Peptide Growth Factor Signaling Research Articles

TrkA + sensory neurons regulate osteosarcoma proliferation and vascularization to promote disease progression.

Bone pain is a presenting feature of bone cancers such as osteosarcoma (OS), relayed by skeletal-innervating peripheral afferent neurons. Potential functions of tumor-associated sensory neurons in bone cancers beyond pain sensation are unknown. To uncover neural regulatory functions, a chemical-genetic approach in mice with a knock-in allele for TrkA was used to functionally perturb sensory nerve innervation during OS growth and disease progression. TrkA inhibition in transgenic mice led to significant reductions in sarcoma-associated sensory innervation and vascularization, tumor growth and metastasis, and prolonged overall survival. Single-cell transcriptomics revealed that sarcoma denervation was associated with phenotypic alterations in both OS tumor cells and cells within the tumor microenvironment, and with reduced calcitonin gene-related peptide (CGRP) and vascular endothelial growth factor (VEGF) signaling. Multimodal and multi-omics analyses of human OS bone samples and human dorsal root ganglia neurons further implicated peripheral innervation and neurotrophin signaling in OS tumor biology. In order to curb tumor-associated axonal ingrowth, we next leveraged FDA-approved bupivacaine liposomes leading to significant reductions in sarcoma growth, vascularity, as well as alleviation of pain. In sum, TrkA-expressing peripheral neurons positively regulate key aspects of OS progression and sensory neural inhibition appears to disrupt calcitonin receptor signaling (CALCR) and VEGF signaling within the sarcoma microenvironment leading to significantly reduced tumor growth and improved survival. These data suggest that interventions to prevent pathological innervation of osteosarcoma represent a novel adjunctive therapy to improve clinical outcomes and survival.

Biomarker Analyses of Response to Cyclin-Dependent Kinase 4/6 Inhibition and Endocrine Therapy in Women with Treatment-Naïve Metastatic Breast Cancer.

Preclinical data identified the cyclin-dependent kinase 4/6 (CDK4/6) inhibitor palbociclib as synergistic with antiestrogens in inhibiting growth of hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2-) human breast cancer models. This observation was validated clinically in the randomized, placebo-controlled, phase III PALOMA-2 study. To determine markers of sensitivity and resistance to palbociclib plus letrozole, we performed comprehensive biomarker analyses, investigating the correlation with progression-free survival (PFS), on baseline tumor tissues from PALOMA-2. Despite a broad biomarker search, palbociclib plus letrozole demonstrated consistent PFS gains versus placebo plus letrozole, with no single biomarker or cassette of markers associated with lack of benefit from combination treatment. Palbociclib plus letrozole confers efficacy on both luminal A and B patients. Higher CDK4 levels were associated with endocrine resistance which was mitigated by the addition of palbociclib, whereas lower PD-1 levels were associated with greater palbociclib plus letrozole benefit. Tumors with more active growth factor signaling, as exemplified by increased expression of FGFR2 and ERBB3 mRNA, appeared to be associated with greater PFS gain from the addition of palbociclib. These data underscore the importance of CDK4/6 signaling in HR+/HER2- breast cancer and suggest that the interplay between steroid hormone and peptide growth factor signaling could drive dependence on CDK4/6 signaling.See related commentary by Anurag et al., p. 3.

Abstract P2-09-10: Comprehensive gene expression biomarker analysis of CDK 4/6 and endocrine pathways from the PALOMA-2 study

Abstract Background: Palbociclib (P) is a potent, oral, highly selective, inhibitor of cyclin-dependent kinase (CDK) 4 and 6. In the Phase 3 PALOMA-2 study, P in combination with letrozole (L) demonstrated a significant improvement in progression-free survival (PFS) versus placebo (PBO) plus L. To further explore predictive markers for sensitivity and resistance to CDK4/6-based therapy or endocrine therapy (ET) alone, we performed a large gene expression analysis of baseline tumor tissues using mature PFS data with extended follow-up. Methods: Postmenopausal women (N=666) with no prior systemic therapy for ER+/HER2- ABC were randomized 2:1 to P+L or PBO+L until disease progression, death, unacceptable toxicity, or consent withdrawal. All patients provided tumor tissues to participate in the study. 455 of 666 intent-to-treat (ITT) patients had tumor tissue appropriate for gene expression evaluation (303 [68%] P+L arm and 152 [68%] PBO+L arm). EdgeSeq Oncology Biomarker Panel (HTG Molecular Diagnostics; Tucson, AZ) was used for mRNA profiling, assessing 2534 genes involved in a variety of cancer-related pathways. Using systematic and unsupervised approaches, gene expression levels were interrogated for identification of potential genes and/or pathways associated with a treatment effect of P+L compared with PBO+L. Results: With median follow-up of 38 months, mPFS of P+L vs L + PBO was 27.6 mos vs 14.5 mos (HR=0.563 [p<0.000001] in ITT). PFS in the biomarker-assessed group was similar. Using a supervised approach, expression of genes involved in the Cyclin D-CDK4/6-RB pathway were analyzed. This demonstrated that patients receiving P+L had a consistent benefit similar to the ITT population irrespective of various expression levels of CCND1, CCNE1/2, CDK2/4/6, RB1, and CDKN2A. These results are consistent with findings from previous IHC data (CCND1, RB, p16). Unsupervised analysis revealed that tumors with higher levels of growth factor receptors (eg. FGFR2 FDR 0.032, interaction p=0.056 and ERBB3 FDR 0.221, interaction p=0.043,) were associated with greater sensitivity to P+L vs L alone. Using parallel gene signature-based analyses, tumors with higher expression of a growth factor signature had longer PFS in the P+L arm. In addition, higher CDK4 expression was identified as a resistance marker for PBO+L arm (FDR 0.095, interaction p=0.016). Gene expression-based molecular subtyping demonstrated that both luminal A and B subtypes benefited equally from P+L vs PBO+L. Patients with lower level of tumor PD-1 expression showed more benefit from P+L (FDR 0.099, interaction p=0.021). Conclusion: These results confirm efficacy of palbociclib + letrozole in ER+/HER2- breast cancer, and support the use of ER+ as a biomarker for sensitivity to CDK 4/6 inhibition. Expression levels (whether high or low) of genes in the Cyclin D-CDK4/6-RB pathway did not correlate with benefit from palbociclib + letrozole. These data provide evidence that the interplay between steroid hormone and peptide growth factor signaling in ER+ breast cancer drives dependence on CDK 4/6 and benefit from CDK 4/6 inhibition with palbociclib. These data can be used to guide additional therapeutic opportunities in ER+/HER2- ABC. Sponsor: Pfizer Citation Format: Finn RS, Liu Y, Martin M, Rugo HS, Dieras V, Im S-A, Gelmon K, Harbeck N, Zhu Z, Lu D, Gauthier E, Bartlett CH, Slamon DJ. Comprehensive gene expression biomarker analysis of CDK 4/6 and endocrine pathways from the PALOMA-2 study [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-10.

Homozygous truncating PTPRF mutation causes athelia.

Athelia is a very rare entity that is defined by the absence of the nipple-areola complex. It can affect either sex and is mostly part of syndromes including other congenital or ectodermal anomalies, such as limb-mammary syndrome, scalp-ear-nipple syndrome, or ectodermal dysplasias. Here, we report on three children from two branches of an extended consanguineous Israeli Arab family, a girl and two boys, who presented with a spectrum of nipple anomalies ranging from unilateral hypothelia to bilateral athelia but no other consistently associated anomalies except a characteristic eyebrow shape. Using homozygosity mapping after single nucleotide polymorphism (SNP) array genotyping and candidate gene sequencing we identified a homozygous frameshift mutation in PTPRF as the likely cause of nipple anomalies in this family. PTPRF encodes a receptor-type protein phosphatase that localizes to adherens junctions and may be involved in the regulation of epithelial cell-cell contacts, peptide growth factor signaling, and the canonical Wnt pathway. Together with previous reports on female mutant Ptprf mice, which have a lactation defect, and disruption of one allele of PTPRF by a balanced translocation in a woman with amastia, our results indicate a key role for PTPRF in the development of the nipple-areola region.

Brivanib: A Review of Development

The development of new agents in oncology has focused on disrupting key pathways in oncogenesis. Both malignant angiogenesis and peptide growth factor signaling have been studied extensively and have been validated for cancer treatment. While antibody-directed therapeutics offer increased specificity, small-molecule tyrosine kinase inhibitors often have the ability to hit multiple targets. Brivanib alaninate (BMS582664) is an oral, potent selective inhibitor of both the FGF and VEGF family of receptors. It is a first-in-class FGF/VEGF inhibitor now in late-phase clinical trials. Besides its antiangiogenic activity from blocking VEGF receptor 1-3, its ability to disrupt FGF receptors 1-3 has been suggested to add additional antiangiogenic activity, overcome resistance from VEGF blockade, and block FGF-dependent tumor proliferation. In this review, we will discuss the preclinical science driving brivanib's development and the clinical data generated to date.

Estrogen Receptor, Progesterone Receptor, Human Epidermal Growth Factor Receptor 2 (HER2), and Epidermal Growth Factor Receptor Expression and Benefit From Lapatinib in a Randomized Trial of Paclitaxel With Lapatinib or Placebo As First-Line Treatment in HER2-Negative or Unknown Metastatic Breast Cancer

Lapatinib is a dual inhibitor of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) with activity in HER2-amplified metastatic breast cancer (MBC). Its role in non-HER2-amplified MBC remains unclear. EGF30001, a phase III trial of lapatinib and paclitaxel versus paclitaxel and placebo, demonstrated lapatinib does not significantly benefit HER2-negative or HER2-unselected patients with MBC. Published data support interactions between steroid hormone and peptide growth factor signaling. We hypothesized that molecular subgroups may exist within EGF30001 that would benefit from lapatinib. A blinded, retrospective biomarker evaluation was performed using immunohistochemistry to semiquantitate estrogen (ER), progesterone (PR), and EGFR expression. HER2 amplification was determined by fluorescent in situ hybridization. Effects of these biomarkers on event-free survival (EFS) were examined in patients with available tissue (n = 493). Lapatinib improved median EFS in HER2-amplified, ER- or PR-positive MBC (n = 36; 5.7 v 4.5 months; P = .351); benefit was greater and statistically significant in HER2-amplified, ER-negative, PR-negative MBC (n = 42; 8.3 v 5.0 months; P = .007). In HER2-negative, ER-positive MBC, median EFS improvement varied by degree of PR expression (H-score): no benefit if PR-strong (n = 133; 9.3 v 7.3 months; P = .373), benefit if PR-weak (n = 50; 7.3 v 2.4 months; P = .026), and potential antagonism if PR-negative (n = 40; 3.7 v 7.2 months; P = .004). No benefit was seen in triple-negative MBC (n = 131; median EFS, 4.6 v 4.8 months; P = .255). EGFR expression was not correlated with benefit from lapatinib. Although subgroups are small, these analyses support the hypothesis that semiquantitative determination of hormone receptor status may be a surrogate for EGFR and/or HER2 dependency.

Progression-free survival (PFS) of patients with HER2-negative, estrogen-receptor (ER)-low metastatic breast cancer (MBC) with the addition of lapatinib to letrozole: Biomarker results of EGF30008

1018 Background: Lapatinib (Lap), an oral, dual inhibitor of EGFR/HER-2, is approved with capecitabine for treatment of HER-2+ MBC for recurrence after taxane, anthracycline, and trastuzumab-based therapy. Letrozole (Let) has activity in hormone-receptor positive (HR+) BC. It has been hypothesized that peptide growth factor signaling and hormone signaling pathways interact and dual targeting of these pathways results in therapeutic synergy. We report a blinded analysis of HER-2, ER, and progesterone receptor (PR) expression for patients (pts) with HR+ MBC at first diagnosis or post-adjuvant relapse and response to Lap + Let versus Let. Methods: 1286 pts were randomized 1:1 to 2.5 mg Let daily plus either 1500 mg Lap daily or placebo. The primary endpoint was PFS in HR+, HER-2+ MBC pts; secondary endpoints included PFS in the intent-to-treat population. Blinded, centralized commerical laboratory analysis of archived tumor tissue for HER-2 (IHC and/or FISH) and academic laboratory semi-quantitative analysis of ER/PR (IHC, H-score) was performed and correlated with clinical outcome. Results: In 219/1286 (17%) HER-2+ (FISH+ or IHC3+) pts, a significant improvement in median PFS was observed for Lap + Let versus Let (8.2 v 3.0 mos, HR = 0.71, 95% CI 0.53, 0.96, p = 0.019). No significant difference in median PFS was seen in 952 (74%) HER-2-negative pts (13.4 v 13.7 mos, HR = 0.90, 95% CI 0.77, 1.05, p = 0.188). 821/952 (86%) HER-2-negative pts had tissue available for quantitative ER and PR analysis. Analysis of ER and PR by quartile identified a subgroup of HER-2-negative pts that benefitted from adding Lap to Let. Pts with the lowest quartile of ER expression (H-score <160, n = 207) had a significant improvement in median PFS (13.6 mos v 6.6 mos, HR = 0.65, 95% CI 0.47, 0.9, p < 0.005). Pts with higher levels of ER did not significantly benefit from adding Lap to Let. Analysis of PR expression did not identfiy a subgroup that benefited from Lap. Conclusions: Pts with HER-2-negative, ER+ MBC and low ER expression may benefit from the addition of lap to let. Additional analyses of EGFR, other biomarkers, and prior hormone therapy will be presented. [Table: see text]

Dual-Agent Molecular Targeting of the ErbB2 Receptor: Killing One Bird With Two Stones

The phase I study by Storniolo et al in this issue of Journal of Clinical Oncology, which evaluates the combination of lapatinib (a selective small-molecule ErbB1 and ErbB2 tyrosine kinase inhibitor) and trastuzumab (an antibody blocking the extracellular domain of ErbB2) in patients with advanced ErbB2-positive breast cancer, is the first published study to our knowledge examining dual-agent molecular targeting of the ErbB2 receptor. The study demonstrated that there was clinical activity in a group of heavily pretreated patients and—using a toxicity end point—determined an optimally tolerated regimen for this combination of agents. The epidermal growth factor family of receptors plays a key role in regulating growth, survival, and resistance to chemotherapy in many epithelial malignancies. Approximately 25% of breast cancers overexpress ErbB2 or demonstrate ErbB2 gene amplification; ErbB2 is a key mediator of tumor cell growth and survival, and a predictor of poor outcome. Potential strategies to block peptide growth factor signaling pathways include (1) blocking or neutralizing the growth factor or its production, (2) blocking the receptor-binding site or prevention of receptor dimerization, (3) blocking the receptor tyrosine kinase domain, and (4) blocking downstream signaling pathways. The mechanism of action of trastuzumab is multifactorial and includes (1) antibody-dependent cellular cytotoxity, (2) modulation of ErbB2 signaling, (3) internalization of ErbB2 cell-surface protein, (4) reduction in proteolytic cleavage of the ErbB2 extracellular domain, and (5) interference with DNA damage repair. Small-molecule inhibitors such as lapatinib have an advantage over antibodies because they inhibit multiple signaling pathways at plasma concentrations achieved in the clinic. This is relevant because the survival of epithelial tumors is often dependent on the presence of redundant signaling pathways. Combining anti-ErbB2 antibodies with small-molecule tyrosine kinase inhibitors is an appealing strategy because both therapies exert their antitumor effects via distinct mechanisms of action and target ErbB2 at different sites. Combining distinct classes of inhibitors may not only potentiate cellular cytotoxicity, but may also assist in overcoming inherent or acquired resistance to one class of inhibitors. Mechanisms of resistance identified to date include activating mutations in the domain of the adenosine triphosphate–binding pocket for small-molecule tyrosine kinase inhibitors and the presence of truncated receptors for monoclonal antibodies. Dual-agent molecular targeting has been examined in preclinical models using the ErbB1 pathway as well as the ErbB2 pathway. With respect to the ErbB1 pathway, Huang et al and Matar et al studied the effect of cetuximab (an antibody that binds to the extracellular domain of ErbB1) in combination with either gefitinib or erlotinib (small-molecule inhibitors of the tyrosine kinase domain or ErbB1) against a panel of human cancer cell lines in culture and in tumor xenografts. The combination of cetuximab plus gefitinib or erlotinib enhances growth inhibition, in culture and in vivo, over that observed with either agent alone. Phosphorylation inhibition of downstream effector molecules (mitogen-activated protein kinase and AKT), and apoptosis was enhanced with the combination. In lung and head and neck tumor cells highly resistant to cetuximab, gefitinib, and erlotinib, but not cetuximab, retained the capacity to inhibit these tumor cells and were also able to further inhibit the activation of downstream effectors of ErbB1 signaling. When tested in vivo, single-agent therapy resulted in transient tumor regression only at the highest doses, whereas suboptimal doses of gefitinib and cetuximab in combination resulted in complete and permanent regression of large tumors. Contradictory results were obtained by Fishel et al who determined that gefitinib and cetuximab, when used in combination, were antagonistic when tested against two ErbB1expressing epidermoid cell lines. The combination caused less apoptosis than gefitinib alone, and this appeared to be the result of overexpression of functional ErbB1 associated with the combination. It is likely that these discordant results were a result of the particular cell lines studied. Most relevant to the study being reviewed are the preclinical models examining dual targeting of the ErbB2 pathway. Xia et al treated ErbB2-overexpressing breast cancer cell lines with a combination of lapatinib and either trastuzumab or a polyclonal anti-ErbB2 antibody. The combination treatments led to enhanced tumor cell apoptosis, whereas treatment with the same doses of lapatinib, trastuzumab, or polyclonal anti-ErbB2 antibody alone led to minimal or no increase in apoptosis. They demonstrated that the enhanced tumor cell apoptosis after combination ErbB2-targeted therapies was most closely associated with downregulation of survivin protein. Konecny et al studied the combination of lapatinib and trastuzumab in a panel of human breast cancer cell lines. Synergistic activity was seen when the combination was tested in ErbB2-overexpressing cell lines. Of JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 26 NUMBER 20 JULY 1

Clinical strategies for rationale combinations of aromatase inhibitors with novel therapies for breast cancer

Improving endocrine responsiveness and preventing the development of resistance is the goal of many current strategies that are looking to combine aromatase inhibitors with novel drugs that target various pathways in estrogen receptor (ER) positive breast cancer. Pre-clinical models of acquired resistance to aromatase inhibitors have suggested an increase in several signaling pathways including peptide growth factor signaling (EGFR, HER2) and activation of the mTOR signaling pathway. These may result in associated ‘cross-talk’ activation of ER-dependent gene transcription, such that dual blockade of ER together with other signaling pathways has become a logical approach to improve endocrine responsivness. Clinical strategies with aromatase inhibitors are looking to prevent activation of these pathways either through combination with the selective ER downregulator fulvestrant, or with various signal transduction inhibitors (STIs) including monoclonal antibodies (trastuzumab), small molecule tyrosine kinase inhibitors (TKIs) against EGFR or HER2 (lapatinib, gefitinib) and mTOR antagonists (temsirolimus). Early clinical data have emerged this year for some of these approaches with mixed results. This article reviews the rationale for these strategies, and discusses the lessons that need to be learnt if we are to successfully integrate these new drugs with aromatase inhibitors in the clinic.

Clinical trials of intracellular signal transductions inhibitors for breast cancer — a strategy to overcome endocrine resistance

Acquired resistance to endocrine therapy in breast cancer is associated with an increase in peptide growth factor signaling that results in cross-talk activation of the estrogen receptor (ER). Small molecule signal transduction inhibitors (STIs) can target components of these intracellular pathways, and may prove effective in anticancer therapy. However, early phase II clinical trials with various STIs as monotherapy in advanced breast cancer have shown only a modest level of efficacy for these intracellular inhibitors. Preclinical data suggest that combinations of tamoxifen with STIs may provide significantly greater growth inhibition than either therapy alone, and, furthermore, may delay the emergence of endocrine resistance. There are now several trials assessing the efficacy of combinations of small molecule tyrosine kinase inhibitors (TKIs), such as gefitinib and lapatinib, with either tamoxifen or aromatase inhibitors both in the second-line, endocrine-resistant and first-line, hormone-sensitive setting. Similar trials continue with both farnesyltransferase inhibitors (FTIs) and mTOR antagonists, where there are strong preclinical data to suggest additive or synergistic effects for either of these agents in combination with endocrine therapies. Biomarker studies in the presurgical setting are also being utilized as an alternative approach to investigate whether combined endocrine/STI therapy is an effective clinical strategy. This article reviews some of the preclinical evidence supporting this strategy, together with the current status of clinical trials in this area.

Aromatase inhibitors: Combinations with fulvestrant or signal transduction inhibitors as a strategy to overcome endocrine resistance

There is an increasing rationale for effective combinations of endocrine therapy with novel drugs that target aberrant signal transduction pathways in estrogen receptor (ER) positive breast cancer. Prolonged endocrine therapy can be associated with an acquired increase in peptide growth factor signaling (EGFR, HER2), together with cross-talk activation of ER-dependent gene transcription and cell growth that leads to endocrine resistance. Current approaches to target these pathways include both the selective ER downregulator fulvestrant, and various signal transduction inhibitors (STIs). Fulvestrant can overcome resistance to tamoxifen (TAM-R) and long-term estrogen deprivation (LTED-R) in experimental models by reducing ER expression, and represents a current option for post-menopausal women with endocrine resistant ER+ve breast cancer. Emerging data suggest that fulvestrant's effect may be greater when combined with estrogen deprivation, and several phase III trials are assessing fulvestrant combined with aromatase inhibitors (AIs). Small molecule STIs such as tyrosine kinase inhibitors (TKIs), farnesyltransferase inhibitors (FTIs) and mTOR antagonists are also active in breast cancer. Pre-clinical data suggest that combined endocrine/STI therapy may result in greater growth inhibition than either therapy alone, and thus delay emergence of resistance. Several clinical trials are now examining STIs combined with AIs both in the tamoxifen-resistant and first-line advanced breast cancer setting, while pre-surgical studies are investigating the efficacy of combined endocrine/STI therapy utilising biological primary endpoints. This article reviews the pre-clinical rationale for this strategy and the clinical trials in this area.

Combinations of Endocrine and Biological Agents: Present Status of Therapeutic and Presurgical Investigations

There is an increasing rationale to develop effective combinations of endocrine agents with novel therapeutics that target aberrant signal transduction pathways in estrogen receptor-positive breast cancer. Acquired resistance to endocrine therapy is associated with an increase in peptide growth factor signaling that results in crosstalk activation of estrogen receptor, and various signal transduction inhibitors (STI) can target these pathways to inhibit hormone-resistant growth. In experimental models of hormone-sensitive breast cancer, combinations of endocrine agents with STIs provide significantly greater growth inhibition than either alone, delaying the emergence of resistance. There are now several trials assessing the efficacy of combinations of tyrosine kinase inhibitors with various endocrine agents in the tamoxifen-resistant/second-line setting, together with five randomized phase II/III trials in the first-line setting. Similar work is ongoing with both farnesyltransferase inhibitors and mTOR antagonists where there are strong preclinical data to suggest additive or synergistic effects for either of these agents in combination with tamoxifen or estrogen deprivation therapies. More recently, presurgical studies with biological primary end points are being utilized as an alternative approach to investigate whether combined endocrine/STI therapy is a more effective strategy than endocrine therapy alone. This article reviews the rationale and current status of clinical trials in this area as well as the challenges that lie ahead for the development of these therapeutic combinations for breast cancer.

Growth factor signaling in lung morphogenetic centers: automaticity, stereotypy and symmetry.

Lung morphogenesis is stereotypic, both for lobation and for the first several generations of airways, implying mechanistic control by a well conserved, genetically hardwired developmental program. This program is not only directed by transcriptional factors and peptide growth factor signaling, but also co-opts and is modulated by physical forces. Peptide growth factors signal within repeating epithelial-mesenchymal temporospatial patterns that constitute morphogenetic centers, automatically directing millions of repetitive events during both stereotypic branching and nonstereotypic branching as well as alveolar surface expansion phases of lung development. Transduction of peptide growth factor signaling within these centers is finely regulated at multiple levels. These may include ligand expression, proteolytic activation of latent ligand, ligand bioavailability, ligand binding proteins and receptor affinity and presentation, receptor complex assembly and kinase activation, phosphorylation and activation of adapter and messenger protein complexes as well as downstream events and cross-talk both inside and outside the nucleus. Herein we review the critical Sonic Hedgehog, Fibroblast Growth Factor, Bone Morphogenetic Protein, Vascular Endothelial Growth Factor and Transforming Growth Factorbeta signaling pathways and propose how they may be functionally coordinated within compound, highly regulated morphogenetic gradients that drive first stereotypic and then non-stereotypic, automatically repetitive, symmetrical as well as asymmetrical branching events in the lung.

Transcriptional hyperactivity of human progesterone receptors is coupled to their ligand-dependent down-regulation by mitogen-activated protein kinase-dependent phosphorylation of serine 294.

Breast cancers often exhibit elevated expression of tyrosine kinase growth factor receptors; these pathways influence breast cancer cell growth in part by targeting steroid hormone receptors, including progesterone receptors (PR). To mimic activation of molecules downstream of growth factor-initiated signaling pathways, we overexpressed mitogen-activated protein kinase (MAPK; also known as extracellular signal-regulated kinase) kinase kinase 1 (MEKK1) in T47D human breast cancer cells expressing the B isoform of PR. MEKK1 is a strong activator of p42 and p44 MAPKs. MEKK1 expression increased progestin-mediated transcription 8- to 10-fold above normal PR-driven transcription levels. This was dependent on the presence of a progesterone response element and functional PR. PR protein levels were unchanged by MEKK1 alone but were extensively down-regulated by MEKK1 plus the progestin R5020. MEKK1 expression resulted in phosphorylation of PR on Ser294, a MAPK consensus site known to mediate ligand-dependent PR degradation. MEK inhibitors blocked phosphorylation of Ser294 and attenuated PR transcriptional hyperactivity in response to MEKK1 plus R5020; stabilization of PR by inhibition of the 26S proteasome produced similar results. T47D cells stably expressing mutant S294A PR, in which serine 294 is replaced by alanine, fail to undergo ligand-dependent down-regulation and are resistant to MEKK1-plus-R5020-induced transcriptional synergy but respond to progestins alone. Similarly, c-myc protein levels are synergistically increased by epidermal growth factor and R5020 in cells expressing wild-type PR, but not S294A PR. Thus, highly stable mutant PR are functional in response to progestins but are incapable of cross talk with MAPK-driven pathways. These studies demonstrate a paradoxical coupling between steroid receptor down-regulation and transcriptional hyperactivity. They also suggest a link between phosphorylation of PR by MAPKs in response to peptide growth factor signaling and steroid hormone control of breast cancer cell growth.

Commitment and differentiation of lung cell lineages

To form a large diffusible interface capable of conducting respiratory gases to and from the circulation, the lung must undergo extensive cell proliferation, branching morphogenesis, and alveolar saccule formation, to generate sufficient surface area. In addition, the cells must differentiate into at least 40 distinct lung cell lineages. Specific transcriptional factors, peptide growth factor receptor-mediated signaling pathways, extracellular matrix components, and integrin-signaling pathways interact to direct lung morphogenesis and lung cell lineage differentiation. Branching mutants of the respiratory tracheae in Drosophila have identified several functionally conserved genes in the fibroblast growth factor signaling pathway that also regulate pulmonary organogenesis in mice and probably also in man. Key transcriptional factors including Nkx2.1, hepatocyte nuclear factor family forkhead homologues, GATA family zinc finger factors, pou and homeodomain proteins, as well as basic helix-loop-helix factors, serve as master genes to integrate the developmental genetic instruction of lung morphogenesis and cell lineage determination. Lung mesenchyme serves as a 'compleat' inducer of lung morphogenesis by secreting soluble peptide growth factors. In general, peptide growth factors signaling through cognate receptors with tyrosine kinase intracellular signaling domains such as epidermal growth factor receptor, fibroblast growth factor receptors, hepatocyte growth factor/scatter factor receptor, c-met, insulin-like growth factor receptor, and platelet-derived growth factor receptor, stimulate lung morphogenesis, while the cognate receptors with serine/threonine kinase intracellular signaling domains, such as the transforming growth factor-beta receptor family are inhibitory. The extracellular matrix also plays a key role in determining branching morphogenesis. Pulmonary neuroendocrine (PNE) cells differentiate earliest in gestation among lung epithelial cells. PNE cells are principally derived from endoderm and not neural crest. PNE cells have been proposed to function as airway chemoreceptors, while PNE cell secretory granules contain many bioactive substances such as GRP which may direct proliferation of adjacent epithelial cells. Mammalian achaete-schute homolog-1 null mutant mice do not develop PNE cells. Candidate molecular switches in the transition from a quiescent to a proliferative alveolar epithelial cell (AEC) phenotype and back again following acute hyperoxia, include autocrine peptide growth factor signaling pathways and cell cycle regulatory elements. AEC type 2 also appear capable of reversible transdifferentiation into AEC type 1 and intermediate phenotypes in response to cues from extracellular matrix and cell shape, as well as soluble factors. Evidence for expression of telomerase by alveolar epithelial stem cells, which correlates with self-renewal potential, is now beginning to emerge. Lung regeneration following lobectomy in juvenile rodents is associated with co-ordinated cell proliferation, re-expression of elastin and formation of alveoli. Retinoic acid has recently shown promise as a stimulator of alveolization in juvenile rats. Our future goal is to devise new rational and gene therapeutic strategies to stimulating lung growth and maturation, ameliorating lung injury, augmenting lung repair, and inducing lung regeneration. The ideal agent or agents would therefore mimic the instructive role of lung mesenchyme, correctly inducing the temporospatial pattern of lung cell lineages necessary to restore pulmonary gas diffusing capacity.

Cross-talk between peptide growth factor and estrogen receptor signaling pathways.

The classical receptor for estradiol is a member of a super-family of nuclear receptors that function as hormone-regulated transcription factors. The ability of the estrogen receptor (ER)-alpha to activate target gene transcription is mediated by two transcriptional activation functions (AF): AF-1 located in the amino-terminal domain and AF-2 found in the carboxyl-terminal portion of the molecule. The ligand binding domain overlaps AF-2, and upon estrogen binding this region undergoes a conformational change that enables it to contribute to the receptor's transcriptional activity. ER activation is accompanied by increased phosphorylation, and in the absence of ligand, activators of protein kinase A or inhibitors of protein phosphatases are able to stimulate ER-dependent gene expression. More importantly, polypeptide growth factors, such as epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I), also stimulate the ER's transcriptional activity in an estrogen-independent manner. The AF-1 domain appears to be required for activation by EGF and IGF-I, and point mutation of a single phosphorylation site located within this domain inhibits the ability of growth factor to activate the ER. Thus, steroid receptor function may be regulated by estrogenic ligands as well as by pathway "cross-talk" from membrane receptors for growth factors.

Cross Talk between Peptide Growth Factor and Estrogen Receptor Signaling Systems

Cross Talk between Peptide Growth Factor and Estrogen Receptor Signaling Systems

Cross talk between peptide growth factor and estrogen receptor signaling systems.

Epidermal growth factor reproduces many of the effects of estrogen on the murine female reproductive tract and may partially mediate estrogen-induced growth and differentiation. The mechanism by which the actions of estrogens and epidermal growth factor (EGF) converge is unknown. The studies described herein were performed to investigate the possibility that some of the actions of EGF may be mediated through the estrogen receptor. A specific estrogen receptor (ER) antagonist inhibited estrogenlike effects of EGF in the mouse uterus, specifically induction of DNA synthesis and phosphatidylinositol turnover. In addition, EGF elicited enhanced nuclear localization of uterine ER and formation of a unique nuclear form of ER that is present after estrogen treatment. These in vivo observations indicated that EGF may elicit some of its actions by activation of nuclear ER. Thus, the effect of peptide growth factors on activation of a consensus estrogen response element was assessed in Ishikawa human endometrial adenocarcinoma cells, which contain negligible ER levels, and in BG-1 human ovarian adenocarcinoma cells, which contain abundant ER. EGF and TGF alpha induced transcriptional activation of a consensus estrogen response element (ERE) in an ER-dependent manner in both cell types. In addition, insulinlike growth factor I (IGF-I) was as potent as 17 beta-estradiol in BG-1 cells. Synergism between growth factors and estrogen was observed in both cell types, although synergism was not observed between the different classes of growth factors [i.e., transforming growth factor alpha (TGF alpha) and IGF-I] in BG-1 cells. The most potent activator of ERE-dependent transcription was a protein kinase C activator (TPA), which acted synergistically with 17 beta-estradiol. A protein kinase C inhibitor abolished the effect of TPA but not that of 17 beta-estradiol, IGF-I, or TGF alpha. A protein kinase A activator elicited ER-dependent activation of transcription and did not synergize with estrogen or growth factors. In conclusion, some physiologic actions of peptide growth factors are dependent on ER. Indeed, growth factors are capable of eliciting ER-dependent activation of an ERE. Both the protein kinase A and protein kinase C pathways can elicit ER-dependent transcriptional activation; however, it is unlikely that these pathways mediate the effects of peptide growth factors on the ER in BG-1 cells.