SOX9 activity is induced by oncogenic Kras to affect MDC1 and MCMs expression in pancreatic cancer.

SRY (sex determining region Y)-box 9 (SOX9) is required for oncogenic Kras-mediated acinar-to-ductal metaplasia (ADM), pancreatic intraepithelial neoplasias (PanINs) and ultimately pancreatic ductal adenocarcinoma (PDAC). However, how oncogenic Kras affects SOX9 activity is not yet understood, and SOX9-associated genes in PDAC are also unknown at all. Here, we investigated the mechanistic link between SOX9 and oncogenic Kras, studied biological function of SOX9, and identified SOX9-related genes and their clinical significance in patients with PDAC. Our studies reveal that oncogenic Kras induces SOX9 mRNA and protein expression as well as phosphorylated SOX9 expression in human pancreatic ductal progenitor cells (HPNE) and pancreatic ductal cells (HPDE). Moreover, oncogenic Kras promoted nuclear translocation and transcriptional activity of SOX9 in these cells. TAK1/IκBα/NF-κB pathway contributed to induction of SOX9 by oncogenic Kras, and SOX9 in turn enhanced NF-κB activation. SOX9 promoted the proliferation of HPNE and PDAC cells, and correlated with minichromosome maintenance complex components (MCMs) and mediator of DNA damage checkpoint 1 (MDC1) expression. The overexpressive MDC1 was associated with less perineural and lymph node invasion of tumors and early TNM-stage of patients. Our results indicate that oncogenic Kras induces constitutive activation of SOX9 in HPNE and HPDE cells, and Kras/TAK1/IκBα/NF-κB pathway and a positive feedback between SOX9 and NF-κB are involved in this inducing process. SOX9 accelerates proliferation of cells and affects MCMs and MDC1 expression. MDC1 is associated negatively with invasion and metastasis of PDAC.

Enhanced I kappa B alpha degradation is responsible for constitutive NF-kappa B activity in mature murine B-cell lines.

Nuclear factor kappa B (NF-kappa B) is a ubiquitous transcription factor which binds to decameric DNA sequences (kappa B sites) and regulates transcription of multiple genes. The activity of NF-kappa B is regulated by an inhibitor protein, I kappa B, which sequesters NF-kappa B in the cytoplasm. Release of I kappa B and subsequent nuclear translocation of NF-kappa B generally require activating signals. However, in mature murine B cells, the DNA-binding activity of NF-kappa B is constitutively nuclear and activates the Ig kappa gene, a marker for mature B cells. To understand the basis for the constitutive NF-kappa B activation, we examined the properties of NF-kappa B and I kappa B in both pre-B and mature B cells, the regulated and constitutive states, respectively. We found that expression of I kappa B alpha and p105, members of the I kappa B family, and Rel, a member of the NF-kappa B family, is augmented in mature B cells. Both I kappa B alpha and p 105 are associated with NF-kappa B proteins and sequester most of these proteins in the cytoplasm of mature B cells. However, rapid I kappa B alpha dissociation and degradation lead to continuous nuclear translocation of a small fraction of NF-kappa B proteins, which represent the constitutively active NF-kappa B in mature B cells. We estimate that the protease activity is at least 35-fold greater in mature B cells than in pre-B cells. Rapid degradation of I kappa B alpha is directly involved in constitutive NF-kappa B activation, because stabilization of I kappa B alpha by a protease inhibitor causes loss of NF-kappa B activity in mature B cells. These results provide evidence that continuous and rapid degradation of I kappa B alpha in the absence pf external stimuli is causally involved in the constitutive activation of NF-kappa B in mature murine B cells.

Overexpression of the tumor suppressor gene Smad4/DPC4 induces p21waf1 expression and growth inhibition in human carcinoma cells.

The Smad4/DPC4 protein functions as a key transcription factor in transforming growth factor beta (TGF-beta) signaling pathways. However, the downstream target genes regulated by Smad4/DPC4 have not been identified until now. We previously demonstrated that the loss of TGF-beta-induced p21waf1 expression and growth inhibition correlates with inactivation of the Smad4/DPC4 gene. Now we show that transient overexpression of Smad4/DPC4 can induce p21waf1 expression, specific Smad4 DNA binding activity, SBE4-luc reporter gene activity, and subsequent growth inhibition in Smad4/DPC4-null cells and other carcinoma cells in the presence or absence of TGF-beta. Taken together, these data show that p21waf1 is one of the Smad4/DPC4-regulated downstream target genes and suggest that overexpression of the Smad4/DPC4 gene can bypass TGF-beta receptor activation and reestablish one of the key regulatory controls of cell proliferation.

Induction of p21waf1 expression and growth inhibition by transforming growth factor beta involve the tumor suppressor gene DPC4 in human pancreatic adenocarcinoma cells.

The tumor suppressor gene deleted in pancreatic cancer locus 4 (DPC4) is inactivated in about 50% of pancreatic adenocarcinomas. DPC4 was found to be homologous to Smad4 and may function as a transcription factor in the transforming growth factor beta (TGF-beta) receptor-mediated signal transduction pathway. We have investigated the role of DPC4 in the TGF-beta receptor-mediated signal transduction cascade in five human pancreatic cancer cell lines (Panc-1, MDAPanc-28, HS766T, Capan-1, and MiaPaCa-2). Our results demonstrate that the loss of responsiveness to TGF-beta-induced growth inhibition correlates with the loss of expression of DPC4. We have shown that TGF-beta induces p21waf1 expression in Panc-1 cells, whereas no induction of p21waf1 expression by TGF-beta was detected in the other four cell lines lacking either DPC4 expression or the TGF-beta type II receptor. No increase in p21waf1 mRNA stability was observed after treatment with TGF-beta, which suggests that the induction of p21waf1 in Panc-1 cells is transcriptionally regulated by TGF-beta. Our data also demonstrate that the expression of DPC4 is directly involved in TGF-beta-mediated induction of the 3TP-lux reporter gene, which contains a known TGF-beta-inducible plasminogen activator inhibitor promoter. These data suggest that: (a) TGF-beta-mediated induction of p21waf1 and subsequent growth inhibition require the expression of DPC4; (b) p21waf1 is a downstream target gene of DPC4; and (c) transfection of the DPC4 gene restores the TGF-beta-inducible gene expression. Inactivation of the tumor suppressor gene DPC4 and other components of the TGF-beta signal cascades may abolish one of the key negative controls of cell proliferation in pancreatic adenocarcinomas.

Overexpression of urokinase-type plasminogen activator in pancreatic adenocarcinoma is regulated by constitutively activated RelA.

The Rel/NF-kappaB transcription factors regulate the expression of many genes. The activity of RelA, a member of the Rel/NF-kappaB transcription factor family, is constitutively activated in the majority of pancreatic adenocarcinomas and cell lines. We report that the urokinase-type plasminogen activator (uPA), one of the critical proteases involved in tumor invasion and metastasis, is overexpressed in pancreatic tumor cells and its overexpression is induced by constitutive RelA activity. The uPA promoter contains an NF-kappaB binding site that directly mediates the induction of uPA expression by RelA. Expression of a dominant-negative IkappaBalpha mutant inhibits kappaB site-dependent transcriptional activation of a uPA promoter-CAT reporter gene. Treating the pancreatic tumor cell lines with the known NF-kappaB inhibitors, dexamethasone and n-tosylphenyalanine chloromethyl ketone (TPCK), abolishes constitutive RelA activity and uPA overexpression. These results show that uPA is one of the downstream target genes induced by constitutively activated RelA in human pancreatic tumor cells, and suggests that constitutive RelA activity may play a critical role in tumor invasion and metastasis. Inhibition of constitutive RelA in pancreatic tumor cells may reduce their invasive and metastatic potential.

Susceptibility to ras oncogene transformation is coregulated with signal transduction through growth factor receptors.

The human teratocarcinoma cell line PA-1 was derived from culturing ascites fluid cells from a patient with an ovarian germ line tumor. We previously described a non-neoplastic variant cloned from the PA-1 human teratocarcinoma cell line, clone 6, which at passage 40 was resistant to transformation by activated ras oncogenes. However, these cells could be transformed by a plasmid containing both myc and ras. Another PA-1 cell variant, clone 1, isolated at passage 63 and used 50 passages later becomes tumorigenic in nude mice after transfection with an activated ras oncogene (Tainsky et al., Anticancer Res., 8, 899-914, 1988). We report here that the progression from ras resistance to ras susceptibility occurs in both clone 1 and clone 6 cells during 25 passages in culture. In the presence of epidermal growth factor, transforming growth factor-alpha, and basic fibroblast growth factor, the ras-transformable cells exhibit anchorage independent growth, whereas the ras-resistant cells can not be growth stimulated by these growth factors. Similarly, ornithine decarboxylase (ODC) activity was inducible in ras susceptible and ras transformed cells by these growth factors, but not in the ras resistant cells. These differences are not due to the level and activity of epidermal growth factor receptor or to the level of expression of 25 proto-oncogenes.

Modulation of differentiation in PA-1 human teratocarcinoma cells after N-ras oncogene-induced tumorigenicity.

We have been studying the effect of oncogenes on differentiation using the human ovarian teratoma-derived cell line PA-1. From this study we have characterized variants representing four stages relevant to multistage carcinogenesis, two non-tumorigenic and two tumorigenic. The two non-tumorigenic cell variants differ in that one is resistant to transformation by ras oncogenes whereas the other can be transformed to tumorigenicity. When these non-tumorigenic PA-1 variants are treated with retinoic acid (RA), a morphogen, they stop dividing, begin to express homeobox genes, and change in morphology. Transfection of an activated N-ras oncogene into ras-resistant non-tumorigenic PA-1 cells does not alter the RA responsiveness of the cells, indicating that expression of the activated oncogene is not sufficient for blocking RA-induced differentiation. Spontaneous activation of an N-ras oncogene leading to tumorigenic transformants and gene transfer-induced N-ras transformants are resistant to these effects of RA. However, another spontaneous transformant of PA-1 cells that does not contain an activated N-ras is responsive to RA. We prepared somatic cell hybrids of the RA-non-responsive, N-ras-transformed and tumorigenic PA-1 cell and the RA-responsive, ras-resistant non-tumorigenic PA-1 cell; the hybrid cell lines continue to express the oncogene but are non-tumorigenic. These non-tumorigenic hybrids are responsive to RA with regard to morphological changes, growth arrest and induction of homeobox gene expression. Tumorigenic revertants of these hybrids arise as a result of the loss of some chromosomes; these hybrid cells express the oncogene but have lost RA responsiveness. These results indicate that tumorigenic transformation in general is not sufficient to induce RA resistance, and resistance to differentiation may be oncogene-specific. In addition, the expression of an activated N-ras oncogene alone is insufficient to induce resistance to RA and ras-induced tumorigenicity is necessary. Therefore, some feature of cellular metabolism that is altered by and discordantly segregates with tumorigenic transformation controls responsiveness to RA. This controlling element is presumably a tumor suppressor.

The nuclear factor-kappa B RelA transcription factor is constitutively activated in human pancreatic adenocarcinoma cells.

Pancreatic adenocarcinoma is a leading cause of adult cancer mortality in the United States. Recent studies have revealed that point mutation of the K-ras oncogene is a common event in pancreatic cancer, and oncogenesis mediated by Ras may also involve activation of Rel/nuclear factor (NF)-kappa B transcription factors. Furthermore, the c-rel member of Rel/NF-kappa B transcription factor family was first identified as a cellular homologue of the v-rel oncogene, suggesting that other members of the Rel/NF-kappa B family are potentially oncogenes. We therefore investigated the possibility that Rel/NF-kappa B transcription factors are activated in pancreatic cancer. Immunohistochemical analysis, Western blot and Northern blot analysis, electrophoretic mobility shift assays, and chloramphenicol acetyltransferase assays were performed to determine RelA activity in human pancreatic adenocarcinomas and normal tissues and nontumorigenic or tumorigenic cell lines. RelA, the p65 subunit of NF-kappa B, was constitutively activated in approximately 67% (16 of 24) of pancreatic adenocarcinomas but not in normal pancreatic tissues. Constitutive RelA activity was also detected in 9 of 11 human pancreatic tumor cell lines but not in nontumorigenic Syrian golden hamster cell lines. I kappa B alpha, a previously identified NF-kappa B-inducible gene, was overexpressed in human pancreatic tumor tissues and cell lines, and RelA activation could be inhibited by curcumin and dominant-negative mutants of I kappa B alpha, raf, and MEKK1. This is the first report demonstrating constitutive activation of RelA in nonlymphoid human cancer. These data are consistent with the possibility that RelA is constitutively activated by the upstream signaling pathway involving Ras and mitogen-activated protein kinases in pancreatic tumor cells. Constitutive RelA activity may play a key role in pancreatic tumorigenesis through activation of its downstream target genes.

drp, a novel protein expressed at high cell density but not during growth arrest.

Contact is a vital mechanism used by cells to interact with their environment. Contact with living and nonliving elements adjacent to a cell is the basis for many common biological events ranging from growth regulation to metastasis to embryonic pattern formation. We describe the cloning and characterization of a novel density-regulated protein (drp) whose expression is increased in cultured cells at high density compared with cells at low density. A drp cDNA was isolated from the human teratocarcinoma cell line PA-1. Northern analysis with a drp probe revealed transcripts of 2.8 and 3.2 kb. The drp RNA was expressed in a variety of tissues, with the highest amounts in skeletal and cardiac muscle. Using antipeptide antisera, increasing amounts of a 70-kDa protein were detected using several experimental approaches in several cells lines as cell density is increased. Conditioned medium from high-density cells was unable to induce expression of drp in cells growing at low density. Similarly, growth arrest by serum starvation or transforming growth factor-beta (TGF-beta) treatment failed to elicit drp expression. We conclude that drp is a novel protein whose expression is increased at high cell density but not growth arrest.

Tumor suppressor gene Smad4/DPC4, its downstream target genes, and regulation of cell cycle.

The tumor suppressor gene deleted in pancreatic cancer locus 4 (Smad4/DPC4) is inactivated in about 50% of pancreatic adenocarcinomas. The role of DPC4 in the transforming growth factor-beta (TGF-beta) receptor-mediated signal transduction cascade in human pancreatic, colon, and breast carcinoma cell lines has been investigated by a number of laboratories. The results demonstrate that Smad4/DPC4 protein functions as a key transcription factor required in regulation of TGF-beta inducible gene expression and subsequent growth inhibition. Many transcription regulators that are involved in cell growth, differentiation, and oncogenesis have been identified and cloned. Yet paradoxically, it is much more difficult to identify the important downstream target genes responsible for the biological effects elicited by these transcription factors. Although numerous attempts have been made and different approaches have been used to identify the target genes, only limited success has been achieved. Our data show that p21waf1 is one of the Smad4/DPC4-regulated downstream target genes and suggest that overexpression of the Smad4/DPC4 gene can bypass TGF-beta receptor activation and reestablish one of the key regulatory controls of cell proliferation. Identification of the Smad-regulated downstream target genes responsible for diverse biological processes that they control will extend our understanding of the mechanism for cell cycle regulation and cell differentiation.

Prognostic factors in resectable pancreatic cancer: p53 and bcl-2.

The p53 tumor suppressor gene and the Bcl-2 proto-oncogene regulate cell cycle progression and apoptosis. We evaluated the expression of these molecular markers with standard pathologic prognostic variables in patients who received multimodality therapy for resectable adenocarcinoma of the pancreas to study the effect of p53 and Bcl-2 on survival duration. Immunohistochemical staining of archival material was performed to determine levels of expression of p53 and Bcl-2 proteins in 70 patients with adenocarcinoma of pancreatic origin. All patients underwent a potentially curative pancreaticoduodenectomy and standardized pathologic analysis of resected specimens. Potential pathologic and molecular prognostic variables were assessed for their effect on survival duration. Nuclear staining for p53 was observed in 33 (47%) of 70 specimens. Immunostaining for Bcl-2 was observed in 23 specimens (33%). A trend toward improved survival duration was seen in patients whose tumors stained positive for either p53 or Bcl-2. Negative staining for both markers predicted short survival (P = 0.01). By univariate and multivariate analyses, no single pathologic factor was associated with survival duration. Immunohistochemical staging using both p53 and Bcl-2 significantly predicted survival duration by univariate and multivariate analysis; patients whose tumors stained positively for p53 and/or overexpressed Bcl-2 had a significantly longer survival than those whose tumors stained negative for both proteins.

PA-1, a human cell model for multistage carcinogenesis: oncogenes and other factors.

We have developed a cell system which utilizes the human teratocarcinoma cell line PA-1, from which we have characterized four stages of tumor progression. Soon after establishment in culture PA-1 cells revert and are no longer tumorigenic in athymic nude mice. Later, PA-1 cells as they are passaged in culture, become tumorigenic at passage 100. The transition from nontumorigenic to tumorigenic is the result of the biological effects of an activated N-ras oncogene and can be reproduced by transfection of the cloned oncogene into preneoplastic PA-1 cells. Certain preneoplastic cells (prior to passage 100) in this series are susceptible to transformation by single oncogenes while others are not. In studying the basis of this susceptibility to single oncogene induced transformation we have found that somatic cell hybrids between preneoplastic cells which can suppress ras-induced transformation and ras-transformed cells are non-tumorigenic. Therefore, we believe that the progression from ras suppressing to ras susceptibility may be due to the inactivation of a trans-dominant suppressor gene. Our system has identified at least three steps which lead to tumorigenicity; establishment of growth past senesence, activation of a ras oncogene, and inactivation of an oncogene suppressor function. Further genetic alterations are necessary for tumor dissemination and metastasis.

Neoadjuvant chemoradiation for adenocarcinoma of the pancreas.

Pancreaticoduodenectomy is performed on carefully selected patients as part of a protocol-based clinical research program emphasizing the importance of multimodality management for patients with potentially resectable adenocarcinoma of the pancreatic head. Treatment schemas emphasize the importance of minimizing toxicity and treatment duration, while attempting to improve therapeutic efficacy. Cytotoxicity is enhanced by combining radiation therapy with more potent radiation-sensitizing agents. Because of the high incidence of liver metastases, systemic therapy is continued after chemoradiation and surgery with systemic agents of low toxicity directed at specific molecular events involved in pancreatic tumorigenesis such as inhibition of angiogenesis, induction of apoptosis, or arrest of the cell cycle.

Autoregulation of I kappa B alpha activity.

Transcription factor NF-kappa B regulates the expression of a plethora of genes. The activity of NF-kappa B proteins is regulated by I kappa B proteins. We report that induction of I kappa B alpha, a member of the I kappa B family of proteins, is preceded by activation of NF-kappa B complex. The promoter of the I kappa B alpha gene contains a kappa B site that is directly involved in its induction by the NF-kappa B complex. Degradation of I kappa B alpha protein precedes activation of NF-kappa B DNA binding activity, whereas newly synthesized I kappa B alpha protein inhibits NF-kappa B activity. If the degradation of I kappa B alpha is prevented, the induction of DNA binding activity of NF-kappa B complex is severely curtailed. These data suggest the existence of an autoregulatory loop whereby I kappa B alpha regulates the activity of transcription factor NF-kappa B, which in turn regulates the I kappa B alpha activity.

The current state of oncogenes and cancer: experimental approaches for analyzing oncogenetic events in human cancer.

The development of cancer is a multistage process. The activation of proto-oncogenes and the inactivation of tumor suppressor genes play a critical role in the induction of tumors. Using human cell model systems of carcinogenesis, we have studied how oncogenes, tumor suppressor genes, and recessive cancer susceptibility genes participate in this multistep process. Normal human cells are resistant to the transforming potential of oncogenes, such as ras oncogenes, which are activated by specific point mutations. Since as many as 40% of some tumor types contain activated ras oncogenes, a preneoplastic transition in multistage carcinogenesis must involve changing from an oncogene-resistant stage to an oncogene-susceptible stage. The analysis of such critical steps in carcinogenesis using rodent systems has usually not represented the human disease with fidelity. In order to study this carcinogenic process, we have developed human cell, in vitro systems that represent some of the genetic changes that occur in cellular genes during human carcinogenesis. Using these systems, we have learned some of the functions of dominant activated-transforming oncogenes, tumor suppressor genes, and cellular immortalization genes and how they influence the carcinogenic process in human cells. Using our understanding of these processes, we are attempting to clone critical genes involved in the etiology of familial cancers. These investigations may help us to develop procedures that allow us to predict, in these cancer families, which individuals are at high risk for developing cancer.

N-ras oncogene causes AP-2 transcriptional self-interference, which leads to transformation.

Genetic alterations in elements of normal signal transduction mechanisms are known to be oncogenic events often resulting in aberrant activation of programs of gene transcription. We have investigated the effect of N-ras oncogene-induced tumorigenic transformation on the transcription factor AP-2. N-ras oncogene-induced transformation of human teratocarcinoma cells PA-1 results in sixfold elevated AP-2 mRNA levels. However, the level of AP-2-mediated trans-activation is dramatically inhibited in these cells. We show here that the high-level expression of AP-2 ultimately results in transcriptional "self-interference". The activation domain of AP-2, when fused to the DNA-binding domain of GAL4, is sufficient for self-interference. Non-N-ras PA-1 cells constitutively expressing AP-2 or GAL4-AP-2 fusion protein from an SV40 promoter exhibit reduced AP-2-mediated transcriptional activation, inhibition of differentiation, and promotion of anchorage-independent growth, properties that are similar to N-ras-transformed PA-1 cells. Thus, AP-2 is placed in the N-ras signal transduction pathway, and many of the biological effects of N-ras can be accomplished by overexpression of AP-2. This is the first evidence that inhibition of the activity of a transcription factor by self-interference contributes to a physiological process.

Elevated expression of the ribosomal protein S2 gene in human tumors.

Differential screening of a cDNA library was used to isolate genes differentially expressed in a nontumorigenic clone and a ras-transformed variant of the human teratocarcinoma cell line PA-1. The RNA transcript for one of the cDNA clones that we identified was expressed at a 25-fold higher level in the ras-transformed PA-1 cells than in the nontumorigenic PA-1 cells. DNA sequence analysis of this clone showed that it had 86% nucleic acid homology to the mouse LLRep3 gene and only differed at a single amino acid codon (codon 198), which is changed from serine in LLRep3 to threonine in this cDNA clone. The rat ribosomal S2 protein is closely related to the yeast omnipotent informational suppressor SUP44, which encodes the yeast ribosomal protein S4; to the mouse protein LLRep3; and to the human cDNA clone we describe in this report. We therefore concluded that this clone codes for the human ribosomal S2 protein. In situ hybridization experiments revealed that expression of this gene was elevated in cultured human head and neck squamous cell carcinomas compared with normal keratinocytes. In situ hybridization experiments also demonstrated that expression of this gene was elevated in histological sections of human premalignant leukoplakia, head and neck squamous cell carcinomas, and colon and breast cancers compared with the adjacent normal tissues. S2 expression may be a useful diagnostic or prognostic marker for grading human tumors.

Activation of ribosomal protein S2 gene expression in a hamster model of chemically induced oral carcinogenesis.

7,12-Dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis of the hamster buccal pouch has been an excellent model for the study of squamous cell carcinogenesis in human head and neck cancer. Using a differential hybridization of cDNA cloning technique, we isolated a cDNA clone that is expressed in N-ras-transformed PA-1 cells but poorly expressed in non-tumorigenic PA-1 cells; the cDNA codes for the human ribosomal S2 gene product. To define the involvement of S2 gene expression during carcinogenesis in this animal model, we used in situ hybridization technique with non-radioactive digoxigenin-11-dUTP-labeled cDNA. S2 gene was expressed at low levels in basal and suprabasal cell layers of the epidermis in the control, but showed marked elevation throughout the epidermis other than the keratin layer in samples treated for 4 or 8 weeks; S2 was highly expressed in all malignant squamous cell carcinoma cells resulting from DMBA treatment for 16 weeks. As tumors progress from normal epithelium to squamous cell carcinomas, mRNA of the S2 gene was not only elevated sequentially, but also demonstrated the marked heterogeneity among transformed populations, particularly in dysplastic lesions and squamous cell carcinomas. The S2 gene was expressed in a stage-specific manner in the hamster tumor model; S2 could be useful as a neoplastic marker for the detection of certain epithelial origin of tumors and premalignant lesions as well.