Progesterone receptor isoform-dependent cross-talk between prolactin and fatty acid synthase in breast cancer.

Progesterone receptor (PR) isoforms can drive unique phenotypes in luminal breast cancer (BC). Here, we hypothesized that PR-B and PR-A isoforms differentially modify the cross-talk between prolactin and fatty acid synthase (FASN) in BC. We profiled the responsiveness of the FASN gene promoter to prolactin in T47Dco BC cells constitutively expressing PR-A and PR-B, in the PR-null variant T47D-Y cell line, and in PR-null T47D-Y cells engineered to stably re-express PR-A (T47D-YA) or PR-B (T47D-YB). The capacity of prolactin to up-regulate FASN gene promoter activity in T47Dco cells was lost in T47D-Y and TD47-YA cells. Constitutively up-regulated FASN gene expression in T47-YB cells and its further stimulation by prolactin were both suppressed by the prolactin receptor antagonist hPRL-G129R. The ability of the FASN inhibitor C75 to decrease prolactin secretion was more conspicuous in T47-YB cells. In T47D-Y cells, which secreted notably less prolactin and downregulated prolactin receptor expression relative to T47Dco cells, FASN blockade resulted in an augmented secretion of prolactin and up-regulation of prolactin receptor expression. Our data reveal unforeseen PR-B isoform-specific regulatory actions in the cross-talk between prolactin and FASN signaling in BC. These findings might provide new PR-B/FASN-centered predictive and therapeutic modalities in luminal intrinsic BC subtypes.

Blockade of a key region in the extracellular domain inhibits HER2 dimerization and signaling.

BACKGROUND: Several treatment strategies target the human epidermal growth factor receptor 2 (HER2) in breast carcinomas, including monoclonal antibodies directed against HER2's extracellular domain (ECD) and small molecule inhibitors of its tyrosine kinase activity. Yet, novel therapies are needed that prevent HER2 dimerization with other HER family members, because current treatments are only partially effective. METHODS: To test the hypothesis that HER2 activation requires a protein sequence in the HER2-ECD that mediates HER2 homo- and heterodimerization, we introduced a series of deletion mutations in the third subdomain of HER2-ECD. These deletion mutants were retrovirally expressed in breast cancer (BC) cells that naturally overexpress HER2 and in noncancerous, HER2-negative breast epithelial cells. One-factor analysis of variance or Student's t test were used to analyze differences. All statistical tests were two-sided. RESULTS: The smallest deletion in the ECD domain of HER2, which removed only 16 amino acids (HER2-ECDΔ451-466), completely disrupted the oncogenic potential of HER2. In contrast to wild-type HER2, the mutant-inhibited anchorage-independent growth (mean number of colonies: mutant, 70, 95% confidence interval [CI] = 55 to 85; wild-type, 400, 95% CI = 320 to 480, P < .001) increased sensitivity to paclitaxel treatment in both transformed and nontransformed cells. Overexpression of HER2Δ451-466 efficiently inhibited activation of HER1, HER2, and HER3 in all cell lines tested. CONCLUSIONS: These findings reveal that an essential "activating" sequence exists in the extracellular domain of HER2. Disruption of this sequence disables the HER2 dimerization loop, blocks subsequent activation of HER2-driven oncogenic signaling, and generates a dominant-negative form of HER2. Reagents specifically against this molecular activation switch may represent a novel targeted approach for the management of HER2-overexpressing carcinomas.

EGFR signaling defines Mcl⁻1 survival dependency in neuroblastoma.

The pediatric solid tumor neuroblastoma (NB) often depends on the anti-apoptotic protein, Mcl(-)1, for survival through Mcl(-)1 sequestration of pro-apoptotic Bim. High affinity Mcl(-)1 inhibitors currently do not exist such that novel methods to inhibit Mcl(-)1 clinically are in high demand. Receptor tyrosine kinases (RTK) regulate Mcl(-)1 in many cancers and play a role in NB survival, yet how they regulate Bcl(-)2 family interactions in NB is unknown. We found that NB cell lines derived to resist the Bcl(-)2/-xl/-w antagonist, ABT-737, acquire a dependence on Mcl(-)1 and show increased expression and activation of the RTK, EGFR. Mcl(-)1 dependent NB cell lines derived at diagnosis and from the same tumor following relapse also have increased EGFR expression compared to those dependent on Bcl(-)2. Inhibition of EGFR by shRNA or erlotinib in Mcl(-)1 dependent NBs disrupts Bim binding to Mcl(-)1 and enhances its affinity for Bcl(-)2, restoring sensitivity to ABT-737 as well as cytotoxics in vitro. Mechanistically treatment of NBs with small molecule inhibitors of EGFR (erlotinib, cetuximab) and ERK (U0126) increases Noxa expression and dephosphorylates Bim to promote Bim binding to Bcl(-)2. Thus, EGFR regulates Mcl(-)1 dependence in high-risk NB via ERK-mediated phosphorylation of Bim such that EGFR/ERK inhibition renders Mcl(-)1 dependent tumors now reliant on Bcl(-)2. Clinically, EGFR inhibitors are ineffective as single agent compounds in patients with recurrent NB, likely due to this transferred survival dependence to Bcl(-)2. Likewise, EGFR or ERK inhibitors warrant further testing in combination with Bcl(-)2 antagonists in vivo as a novel future combination to overcome therapy resistance in the clinic.

PTEN and PI-3 kinase inhibitors control LPS signaling and the lymphoproliferative response in the CD19+ B cell compartment.

Pattern recognition receptors (PRRs), e.g. toll receptors (TLRs) that bind ligands within the microbiome have been implicated in the pathogenesis of cancer. LPS is a ligand for two TLR family members, TLR4 and RP105 which mediate LPS signaling in B cell proliferation and migration. Although LPS/TLR/RP105 signaling is well-studied; our understanding of the underlying molecular mechanisms controlling these PRR signaling pathways remains incomplete. Previous studies have demonstrated a role for PTEN/PI-3K signaling in B cell selection and survival, however a role for PTEN/PI-3K in TLR4/RP105/LPS signaling in the B cell compartment has not been reported. Herein, we crossed a CD19cre and PTEN(fl/fl) mouse to generate a conditional PTEN knockout mouse in the CD19+ B cell compartment. These mice were further crossed with an IL-14α transgenic mouse to study the combined effect of PTEN deletion, PI-3K inhibition and expression of IL-14α (a cytokine originally identified as a B cell growth factor) in CD19+ B cell lymphoproliferation and response to LPS stimulation. Targeted deletion of PTEN and directed expression of IL-14α in the CD19+ B cell compartment (IL-14+PTEN-/-) lead to marked splenomegaly and altered spleen morphology at baseline due to expansion of marginal zone B cells, a phenotype that was exaggerated by treatment with the B cell mitogen and TLR4/RP105 ligand, LPS. Moreover, LPS stimulation of CD19+ cells isolated from these mice display increased proliferation, augmented AKT and NFκB activation as well as increased expression of c-myc and cyclinD1. Interestingly, treatment of LPS treated IL-14+PTEN-/- mice with a pan PI-3K inhibitor, SF1126, reduced splenomegaly, cell proliferation, c-myc and cyclin D1 expression in the CD19+ B cell compartment and normalized the splenic histopathologic architecture. These findings provide the direct evidence that PTEN and PI-3K inhibitors control TLR4/RP105/LPS signaling in the CD19+ B cell compartment and that pan PI-3 kinase inhibitors reverse the lymphoproliferative phenotype in vivo.

The PI-3 kinase-Akt-MDM2-survivin signaling axis in high-risk neuroblastoma: a target for PI-3 kinase inhibitor intervention.

PURPOSE: Studies of SF1126, an RGDS targeted, water-soluble prodrug of LY294002, are currently nearing completion in two adult Phase I trials. Herein, we performed a preclinical evaluation of SF1126 as a PI-3K inhibitor for Phase I trials in the treatment of recurrent neuroblastoma (NB). METHODS: The effects of SF1126 on pAkt-MDM2 cell signaling, proliferation, apoptosis, and migration were determined using a panel of NB cell lines, and anti-tumor activity was determined using a xenograft model of NB. RESULTS: SF1126 blocks MDM2 activation, IGF-1 induced activation of Akt, and the upregulation of survivin induced by IGF-1. It also increases sensitivity to doxorubicin in vitro and was found to exhibit marked synergistic activity in combination with doxorubicin. Treatment disrupts the integrin αvß3/αvß5-mediated organization of the actin cytoskeleton as well as the α4ß1/α5ß1-mediated processes essential to metastasis. In vivo, SF1126 markedly inhibits tumor growth in NB xenografted mice (P < 0.05). CONCLUSIONS: A pan PI-3 kinase inhibitor has potent antitumor activity and induces apoptosis in multiple neuroblastoma cell lines. The observed effects of SF1126 on the p-Akt-MDM2-survivin axis suggest a patient selection paradigm in which NB tumors with increased pAkt-MDM2-survivin signaling may predict response to SF1126 alone or in combination with standard chemotherapy regimens that contain anthracyclines.

High level MycN expression in non-MYCN amplified neuroblastoma is induced by the combination treatment nutlin-3 and doxorubicin and enhances chemosensitivity.

MYCN gene amplification is a negative prognostic indicator in neuroblastoma and high level MycN expression in Stage IV neuroblastoma is generally a hallmark of poor patient outcome. However, high level expression of the MycN protein in neuroblastoma cells lacking MYCN amplification suppresses growth and drives apoptosis; this, in part, explains the absence of clinical observations of high level MycN in neuroblastoma lacking MYCN amplification. In the current study, we found that combination treatment with nutlin-3 and doxorubicin upregulated MycN expression in non-MYCN-amplified neuroblastoma cells at both the protein and mRNA levels. The induced expression of MycN in non-MYCN-amplified cells inhibited cell proliferation and increased apoptosis. MycN induction also upregulated p53, p21 and Bax protein levels, as well as mRNA levels for the positive neuroblastoma prognostic factors CD44 and EFNB3. Blocking MycN reversed these effects. These results were corroborated by findings using a MycN-inducible system in SHEP cells, another MYCN non-amplified neuroblastoma cell line. Our results indicate that doxorubicin/nutlin-3 combination treatment both induces expression of MycN in a non-MYCN-amplified background and sensitizes neuroblastoma cells to chemotherapy. These findings support the idea that induction of MycN in non-MYCN-amplified cells drives neuroblastoma cells toward apoptosis and suggest that combination nutlin-3/doxorubicin treatment may be clinically important.

MycN is a transcriptional regulator of livin in neuroblastoma.

Our previous studies have suggested that MycN may have a role in regulating livin expression in neuroblastoma. Here, we show that siRNA-mediated repression of MycN in neuroblastoma cells with both elevated MycN and livin expression resulted in significant downregulation of livin. Conversely, induction of MycN in neuroblastoma cells with low endogenous levels of MycN and livin protein upregulated livin expression. MycN directly associated with its regulatory motif (E-box) within the putative livin promoter. Based on these results, we hypothesize that MycN is required for livin expression and that livin may counteract the proapoptotic effects of MycN.

The MDM2 antagonist nutlin-3 sensitizes p53-null neuroblastoma cells to doxorubicin via E2F1 and TAp73.

Neuroblastoma (NB) is a primitive neuroectodermal tumor and the second most common solid tumor in children. NB exhibits heterogeneous behavior and spontaneous regression can occur in patients under 12 months of age. Response to treatment is both age- and stage-specific; however, patients over 1 year of age are generally considered high risk. NB tumors from these patients are often characterized by alterations in p53 expression and murine double minute (MDM2) activity with concomitant resistance to chemotherapy. We evaluated the ability of nutlin-3 to sensitize a p53-null and doxorubicin-resistant NB cell line, LA155N, to doxorubicin. Nutlin-3 treatment upregulated TAp73 and E2F1 protein levels. It potentiated the ability of doxorubicin to block cell proliferation and activate apoptosis and TAp73 knockdown resulted in a reduction of this sensitization. Additionally, PUMA expression was induced by the combination treatment, but reduced by knockdown of either TAp73 or E2F1. We conclude that, following nutlin-3 treatment, TAp73 and E2F1 are released from MDM2 and activated by doxorubicin to induce PUMA and apoptosis. This study addresses p53-independent mechanisms of nutlin-3 action in chemoresistant NB, especially in combination with chemotherapeutics. We believe that this model has strong clinical relevance for chemoresistant and p53 dysfunctional NB.

Human prolactin and its antagonist, hPRL-G129R, regulate bax and bcl-2 gene expression in human breast cancer cells and transgenic mice.

To gain insight into the molecular mechanisms involved in human prolactin receptor antagonist (hPRL-G129R)-induced apoptosis, we used real-time reverse transcription-polymerase chain reaction to measure bax and bcl-2 gene expression in 11 human breast cancer cell lines following treatment with hPRL and hPRL-G129R. We also measured bax and bcl-2 gene expression in the mammary glands of transgenic mice expressing hPRL or hPRL-G129R. A time-course study of hPRL and antagonist treatment in T-47D cells indicated changing bax/bcl-2 mRNA ratios beginning at 24 h. We found that bax/bcl-2 mRNA ratios were significantly elevated in seven of the 11 hPRL-G129R-treated cell lines, as well as in the hPRL-G129R transgenic mice. To confirm these results, Bax and Bcl-2 proteins were analysed by Western blot methods in mammary gland tissue homogenates of transgenic mice. Bax/Bcl-2 ratios were highest in the 6-month group of hPRL-G129R transgenics, and lowest in the 6-month group of hPRL transgenics. We expanded our findings by examining the release of a downstream Bax-induced protein, cytochrome c, a hallmark protein of apoptosis, in transgenic mice. Again, cytochrome c levels were highest in the 6-month hPRL-G129R transgenic group. Thus, hPRL-G129R-induced breast cancer cell and/or mammary gland apoptosis is mediated, at least in part, through the regulation of Bax and Bcl-2 gene expression.

Regulation of bcl-2 gene expression in human breast cancer cells by prolactin and its antagonist, hPRL-G129R.

To gain insight into the molecular basis of human prolactin (hPRL) antagonist induced apoptosis, we compared the differential gene expression profile of four human breast cancer cell lines following treatment with hPRL and its antagonist (hPRL-G129R). Among the genes identified, the bcl-2 gene was of particular interest. We found that bcl-2 mRNA was up regulated in three of the four cell lines that were treated with hPRL. To further confirm these results, real time RT-PCR and ELISA analyses were used to detect bcl-2 mRNA and Bcl-2 protein, respectively, in 11 different breast cancer cell lines after hPRL or hPRL-G129R treatment. Our data suggests that Bcl-2 is up-regulated in response to hPRL stimulation and is competitively inhibited by hPRL-G129R in the majority of the cell lines tested. Thus, we propose that the anti-apoptotic role of hPRL in breast cancer is mediated, at least in part, through regulation of Bcl-2.