Ductal Dysplasia Research Articles

DEVELOPMENT OF METAPLASTIC PRECURSOR LESIONS IN MURINE PANCREAS FOLLOWING MUTANT KRAS EXPRESSION IN ADULT PDX1-POSITIVE CELLS

The particular cell type(s) that gives rise to cancer are not always easily distinguished. In an attempt to identify progenitor and/or stem cells as the origin of cancer, we utilized nestin and pdx1 regulatory elements to manage target transgene expression. Previous findings employed a marker gene (hPAP), under the control of nestin and pdx1 regulatory elements, to identify target cells and tissues. This work was expanded to inducibly target mutant Kras to nestin- and pdx1-positive cells and characterize the cellular and tissue phenotypes. Nestin is an intermediate filament with expression in pancreas, brain, intestine, and kidney. Previous findings have demonstrated that nestin expression in the pancreas may be localized to parenchymal and/or mesenchymal cells. Our results verified expression of hPAP in regions around the perimeter of islets and some ducts of the pancreas. When mutant Kras was employed, we observed mild ductal dysplasia and tubular complexes in the pancreas of Nest-tTA/TRE-Kras mice at 3-9 months of age (never administered doxycycline or DOX). These results indicate that nestin regulatory elements can target potential progenitor cells in the pancreas but fail to recapitulate published results where mutant Kras induced PanINs and cancer from nestin-positive cells. Pdx1 is a transcription factor absolutely required for the development of the pancreas. In one of three Pdx1-tTA/TRE-Kras bitransgenic mice at 13 months of age (off DOX at 1 month of age), multiple acinar-to-ductal mucinous metaplastic lesions, considered putative PanIN precursors, were observed. This is the first demonstration of mutant Kras-induced lesions being generated from adult Pdx1-positive cells with the possibility of expanding into PanIN and/or pancreatic cancer. Targeting unique cell types that produce nestin and pdx1 have provided an inducible oncogene system that generates expression of mutant Kras in potential progenitor and adult parenchymal cells in the pancreas. Future analysis will evaluate the dependence of developing neoplasia and/or cancer on continual expression of mutant Kras by abrogating mutant Kras expression (readministration of DOX) following the onset of neoplasia or cancer in the pancreas.

Constitutive activation of smoothened (SMO) in mammary glands of transgenic mice leads to increased proliferation, altered differentiation and ductal dysplasia

The hedgehog signaling network regulates pattern formation, proliferation, cell fate and stem/progenitor cell self-renewal in many organs. Altered hedgehog signaling is implicated in 20-25% of all cancers, including breast cancer. We demonstrated previously that heterozygous disruption of the gene encoding the patched-1 (PTCH1) hedgehog receptor, a negative regulator of smoothened (Smo) in the absence of ligand, led to mammary ductal dysplasia in virgin mice. We now show that expression of activated human SMO (SmoM2) under the mouse mammary tumor virus (MMTV) promoter in transgenic mice leads to increased proliferation, altered differentiation, and ductal dysplasias distinct from those caused by Ptch1 heterozygosity. SMO activation also increased the mammosphere-forming efficiency of primary mammary epithelial cells. However, limiting-dilution transplantation showed a decrease in the frequency of regenerative stem cells in MMTV-SmoM2 epithelium relative to wild type, suggesting enhanced mammosphere-forming efficiency was due to increased survival or activity of division-competent cell types under anchorage-independent growth conditions, rather than an increase in the proportion of regenerative stem cells per se. In human clinical samples, altered hedgehog signaling occurs early in breast cancer development, with PTCH1 expression reduced in approximately 50% of ductal carcinoma in situ (DCIS) and invasive breast cancers (IBC). Conversely, SMO is ectopically expressed in 70% of DCIS and 30% of IBC. Surprisingly, in both human tumors and MMTV-SmoM2 mice, SMO rarely colocalized with the Ki67 proliferation marker. Our data suggest that altered hedgehog signaling may contribute to breast cancer development by stimulating proliferation, and by increasing the pool of division-competent cells capable of anchorage-independent growth.

The transmembrane protein meckelin (MKS3) is mutated in Meckel-Gruber syndrome and the wpk rat

Meckel-Gruber syndrome is a severe autosomal, recessively inherited disorder characterized by bilateral renal cystic dysplasia, developmental defects of the central nervous system (most commonly occipital encephalocele), hepatic ductal dysplasia and cysts and polydactyly. MKS is genetically heterogeneous, with three loci mapped: MKS1, 17q21-24 (ref. 4); MKS2, 11q13 (ref. 5) and MKS3 (ref. 6). We have refined MKS3 mapping to a 12.67-Mb interval (8q21.13-q22.1) that is syntenic to the Wpk locus in rat, which is a model with polycystic kidney disease, agenesis of the corpus callosum and hydrocephalus. Positional cloning of the Wpk gene suggested a MKS3 candidate gene, TMEM67, for which we identified pathogenic mutations for five MKS3-linked consanguineous families. MKS3 is a previously uncharacterized, evolutionarily conserved gene that is expressed at moderate levels in fetal brain, liver and kidney but has widespread, low levels of expression. It encodes a 995-amino acid seven-transmembrane receptor protein of unknown function that we have called meckelin.

Differential Sensitivity of Mouse Epithelial Tissues to the Polyomavirus Middle T Oncogene

To determine how different epithelial cell types respond to the same oncogenic stimulation, we have used a modified human keratin 18 gene to conditionally express the polyomavirus middle T antigen (PyMT) oncogene in simple epithelial tissues of transgenic mice. Activation of PyMT expression by transgenic Cre recombinase in mammary epithelial cells resulted in carcinomas in all bitransgenic females. PyMT expression induced by K18-driven Cre in internal epithelial organs resulted in pancreatic acinar metaplasia and ductal dysplasia with remarkable desmoplastic stromal responses in all 25 bitransgenic mice. Hepatoma formation with altered lipid metabolism and gastric adenocarcinoma occurred in 96 and 54% of these mice, respectively. Elevated PyMT RNA expression also correlated with intraepithelial neoplasia in the prostate. Activated Erk2 was found in mammary tumors, pancreatic tissues, and affected livers. Hes1 RNA, a target of Notch signaling that has been implicated downstream of Ras pathway activation, was elevated in pancreatic and liver lesions. The variety of responses of different epithelia to PyMT demonstrates the importance of the differentiated state in interpreting oncogenic signals.

Two Cytologic Patterns in Invasive Ductal Adenocarcinoma of the Pancreas

To clarify 2 cytologic patterns: severe ductal dysplasia (SD)/carcinoma in situ (CIS) and invasive components of invasive ductal adenocarcinoma of the pancreas (IDAP).Tumor samples from 12 patients with IDAP were examined cytologically and histologically. Cytologic specimens were obtained by fine needle aspiration (ENA) and imprint smears from resected pancreases.In the 12 IDAP cases, roughly 2 cell populations, SD/CIS-type cells and invasive component-type cells, were detected cytologically. The former composed a small portion of the tumor cells, exhibited a small nuclei without anisonucleosis and had a decreased total quantity of chromatin as compared with invasive component-type cells. SD/CIS-type cells were found in small papillary-cohesive and compactly packed clusters and had clearly defined cytoplasmic borders, a nucleus individually enveloped in well-preserved cytoplasm and small amount of cytoplasm (< 15 microm in short diameter) without prominent anisocytosis. The latter comprised mostly of the tumor cells, were present characteristically as a loose and discohesive population and contained a combination of hyperchromatin, large nuclei (> 15 microm) and abundant cytoplasm (>21 microm). Histologically, these 12 cases exhibited IDAP with SD/CIS. Hence, the invasive component-type cells appeared to originate with ordinary invasive spread of IDAP, and the SD/CIS type cells appeared to originate with noninvasive intraductal spread.Two cytologic patterns originating with SD/CIS and invasive components of IDAP could be differentiated.

Hedgehog signaling pathway as a therapeutic target in breast cancer

The Hedgehog (Hh) signaling pathway, which is well conserved even in mammals and other vertebrate species, has long been known to direct growth and patterning during embryonic development. It has been shown that the Hh pathway also plays a critical role in mouse normal mammary gland development. Namely, it has been shown that disruption of the Hh pathway-related genes such as Patched-1 and Gli2 leads to ductal dysplasias that closely resemble some hyperplasia of human breast. In addition, it has been reported that breast carcinoma cells have disruption of these genes. These findings strongly indicate a contribution of the Hh pathway to development of human breast carcinoma. In fact, constitutive activation of the Hh pathway was found in most of 52 surgically resected breast carcinoma specimens. Interestingly, exposure to cyclopamine, a steroidal alkaroid that blocks the Hh pathway, suppressed the growth of the Hh pathway-activated breast carcinoma cells. Thus, the Hh pathway may function in progression of breast carcinoma. In this short review, possibilities of the Hh pathway as a new therapeutic target in breast carcinoma will be mainly discussed.

A novel locus for Meckel-Gruber syndrome, MKS3, maps to chromosome 8q24.

Meckel-Gruber syndrome (MKS), the most common monogenic cause of neural tube defects, is an autosomal recessive disorder characterised by a combination of renal cysts and variably associated features, including developmental anomalies of the central nervous system (typically encephalcoele), hepatic ductal dysplasia and cysts, and polydactyly. Locus heterogeneity has been demonstrated by the mapping of the MKS1locus to 17q21-24 in Finnish kindreds, and of MKS2 to 11q13 in North African-Middle Eastern cohorts. In the present study, we have investigated the genetic basis of MKS in eight consanguineous kindreds, originating from the Indian sub-continent, that do not show linkage to either MKS1 or MKS2. We report the localisation of a third MKS locus ( MKS3) to chromosome 8q24 in this cohort by a genome-wide linkage search using autozygosity mapping. We identified a 26-cM region of autozygosity between D8S586 and D8S1108 with a maximum cumulative two-point LOD score at D8S1179 ( Z(max)=3.04 at theta=0.06). A heterogeneity test provided evidence of one unlinked family. Exclusion of this family from multipoint analysis maximised the cumulative multipoint LOD score at locus D8S1128 ( Z(max)=5.65). Furthermore, a heterozygous SNP in DDEF1, a putative candidate gene, suggested that MKS3 mapped within a 15-cM interval. Comparison of the clinical features of MKS3-linked cases with reports of MKS1- and MKS2-linked kindreds suggests that polydactyly (and possibly encephalocele) appear less common in MKS3-linked families.

The Gli2 Transcription Factor Is Required for Normal Mouse Mammary Gland Development

The hedgehog signal transduction network performs critical roles in mediating cell–cell interactions during embryogenesis and organogenesis. Loss-of-function or misexpression mutation of hedgehog network components can cause birth defects, skin cancer, and other tumors. The Gli gene family (Gli1, Gli2, and Gli3) encodes zinc finger transcription factors that act as mediators of hedgehog signal transduction. In this study, we investigate the role of Gli2 in mammary gland development. Mammary expression of Gli2 is developmentally regulated in a tissue compartment-specific manner. Expression is exclusively stromal during virgin stages of development but becomes both epithelial and stromal during pregnancy and lactation. The null phenotype with respect to both ductal and alveolar development was examined by transplantation rescue of embryonic mammary glands into physiologically normal host females. Glands derived from both wild type and null embryo donors showed ductal outgrowths that developed to equivalent extents in virgin hosts. However, in null transplants, ducts were frequently distended or irregularly shaped and showed a range of histological alterations similar to micropapillary ductal hyperplasias in the human breast. Alveolar development during pregnancy was not overtly affected by loss of Gli2 function. Ductal defects were not observed when homozygous null epithelium was transplanted into a wild type stromal background, indicating that Gli2 function is required primarily in the stroma for proper ductal development. ΔGli2 heterozygotes also demonstrated an elevated frequency and severity of focal ductal dysplasia relative to that of wild type littermate- and age-matched control animals.

Radiation induces genomic instability and mammary ductal dysplasia in Atm heterozygous mice.

Ataxia-telangiectasia (AT) is a genetic syndrome resulting from the inheritance of two defective copies of the ATM gene that includes among its stigmata radiosensitivity and cancer susceptibility. Epidemiological studies have demonstrated that although women with a single defective copy of ATM (AT heterozygotes) appear clinically normal, they may never the less have an increased relative risk of developing breast cancer. Whether they are at increased risk for radiation-induced breast cancer from medical exposures to ionizing radiation is unknown. We have used a murine model of AT to investigate the effect of a single defective Atm allele, the murine homologue of ATM, on the susceptibility of mammary epithelial cells to radiation-induced transformation. Here we report that mammary epithelial cells from irradiated mice with one copy of Atm truncated in the PI-3 kinase domain were susceptible to radiation-induced genomic instability and generated a 10% incidence of dysplastic mammary ducts when transplanted into syngenic recipients, whereas cells from Atm(+/+) mice were stable and formed only normal ducts. Since radiation-induced ductal dysplasia is a precursor to mammary cancer, the results indicate that AT heterozygosity increases susceptibility to radiogenic breast cancer in this murine model system.

Hedgehog signaling in mouse mammary gland development and neoplasia.

Genetic analyses of two hedgehog signal transduction network genes, Patched-1 and Gli2, has demonstrated a critical role for hedgehog signaling in mediating epithelial-stromal tissue interactions during ductal development. Disruption of either gene leads to similar, yet distinct, defects in ductal morphogenesis. Defects are mainly ductal dysplasias that closely resemble some hyperplasias of the human breast. Phenotypic analyses have been coupled with in situ hybridization, transplantation and tissue recombination analyses to formulate a model for tissue compartment-specific control of mouse mammary gland development by hedgehog signaling. In addition, the similarities among hedgehog mutation-induced ductal dysplasias and human breast pathologies suggest a role for altered hedgehog signaling in the development of mammary cancer.

Modifying effects of 4-phenylbutyl isothiocyanate on N-nitrosobis(2-oxopropyl)amine-induced tumorigenesis in hamsters

The modifying effects of dietary 4-phenylbutyl isothiocyanate (PBITC), given during the initiation stage of carcinogenesis, were investigated in hamsters treated with N-nitrosobis(2-oxopropyl)amine (BOP). A total of 120 female 5-week-old hamsters were divided into six groups. Animals in groups 1–3, each consisting of 30 hamsters, were given BOP by two subcutaneous injections, 1 week apart, at a dose of 20 mg/kg body weight, plus 0, 10 or 100 μmol/animal of PBITC in corn oil by gavage 2 h prior to each carcinogen treatment. Ten animals in group 4 served as a vehicle control, and animals in groups 5 and 6, each consisting of ten hamsters, were given 10 and 100 μmol of PBITC alone in corn oil. Sacrifice was 52 weeks after the first BOP injection. The PBITC treatments significantly ( P<0.05) inhibited the development of pancreatic ductal dysplasias and adenocarcinomas. Also, lung tumors (adenomas and adenocarcinomas) were significantly ( P<0.05) reduced in a dose-dependent manner. In contrast, both hepatocellular and cholangiocellular tumors (adenomas and carcinomas) tended to be or were significantly increased by PBITC. These results, taken together with our previous findings, indicate that the natural isothiocyanate, phenethyl isothiocyanate (PEITC), has a more potent chemopreventive action against BOP-induced tumorigenesis than synthetic isothiocyanates with longer alkyl chains, such as 3-phenylpropyl isothiocyanate (PPITC) and PBITC. Thus, their lipophilicity does not necessarily reflect the chemopreventive potential because the strength of lipophilicity is PEITC<PPITC<PBITC.

Genetic Progression and Divergence in Pancreatic Carcinoma

Genetic alterations of pancreatic intraductal lesions adjacent to invasive ductal carcinoma were investigated. We submitted nine foci of ordinary epithelium, 12 foci of nonpapillary hyperplasia, 12 foci of papillary hyperplasia (pap HP), 66 foci of severe ductal dysplasia, and 27 invasive foci from a total of 10 pancreatic carcinomas for genetic analysis. All foci were individually microdissected and allelic losses of 3p, 4q, 5q, 6q, 8p, 9p, 10q, 11q, 13q, 16q, 17p, and 18q were studied. All invasive and severely dysplastic intraductal foci exhibited loss of heterozygosity (LOH) at more than one chromosomal locus. For each case, allelic loss was frequently observed on 9p (severe ductal dysplasia 90%, invasion 100%), 17p (severe ductal dysplasia 80%, invasion 80%), and 18q (severe ductal dysplasia 88%, invasion 88%). Ninety-four percent of severe ductal dysplasia and 96% of invasive foci had multiple LOH. Seventeen percent of nonpapillary hyperplasia and 33% of pap HP showed LOH. Only one focus of pap HP showed multiple LOH. The patterns of allelic loss identified in severe ductal dysplasia were generally conserved in synchronous infiltrating tumors, supporting the paradigm that infiltrating tumors are clonally derived from severe ductal dysplasia. In eight of 10 cases, however, we found frequent genetic heterogeneity in the intraductal lesion, suggestive of genetic progression or diversion. These findings indicate that invasive pancreatic carcinoma evolves through successive and divergent genetic changes with selection of aggressive subclones in the intraductal component.

Salivary gland duct involvement in oral epithelial dysplasia and squamous cell carcinoma

The clinical implications and prognostic significance of oral dysplastic or cancerous epithelium involving salivary gland ducts have not been previously investigated. Screened routine tissue sections of 1216 cases of oral epithelial dysplasias and squamous cell carcinomas revealed 26 examples (2.14%) that exhibited unequivocal ductal involvement. Ductal involvement was more likely to occur in floor of mouth lesions and in lesions exhibiting severe dysplasia or carcinoma in situ. Clinical follow-up on 23 cases showed that the recurrence rate of the preinvasive lesions that exhibited ductal involvement was equal to that of the squamous cell carcinomas. The depth of ductal dysplasia did not correlate with recurrence rate. These results suggest that the involvement of salivary gland ducts by oral epithelial dysplasias and carcinomas in situ is an uncommon but significant finding. Surgical stripping or ablation of such lesions should extend at least 3 mm below the surface to ensure eradication of these reservoirs of dysplastic cells.

DNA quantitation of intraepithelial neoplasia and invasive carcinoma of the prostate.

The relation of prostatic intraepithelial neoplasia (PIN) or ductal dysplasia and the development of invasive prostate cancer is not clear. PIN, especially high grade, is usually associated with coexisting invasive cancer. Although some investigators have identified micro foci of invasive cancer evolving from PIN, the two are usually anatomically separated. Because of these distinct anatomic patterns, many investigators have concluded that PIN represents a "field effect" or marker of potential cancer progression, and is not directly involved in or leads to the development of invasive prostate cancer. We measured the DNA content in 49 foci of invasive cancer and 87 foci of PIN identified in 34 radical prostatectomies containing both PIN and invasive cancer. In addition, we examined 13 prostatectomies and 5 TUR specimens containing only PIN. We found that the majority of low grade PIN had normal or diploid range DNA and that approximately half of the high grade PIN were abnormal or aneuploid. Prostates with coexisting diploid range PIN and invasive cancer had an approximately equal number of diploid range and aneuploid invasive cancers. Conversely, almost all of the aneuploid PIN (usually high grade) had coexisting aneuploid invasive cancers. This would support the hypothesis that events in the progression of prostate cancer may be operative in both the development of PIN and invasive cancer.

The Rate of Progression of Radiation-Transformed Mammary Epithelial Cells Is Enhanced after Low-Dose-Rate Neutron Irradiation

Studies in this laboratory have shown enhancement of the mammary tumorigenic effects of neutron irradiation after low-dose-rate neutron exposures. To investigate possible reasons, a mammary cell system was used which allows quantitation of initiated mammary epithelial cells and examination of the progression of these radiation-altered cells toward the neoplastic phenotype. Female BALB/c mice were irradiated with fission-spectrum neutrons at dose rates of 1 rad/min or 1 rad/day. Twenty-four hours or 16 weeks after irradiation, mammary cells were obtained by enzymatic dissociation. Mammary outgrowths were derived by injection of 10(4) cells into gland-free fat pads of 3-week-old female BALB/c mice. The frequency of ductal dysplasias in outgrowths from cells irradiated at high or low dose rates was similar. Persistence of dysplasias differed markedly. Few of the dysplasias in outgrowths derived from cells irradiated at the high dose rate persisted, while a large fraction of the dysplasias in outgrowths derived from cells irradiated at low dose rate persisted. When cells remained in situ for 16 weeks prior to dissociation a higher frequency of persistent altered cells was also observed in outgrowths derived from cells irradiated at low neutron dose rates. These data suggest that low-dose-rate neutron exposures enhance the probability of progression of carcinogen-altered cells rather than increase the numbers of initiated cells.

Abnormalities of the pancreatic and biliary ducts in adult patients with choledochal cysts.

A choledochal cyst in the adult patient is not just an aneurysmal dilatation of the common bile duct, but it is the most obvious manifestation of a more widespread pancreaticobiliary ductal dysplasia. Most patients have abnormalities of the pancreatic ducts and, frequently, multiple areas of dilatation involving the intrahepatic bile ducts. Of our nine patients, six had pancreatograms, five of which were distinctly abnormal. Two of the five had recurrent pancreatitis. Six patients were found to have marked stenosis of the outflow tract from the cyst. Because the mucosa lining the choledochal cyst was chronically ulcerated, anastomoses to the cyst formed strictures in seven (64%) of 11 patients, and the long-term risk of carcinoma arising in the cyst was increased. The preferred therapy was excision of the cyst with Roux-en-Y hepaticojejunostomy.

Serial Transplantation of Chemical Carcinogen-Induced Mouse Mammary Ductal Dysplasias&lt;xref ref-type="fn" rid="fn2"&gt;2&lt;/xref&gt;

BALB/c and (C57BL times DBAf)F1 mice were treated with 7,12-dimethylbenz[a]anthracene and urethan, respectively, to induce mammary dysplasias. Nine transplantable outgrowth lines of dysplastic tissue were established by transplantation of the primary lesions into the cleared mammary fat pads of syngeneic mice; 8 of the 9 lesions were ductal in origin. The growth and tumorigenic potentials of these 9 lines were followed over 6-9 transplant generations. Most outgrowth lines exhibited a rapid decline in growth potential and/or a progression to papillomatosis and subsequent carcinoma by transplant generation 7. The ductal outgrowth lines and mammary tumors established from urethan-induced dysplasias in hybrid mice were ovary-dependent for tumorigenesis and tumor growth. The transplantation experiments confirmed the hypothesis that ductal dysplasias have high tumorigenic potentials and can be classified as "high-risk" lesions, similar to murine mammary tumor virus-induced hyperplastic alveolar nodules.

A Case of Colic in a Gelding Caused By an Unusual Bowel Condition

A 16-year-old Friesian gelding with relapsing colic was humanely destroyed during diagnostic laparotomy due to suspected abdominal neoplasia. On post-mortem examination, the pancreas appeared as a firm mass (20 × 8 × 8 cm). The cut surface had a lobular structure with multiple cavities. Histological examination revealed severe chronic fibrosing pancreatitis with acinar–ductal metaplasia and duct dysplasia, which was considered to be the cause of the recurrent colic. Formation of tubular complexes within a background of acinar–ductal metaplasia is similar to the regressive lesions detected in the human pancreas in the context of inflammation, duct obstruction, cystic fibrosis and neoplasia. Pancreatic acinar–ductal metaplasia and ductal dysplasia are considered to be preneoplastic conditions in man and in the mouse.