STAT3 attenuates EGFR-mediated ERK activation and cell survival during oxidant stress in mouse proximal tubular cells.

We have shown that renal epithelial cell survival depends on the sustained activation of the extracellular signal-regulated protein kinase (ERK) and lack of this activation was associated with death during oxidative stress. ERK is activated via the canonical epidermal growth factor receptor (EGFR)-Ras-MEK pathway, which could be attenuated by oxidants. We now show that the failure to activate ERK in a sustained manner during severe oxidative stress is owing to the activation of the signal transducer and activator of transcription-3 (STAT3) rather than the failure to activate the EGFR. Tyrosine phosphorylation of the EGFR and STAT3 was studied in hydrogen peroxide (H(2)O(2))-treated mouse proximal tubule (TKPTS) cells or in mouse kidney after ischemia/reperfusion (I/R) injury by Western blotting. STAT3 activation was inhibited by either pharmacologically (AG490) through its upstream janus kinase (JAK2) or by a dominant-negative STAT3 adenovirus. EGFR was inhibited by AG1478. Survival was determined by fluorescence-activated cell sorter analysis and trypan blue exclusion. We found that the EGFR was phosphorylated on its major autophosphorylation site (Tyr1173) regardless of the H(2)O(2) dose. On the other hand, both I/R and severe oxidative stress - but not moderate stress - increased tyrosine phosphorylation of STAT3 in an EGFR and JAK2-dependent manner. Inhibition of JAK2 or STAT3 lead to increased ERK activation and survival of TKPTS cells during severe oxidative stress. Our data suggest a role of tyrosine-phosphorylated STAT3 in the suppression of ERK activation. These data suggest that the STAT3 pathway might represent a new target for improved survival of proximal tubule cells exposed to severe oxidant injury.

Mammalian Scratch participates in neuronal differentiation in P19 embryonal carcinoma cells.

Mammalian Scratch (Scrt) is a Snail family zinc finger transcription factor that is specifically expressed in newly differentiating, post-mitotic central nervous system neurons. While Scrt-related genes appear essential for invertebrate neurogenesis, the role of Scrt in mammalian neural development is unknown. In this study, we found that neural differentiation of multipotent mouse P19 embryonal carcinoma cells by retinoic acid led to the appearance of Scrt together with neuron-specific class III beta-tubulin (Tuj1), following the earlier elaboration of Mash1. Transient co-transfection in P19 cells with either Mash1 or NeuroD2 plus E12 also induced Scrt gene expression. Moreover, overexpression of Scrt alone was sufficient to confer Tuj1 immunoreactivity and neuronal morphology in a subset of P19 cells. Scrt thus appears to function downstream of proneural bHLH proteins in promoting mammalian neural differentiation in this model system.

BARX2 induces cadherin 6 expression and is a functional suppressor of ovarian cancer progression.

The human homeobox BARX2 is located at 11q24-q25, within a minimal region associated with frequent loss of heterozygosity and adverse survival in epithelial ovarian cancer. BARX2 is a transcription factor that regulates transcription of specific cell adhesion molecules in the mouse. We show that BARX2 and cadherin 6 are expressed in normal human ovarian surface epithelium. BARX2 and cadherin 6 both have significantly lower expression in a clinical sample of endometrioid and clear cell ovarian cancers, as compared with serous or mixed mesodermal tumors. In a series of ovarian cancer cell lines, BARX2 expression showed a significant direct correlation with cadherin 6 expression. In OAW42, an ovarian cancer cell line that does not endogenously express BARX2, in vitro transfection of human BARX2 cDNA induced cadherin 6 expression. Transfection of BARX2 into OAW42 inhibited Matrigel invasion, haptotactic cellular migration to a collagen IV signal, and adhesion to collagen IV-coated plates. Our data demonstrate that BARX2 is expressed in the ovarian surface epithelium and has functional suppressor properties in ovarian cancer cells.

Notch signaling induces cell cycle arrest in small cell lung cancer cells.

Among the various forms of human lung cancer, small cell lung cancer (SCLC) exhibits a characteristic neuroendocrine (NE) phenotype. Neural and NE differentiation in SCLC depend, in part, on the action of the basic-helix-loop-helix (bHLH) transcription factor human achaete-scute homologue-1 (hASH1). In nervous system development, the Notch signaling pathway is a critical negative regulator of bHLH factors, including hASH1, controlling cell fate commitment and differentiation. To characterize Notch pathway function in SCLC, we explored the consequences of constitutively active Notch signaling in cultured SCLC cells. Recombinant adenoviruses were used to overexpress active forms of Notch1, Notch2, or the Notch effector protein human hairy enhancer of split-1 (HES1) in DMS53 and NCI-H209 SCLC cells. Notch proteins, but not HES1 or control adenoviruses, caused a profound growth arrest, associated with a G1 cell cycle block. We found up-regulation of p21(waf1/cip1) and p27kip1 in concert with the cell cycle changes. Active Notch proteins also led to dramatic reduction in hASH1 expression, as well as marked activation of phosphorylated extracellular signal-regulated kinase (ERK)1 and ERK2, findings that have been shown to be associated with cell cycle arrest in SCLC cells. These data suggest that the previously described function of Notch proteins as proto-oncogenes is highly context-dependent. Notch activation, in the setting of a highly proliferative hASH1-dependent NE neoplasm, can be associated with growth arrest and apparent reduction in neoplastic potential.

Mammalian Scratch: a neural-specific Snail family transcriptional repressor.

Members of the Snail family of zinc finger transcription factors are known to play critical roles in neurogenesis in invertebrates, but none of these factors has been linked to vertebrate neuronal differentiation. We report the isolation of a gene encoding a mammalian Snail family member that is restricted to the nervous system. Human and murine Scratch (Scrt) share 81% and 69% identity to Drosophila Scrt and the Caenorhabditis elegans neuronal antiapoptotic protein, CES-1, respectively, across the five zinc finger domain. Expression of mammalian Scrt is predominantly confined to the brain and spinal cord, appearing in newly differentiating, postmitotic neurons and persisting into postnatal life. Additional expression is seen in the retina and, significantly, in neuroendocrine (NE) cells of the lung. In a parallel fashion, we detect hScrt expression in lung cancers with NE features, especially small cell lung cancer. hScrt shares the capacity of other Snail family members to bind to E-box enhancer motifs, which are targets of basic helix--loop--helix (bHLH) transcription factors. We show that hScrt directly antagonizes the function of heterodimers of the proneural bHLH protein achaete-scute homolog-1 and E12, leading to active transcriptional repression at E-box motifs. Thus, Scrt has the potential to function in newly differentiating, postmitotic neurons and in cancers with NE features by modulating the action of bHLH transcription factors critical for neuronal differentiation.

Combretastatin A-4 and doxorubicin combination treatment is effective in a preclinical model of human medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC), both in patients and in preclinical models, is resistant to chemotherapy. In this study, we show that the anti-neovascular agent combretastatin A-4 phosphate prodrug (CA4P) in combination with doxorubicin was effective in curtailing tumor growth in a preclinical model of human MTC. This combination of combretastatin and doxorubicin extended the doubling time of established MTC tumors in nude mice to 29 days, compared to 12 days in untreated controls. This suggests that a combination of combretastatin and a cytotoxic chemotherapeutic agent may be an effective treatment for MTC.

Reactive oxygen species are critical for the growth and differentiation of medullary thyroid carcinoma cells.

Reactive oxygen species have recently been demonstrated to play a role in numerous cellular signal transduction pathways. Here we investigate the involvement of H2O2 in Raf-1-mediated differentiation in the human medullary thyroid carcinoma (MTC) cell line TT:deltaRaf-1:ER. Catalase, but not Cu/Zn superoxide dismutase, completely inhibited Raf-1-induced differentiation of beta-estradiol-treated TT: deltaRaf-1:ER. In addition, catalase treatment down-regulated RET expression at both the mRNA and protein levels and induced apoptosis in the parental TT cell line and uninduced TT:deltaRaf-1:ER human MTC cells. These results implicate H2O2 as a downstream mediator of c-Raf-1-induced differentiation and as a survival factor in MTC cells.

Constitutive achaete-scute homologue-1 promotes airway dysplasia and lung neuroendocrine tumors in transgenic mice.

The transcription factor achaete-scute homologue-1 (ASH1) is essential for neural differentiation during fetal development and is a cardinal feature of neuroendocrine (NE) tumors such as small cell lung cancer. To explore the potential of ASH1 to promote NE differentiation and tumorigenesis in the lung, we constitutively expressed the factor in nonendocrine airway epithelial cells using transgenic mice. Progressive airway hyperplasia and metaplasia developed beginning at 3 weeks of life. ASH1 potently enhanced the tumorigenic effect of SV40 large T antigen in airway epithelium. These doubly transgenic animals developed massive NE lung tumors, implying that ASH1 may cooperate with defects in p53, pRb, or related pathways in promoting NE lung carcinogenesis.

Cloning and chromosomal localization of the human BARX2 homeobox protein gene.

The human BARX2 gene encodes a homeodomain-containing protein of 254 amino acids, which binds optimally to the DNA consensus sequence YYTAATGRTTTTY. BARX2 is highly expressed in adult salivary gland and is expressed at lower levels in other tissues, including mammary gland, kidney, and placenta. The BARX2 gene consists of four exons, and is located on human chromosome 11q25. This chromosomal location is within the minimal deletion region for Jacobsen syndrome, a syndrome including craniosynostosis and other developmental abnormalities. This chromosomal location, along with the reported expression of murine barx2 in craniofacial development, suggests that BARX2 may be causally involved in the craniofacial abnormalities in Jacobsen syndrome.

Roles of trk family neurotrophin receptors in medullary thyroid carcinoma development and progression.

Although initiating mutations in the ret protooncogene have been found in familial and sporadic medullary thyroid carcinoma (MTC), the molecular events underlying subsequent tumor progression stages are unknown. We now report that changes in trk family neurotrophin receptor expression appear to be involved in both preneoplastic thyroid C cell hyperplasia and later tumor progression. Only a subset of normal C cells expresses trk family receptors, but, in C cell hyperplasia, the affected cells consistently express trkB, with variable expression of trkA and trkC. In later stages of gross MTC tumors, trkB expression was substantially reduced, while trkC expression was increased and often intense. In a cell culture model of MTC, exogenous trkB expression resulted in severely impaired tumorigenicity and was associated with 11-fold lower levels of the angiogenesis factor vascular endothelial growth factor. These results suggest that trk family receptor genes participate in MTC development and progression, and, in particular, that trkB may limit MTC tumor growth by inhibition of angiogenesis.

Raf-1 causes growth suppression and alteration of neuroendocrine markers in DMS53 human small-cell lung cancer cells.

Ras mutations are common in lung adenocarcinomas and squamous-cell cancers, which are non-small-cell lung cancers (NSCLCs). However, small-cell lung cancers (SCLCs) rarely have ras mutations, suggesting that ras activation may not confer a growth advantage in these cells. In one SCLC cell line DMS53, activated ras expression induced increased neuroendocrine differentiation and decreased cell proliferation. We show here that DMS53 cells undergo differentiation and G1-specific growth arrest in response to ras/raf/ mitogen-activated protein kinase kinase (MEK)/mitogen-activated protein kinase (MAPK) pathway activation. To assess the consequences of activating the raf/MEK/MAPK pathway downstream of ras, we transfected a DMS53 cell line with DeltaRaf-1:ER, an activatable form of c-raf-1. DeltaRaf-1:ER activation suppressed cell proliferation and cloning on soft agar by 90% without evidence of apoptosis. Cell cycle analysis showed a reduced proportion of cells in S phase, and was associated with induction of the cyclin-dependent kinase (cdk) inhibitor p16(INK4). Expression of the cell cycle-specific proteins pRb, Rb2/p130, p107, cyclin A, cdc-2, and E2F-1 was decreased after DeltaRaf-1:ER activation in DMS53 cells. The activity cdk4 and cdk2 was also reduced, as consistent with cell cycle arrest in cells with activated DeltaRaf-1:ER cells. In addition, DeltaRaf-1:ER reduced the expression of neuroendocrine markers, gastrin releasing peptide, and ret gene in DMS53:DeltaRaf-1:ER cells. These results provide further evidence that activation of the raf/MEK/ MAPK signaling pathway, which is associated with transformation in many circumstances, can reduce the growth of SCLC cells, and suggest that activation of this pathway might be clinically efficacious in some settings.

Retinoic acid-mediated growth inhibition of small cell lung cancer cells is associated with reduced myc and increased p27Kip1 expression.

Human lung cancer cells, including small cell lung carcinoma (SCLC), frequently lose expression of retinoic acid receptor beta (RAR-beta) and are resistant to the growth inhibitory activity of all-trans retinoic acid (RA). To elucidate the role of RAR-beta in the growth regulation of SCLC by retinoids, we restored RAR-beta expression in RAR-beta-negative H209 SCLC cells by retroviral transduction (H209-RAR-beta). We found that H209-RAR-beta, but not parental H209 cells, underwent growth inhibition upon RA treatment. RA-treated H209-RAR-beta cells arrested in G1 and displayed reduced L-myc expression and cyclin-dependent kinase 2 (cdk2) activity compared with untreated cells. RA treatment of H209-RAR-beta cells was also accompanied by increased expression of the cdk inhibitor p27Kip1, whereas no differences in the expression of L-myc or p27Kip1 were detected upon RA treatment of parental H209 cells. The RA-induced growth arrest of H82 SCLC cells, which express endogenous RAR-beta, was also associated with reduced c-myc and increased p27Kip1 expression. We found that ectopic expression of p27Kip1 induced growth inhibition in both H209 and H82 cells, and that sustained myc expression in H209-RAR-beta cells promoted the induction of apoptosis upon RA addition. Our observations indicate that RAR-beta gene transfer can restore RA sensitivity in SCLC cells and suggest that myc and p27Kip1 may represent critical mediators of the RA-induced cell cycle arrest in SCLC cells expressing RAR-beta.

Raf-1-induced cell cycle arrest in LNCaP human prostate cancer cells.

Prostate cancer is the most commonly diagnosed neoplasm in men. LNCaP cells continue to possess many of the molecular characteristics of in situ prostate cancer. These cells lack ras mutations, and mitogen-activated protein kinase (MAPK) is not extensively phosphorylated in these cells. To determine the effects of ras/raf/MAPK pathway activation in these cells, we transfected LNCaP cells with an activatable form of c-raf-1(deltaRaf-1:ER). Activation of deltaRaf-1:ER, with resultant MAPK activation, reduced plating efficiency and soft agarose cloning efficiency 30-fold in LNCaP cells. Cell cycle distribution showed an accumulation of cells in G1 and was associated with the induction of CDK inhibitor p21WAF1/CIP1 at the protein and mRNA levels. p21WAF1/CIP1 mRNA stability was increased after deltaRaf-1:ER activation. In addition, activated deltaRaf-1:ER induced the senescence associated-beta-galactosidase in LNCaP cells. These data demonstrate that raf activation can activate growth inhibitory pathways leading to growth suppression in prostate carcinoma cells and also suggest that raf/MEK/MAPK pathway activation, rather than inhibition, may be a therapeutic target for some human prostate cancer cells.

The identification of a cis-element and a trans-acting factor involved in the response to polyamines and polyamine analogues in the regulation of the human spermidine/spermine N1-acetyltransferase gene transcription.

The superinduction of spermidine/spermine N1-acetyltransferase (SSAT) gene has been associated with a cytotoxic response to a new class of antineoplastic polyamine analogues. The initial mechanism of SSAT superinduction is an increase in transcription in response to analogue exposure. This increased transcription appears to be modulated through the association between a nuclear protein factor and a cis-element described here as the polyamine-responsive element (PRE). The PRE was identified as a 9-base pair sequence, 5'-TATGACTAA-3', in the context of a 31-base pair stretch from -1522 to -1492 base pairs with respect to the SSAT transcriptional start site. This element binds a nuclear factor from polyamine analogue-responsive cells, but not from polyamine analogue-insensitive cells. The labeled PRE was used to clone and identify the transcription factor, Nrf-2, that binds constitutively to the PRE sequence. Although the PRE sequence shares homology to the originally identified Nrf-2 recognition sequence, the two sequences are not identical. The Nrf-2 transcription factor appears only to be present in cell types that are capable of expressing high amounts of SSAT. The results of these studies suggest that Nrf-2, bound to the PRE, plays an important regulatory role of expression of the human SSAT gene.

Post-transcriptional silencing of RET occurs, but is not required, during raf-1 mediated differentiation of medullary thyroid carcinoma cells.

Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor of the calcitonin secreting thyroid C-cells. Somatic and germline mutations in the RET proto-oncogene are associated with sporadic and inherited cases of MTC, respectively. The human MTC cell line, TT, can be differentiated by activated raf-1. This differentiation is characterized, in part, by down-regulation of the RET proto-oncogene. We now show that raf-1 induction is followed by activation of the downstream kinases MEK1/2 and ERK1/2 and that differentiation is dependent on activation of MEK1/2. The concurrent down-regulation of RET appears to involve altered nuclear compartmentalization and transport of RET mRNA. Although RET is down-regulated during raf-1 mediated differentiation, overexpression of activated RET alleles which resist down-regulation does not alter the raf-1 mediated differentiation response. These data suggest that RET down-regulation is associated with, but not required, for raf-1 mediated MTC cell differentiation and that the raf-1 signal transduction pathway plays a dominant role in promoting MTC cell differentiation.

Induction of gastrin releasing peptide by all-trans retinoic acid in small cell lung cancer cells.

All-trans retinoic acid (RA) has been shown to inhibit cell proliferation while increasing neuroendocrine differentiation in small cell lung cancer (SCLC) cells. RA and related compounds are rapidly becoming integrated into clinical trials to prevent lung cancers and other aerodigestive neoplasms. We found that expression of gastrin releasing peptide (GRP), which can promote lung tumorigenesis in model systems, was increased by RA in SCLC cells which have functional retinoid signaling. In SCLC cells that possess functional GRP receptors, ectopic expression of RARc increased GRP expression and augmented cloning efficiency, demonstrating that these maneuvers result in biologically active GRP. SCLC cells with defects in RA pathway signaling did not efficiently induce GRP upon RA exposure. In these cells, transfection of RARs rendered the cells competent to induce GRP upon RA exposure. These data show that activation of intact retinoid signaling by RA can induce GRP, a growth factor that can act as a tumor promoter. Our findings suggest the possibility that retinoids may increase, rather than decrease, lung cancer risks in some individuals.

Activated Raf-1 causes growth arrest in human small cell lung cancer cells.

Small cell lung cancer (SCLC) accounts for 25% of all lung cancers, and is almost uniformly fatal. Unlike other lung cancers, ras mutations have not been reported in SCLC, suggesting that activation of ras-associated signal transduction pathways such as the raf-MEK mitogen-activated protein kinases (MAPK) are associated with biological consequences that are unique from other cancers. The biological effects of raf activation in small cell lung cancer cells was determined by transfecting NCI-H209 or NCI-H510 SCLC cells with a gene encoding a fusion protein consisting of an oncogenic form of human Raf-1 and the hormone binding domain of the estrogen receptor (DeltaRaf-1:ER), which can be activated with estradiol. DeltaRaf-1:ER activation resulted in phosphorylation of MAPK. Activation of this pathway caused a dramatic loss of soft agar cloning ability, suppression of growth capacity, associated with cell accumulation in G1 and G2, and S phase depletion. Raf activation in these SCLC cells was accompanied by a marked induction of the cyclin-dependent kinase (cdk) inhibitor p27(kip1), and a decrease in cdk2 protein kinase activities. Each of these events can be inhibited by pretreatment with the MEK inhibitor PD098059. These data demonstrate that MAPK activation by DeltaRaf-1:ER can activate growth inhibitory pathways leading to cell cycle arrest. These data suggest that raf/MEK/ MAPK pathway activation, rather than inhibition, may be a therapeutic target in SCLC and other neuroendocrine tumors.

Tissue-specific expression of human achaete-scute homologue-1 in neuroendocrine tumors: transcriptional regulation by dual inhibitory regions.

Malignancies with neuroendocrine (NE) features such as medullary thyroid cancer (MTC) and small cell lung cancer (SCLC) are prototypic neoplasms arising from peripheral endocrine cells. The mechanisms that regulate the NE phenotype in these tumors and their cellular precursors are not well understood. However, a basic helix-loop-helix transcription factor that is homologous to Drosophila neural fate determination proteins may have a central role. Human achaete-scute homologue-1 (hASH1), a human homologue of the Drosophila achaete-scute complex, is highly expressed in MTC, SCLC, and pheochromocytomas. To determine what mechanisms allow constitutive expression of hASH1 in NE tumors, we cloned human genomic DNA fragments containing the hASH1 gene and characterized its promoter region. We show that hASH1 expression is restricted to NE cell lines by a transcriptionally regulated mechanism. Dual promoters initiate hASH1 transcription, with the predominant site being an evolutionarily conserved initiator (INR) element. Transient transfection studies provide evidence for a generalized enhancer region that has high activity in all cell lines tested. Restriction of hASH1 expression to NE tumor cells depends on two tissue-specific repressor regions, present in the proximal and distal (> 13.5 kb) 5'-flanking region. Understanding the mechanisms of tissue-specific control of hASH1 gene expression provides a useful model to explore regulatory cascades influencing both normal nervous system development and the NE phenotype of tumors such as MTC and SCLC.

Conservation of the Drosophila lateral inhibition pathway in human lung cancer: a hairy-related protein (HES-1) directly represses achaete-scute homolog-1 expression.

The achaete-scute genes encode essential transcription factors in normal Drosophila and vertebrate nervous system development. Human achaete-scute homolog-1 (hASH1) is constitutively expressed in a human lung cancer with neuroendocrine (NE) features, small cell lung cancer (SCLC), and is essential for development of the normal pulmonary NE cells that most resemble this neoplasm. Mechanisms regulating achaete-scute homolog expression outside of Drosophila are presently unclear, either in the context of the developing nervous system or in normal or neoplastic cells with NE features. We now provide evidence that the protein hairy-enhancer-of-split-1 (HES-1) acts in a similar manner as its Drosophila homolog, hairy, to transcriptionally repress achaete-scute expression. HES-1 protein is detected at abundant levels in most non-NE human lung cancer cell lines which lack hASH1 but is virtually absent in hASH1-expressing lung cancer cells. Moreover, induction of HES-1 in a SCLC cell line down-regulates endogenous hASH1 gene expression. The repressive effect of HES-1 is directly mediated by binding of the protein to a class C site in the hASH1 promoter. Thus, a key part of the process that determines neural fate in Drosophila is conserved in human lung cancer cells. Furthermore, modulation of this pathway may underlie the constitutive hASH1 expression seen in NE tumors such as SCLC, the most virulent human lung cancer.

An achaete-scute homologue essential for neuroendocrine differentiation in the lung.

In Drosophila and in vertebrates, the achaete-scute family of basic helix-loop-helix transcription factors plays a critical developmental role in neuronal commitment and differentiation. Relatively little is known, however, about the transcriptional control of neural features in cells outside a neuronal context. A minority of normal bronchial epithelial cells and many lung cancers, especially small-cell lung cancer, exhibit a neuroendocrine phenotype that may reflect a common precursor cell population. We show here that human achaete-scute homologue-1 (hASH1) is selectively expressed in normal fetal pulmonary neuroendocrine cells, as well as in the diverse range of lung cancers with neuroendocrine features. Strikingly, newborn mice bearing a disruption of the ASH1 gene have no detectable pulmonary neuroendocrine cells. Depletion of this transcription factor from lung cancer cells by antisense oligonucleotides results in a significant decrease in the expression of neuroendrocrine markers. Thus, a homologue of Drosophila neural fate determination genes seems to be necessary for progression of lung epithelial cells through a neuroendocrine differentiation pathway that is a feature of small-cell lung cancer, the most lethal form of human lung cancer.