preferentially bound to the -1 kilobase of the EGFR promoter, whereas this did not occur in the 184htrt cells based on chromatin immunoprecipitation. When the cells were exposed to OSU-03012 for 6 h, YB-1/EGFR promoter binding was significantly attenuated. To further confirm this observation, gel-shift assays showed that the drug inhibits YB-1/EGFR promoter binding. The inhibitory effect of OSU-03012 on EGFR was also observed at the mRNA and protein levels. OSU-03012 ultimately inhibited the growth of BLBC in monolayer and soft agar coordinate with the induction of apoptosis using an Array-Scan VTI high-content screening system. Furthermore, OSU-03012 inhibited the expression of EGFR by 48% in tumor xenografts derived from MDA-MB-435/Her-2 cells. This correlated with loss of YB-1 binding to the EGFR promoter. Hence, we find that OSU-03012 inhibits YB-1 resulting in a loss of EGFR expression in vitro and in vivo.

Insulin-like growth factor-1 inscribes a gene expression profile for angiogenic factors and cancer progression in breast epithelial cells.

Activation of the insulin-like growth factor-1 receptor (IGF-1R) by IGF-1 is associated with the risk and progression of many types of cancer, although despite this it remains unclear how activated IGF-1R contributes to cancer progression. In this study, gene expression changes elicited by IGF-1 were profiled in breast epithelial cells. We noted that many genes are functionally linked to cancer progression and angiogenesis. To validate some of the changes observed, the RNA and/or protein was confirmed for c-fos, cytochrome P450 1A1, cytochrome P450 1B1, interleukin-1 beta, fas ligand, vascular endothelial growth factor, and urokinase plasminogen activator. Nuclear proteins were also temporally monitored to address how gene expression changes were regulated. We found that IGF-1 stimulated the nuclear translocation of phosphorylated AKT, hypoxic-inducible factor-1 alpha, and phosphorylated cAMP-responsive element-binding protein, which correlated with temporal changes in gene expression. Next, the promoter regions of IGF-1-regulated genes were searched in silico. The promoters of genes that clustered together had similar regulatory regions. In summary, IGF-1 inscribes a gene expression profile relevant to cancer progression, and this study provides insight into the mechanism(s) whereby some of these changes occur.

Nerve activity-dependent modulation of calcineurin signaling in adult fast and slow skeletal muscle fibers.

This study tested the hypothesis that calcineurin signaling is modulated in skeletal muscle cells by fluctuations in nerve-mediated activity. We show that dephosphorylation of NFATc1, MEF2A, and MEF2D transcription factors by calcineurin in all muscle types is dependent on nerve activity and positively correlated with muscle usage under normal weightbearing conditions. With increased nerve-mediated activity, calcineurin dephosphorylation of these targets was found to be potentiated in a way that paralleled the higher muscle activation profiles associated with functional overload or nerve electrical stimulation conditions. We also establish that muscle activity must be sustained above native levels for calcineurin-dependent dephosphorylation of MEF2A and MEF2D to be transduced into an increase in MEF2 transcriptional function, suggesting that calcineurin cooperates with other activity-linked events to signal via these proteins. Finally, examination of individual fiber responses to overload and nerve electrical stimulation revealed that calcineurin-MEF2 signaling occurs in all fiber types but most readily in fibers that are normally least active (i.e. those expressing IIx and IIb myosin heavy chain (MHC)), suggesting that signaling via this phosphatase is also dependent upon the activation history of the muscle cell.

Up-regulation of urokinase-type plasminogen activator by insulin-like growth factor-I depends upon phosphatidylinositol-3 kinase and mitogen-activated protein kinase kinase.

Elevated levels of urokinase plasminogen activator-1 (uPA) and the insulin-like growth factor-I receptor (IGF-IR) are associated with breast cancer recurrence and decreased survival. It is possible that activation of IGF-IR and elevations in uPA are mechanistically linked. Our laboratory recently showed that insulin-like growth factor-I (IGF-I) induces uPA protein and mRNA in the breast cancer cell line MDA-MB-231. We also found that IGF-IR and uPA were commonly overexpressed in primary breast cancers. In this study, we investigated the signal transduction pathway through which IGF-I regulates uPA. Phosphatidylinositol 3-kinase, mitogen-activated protein kinase kinase, and p70 kinase were inhibited with LY294002, PD98059, and rapamycin, respectively. Induction of uPA protein by IGF-I was partially inhibited by LY294002 (60% inhibition) or PD98059 (30% inhibition) but not by rapamycin. The production of uPA protein induced by IGF-I was blocked up to 90% by the tyrosine kinase inhibitor herbimycin A. Furthermore, herbimycin A suppressed the phosphorylation of AKT and Erk1/2. Next, we tested the impact of the signal transduction inhibitors on uPA gene expression. Both LY294002 and PD98059 were required to completely inhibit uPA mRNA expression, whereas each drug alone resulted in approximately 50% reduction in uPA expression. Next, using a minimal uPA-luciferase promoter construct containing the binding sites for the AP-1 and Ets transcription factors, we observed that IGF-I stimulated the uPA promoter via these sites. Furthermore, both Ly294002 and PD98059 were necessary to block IGF-I-stimulated uPA-Luc activity. In summary, we conclude that IGF-I requires both phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase-dependent pathways to optimally induce uPA expression. These findings suggest that the development of drugs targeting these pathways may benefit breast cancer patients at a high risk of recurrence, such as those who have primary tumors overexpressing IGF-IR and uPA.

Matching of calcineurin activity to upstream effectors is critical for skeletal muscle fiber growth.

Calcineurin-dependent pathways have been implicated in the hypertrophic response of skeletal muscle to functional overload (OV) (Dunn, S.E., J.L. Burns, and R.N. Michel. 1999. J. Biol. Chem. 274:21908-21912). Here we show that skeletal muscles overexpressing an activated form of calcineurin (CnA*) exhibit a phenotype indistinguishable from wild-type counterparts under normal weightbearing conditions and respond to OV with a similar doubling in cell size and slow fiber number. These adaptations occurred despite the fact that CnA* muscles displayed threefold higher calcineurin activity and enhanced dephosphorylation of the calcineurin targets NFATc1, MEF2A, and MEF2D. Moreover, when calcineurin signaling is compromised with cyclosporin A, muscles from OV wild-type mice display a lower molecular weight form of CnA, originally detected in failing hearts, whereas CnA* muscles are spared this manifestation. We also show that OV-induced growth and type transformations are prevented in muscle fibers of transgenic mice overexpressing a peptide that inhibits calmodulin from signaling to target enzymes. Taken together, these findings provide evidence that both calcineurin and its activity-linked upstream signaling elements are crucial for muscle adaptations to OV and that, unless significantly compromised, endogenous levels of this enzyme can accommodate large fluctuations in upstream calcium-dependent signaling events.

The insulin-like growth factor-1 elevates urokinase-type plasminogen activator-1 in human breast cancer cells: a new avenue for breast cancer therapy.

Tumor recurrence is a common problem in the treatment of breast cancer. In breast cancer, the expression of high protein levels of the insulin-like growth factor-1 receptor (IGF-1R) and urokinase-type plasminogen activator-1 (uPA) is strongly associated with breast cancer recurrence and decreased survival. The expression of uPA by tumors is thought to not only stimulate tumor invasion but also facilitate angiogenesis. In this study, our goal was to address whether IGF-1R could influence the expression of the extracell ular matrix proteases, matrix metalloproteinase (MMP), or uPA thus allowing a selective advantage for tumor invasion and concomitant neovascularization. Initially, we determined whether or not insulin-like growth factor (IGF)-1 regulated the production MMP or uPA in the human breast cancer MDA-MB-231 cells. There was no increase in MMP activity when the cells were treated with IGF-1 (10 ng/mL) for 24 h. In contrast, uPA mRNA and protein were induced in a time-dependent manner. Furthermore, clones expressi ng a dominant negative inhibitor of IGF-1R termed 486stop had less uPA mRNA, and the clones were less invasive through Matrigel. Taken together, these data illustrate that IGF-1R stimulates uPA production. Hence, these two prognostic indicators may be interrelated, suggesting they may function in a synergistic manner to facilitate local tumor invasion as well as angiogenesis. Our data suggest that disruption of IGF-1 signaling in breast cancer may lead to breast cancer prevention and intervention by decreasing uPA expression.

Calcineurin is required for skeletal muscle hypertrophy.

Molecular signaling pathways linking increases in skeletal muscle usage to alterations in muscle size have not been identified. In the present study, we tested the hypothesis that calcineurin, a calcium-regulated phosphatase recently implicated in the signaling of some forms of cardiomyopathic growth, is required to induce skeletal muscle hypertrophy and muscle fiber type conversions associated with functional overload in vivo. Administration of the specific calcineurin inhibitors cyclosporin (CsA) or FK506 to mice, for which the fast plantaris muscle was overloaded for 1-4 weeks, prevented the rapid doubling of mass and individual fiber size and the 4-20-fold increase in the number of slow fibers that characterize this condition. CsA treatment influenced the expression of muscle myofibrillar protein genes in a way reflective of fiber phenotype transformations but only in the long term of the overload condition, suggesting that the control of this growth response by calcineurin is not limited to the transcriptional activation of these muscle-specific genes. Clinically, these results provide insight to the post-surgical muscle wasting and weakness observed in recovering transplant recipients administered therapeutic dosages of these immunosuppressants.