The age-dependent association of risk factors with pancreatic cancer.

BACKGROUND: Pancreatic cancer presents as advanced disease in >80% of patients; yet, appropriate ages to consider prevention and early detection strategies are poorly defined. We investigated age-specific associations and attributable risks of pancreatic cancer for established modifiable and non-modifiable risk factors. PATIENTS AND METHODS: We included 167 483 participants from two prospective US cohort studies with 1190 incident cases of pancreatic cancer during >30 years of follow-up; 5107 pancreatic cancer cases and 8845 control participants of European ancestry from a completed multicenter genome-wide association study (GWAS); and 248 893 pancreatic cancer cases documented in the US Surveillance, Epidemiology, and End Results (SEER) Program. Across different age categories, we investigated cigarette smoking, obesity, diabetes, height, and non-O blood group in the prospective cohorts; weighted polygenic risk score of 22 previously identified single nucleotide polymorphisms in the GWAS; and male sex and black race in the SEER Program. RESULTS: In the prospective cohorts, all five risk factors were more strongly associated with pancreatic cancer risk among younger participants, with associations attenuated among those aged >70 years. The hazard ratios comparing participants with three to five risk factors with those with no risk factors were 9.24 [95% confidence interval (CI) 4.11-20.77] among those aged ≤60 years, 3.00 (95% CI 1.85-4.86) among those aged 61-70 years, and 1.46 (95% CI 1.10-1.94) among those aged >70 years (Pheterogeneity = 3×10-5). These factors together were related to 65.6%, 49.7%, and 17.2% of incident pancreatic cancers in these age groups, respectively. In the GWAS and the SEER Program, the associations with the polygenic risk score, male sex, and black race were all stronger among younger individuals (Pheterogeneity ≤0.01). CONCLUSIONS: Established risk factors are more strongly associated with earlier-onset pancreatic cancer, emphasizing the importance of age at initiation for cancer prevention and control programs targeting this highly lethal malignancy.

The effect of mismatch repair deficiency on tumourigenesis; microsatellite instability affecting genes containing short repeated sequences.

We have investigated microsatellite instability (MSI) in colorectal, gastric, endometrial and ovarian cancer as a result of mismatch repair (MMR) deficiency. We detected frameshift mutations in several genes that carry short repeated sequences and are important in cell fidelity and growth control; hMSH3, hMSH6, BAX, IGFIIR, TGFbetaIIR, E2F4 and BRCA2. Accumulation of mutations was heterogeneous and mainly restricted to tumours showing MSI at several loci (MSI-H). Both insertions and deletions were evident and occasional intratumour heterogeneity was evident with more than one different additional allele in the tumour. Most MSI-H tumours had acquired mutations in more than one gene and longer repeated sequences were more frequently targets for mutations. The TGFbetaIIR gene was mutated in 62%, the hMSH3 gene in 43%, the E2F4 gene in 35%, the hMSH6 in 32%, the BAX gene in 32%, the IGFIIR gene in 26%, and the BRCA2 gene in 2% of the MSI-H tumours. Homozygous mutations or mutation of both alleles were evident in all genes except BRCA2, in total 23/105 mutated cases, varying from 7% for BAX to 50% for E2F4. E2F4 mutations were exclusively found in colon tumours and E2F4 polymorphisms was found in 8% of cases. No difference in mutation prevalence was noted between cancer types apart from TGFbetaIIR mutations, which were frequently found in colon and gastric tumours but not in endometrial tumours, suggesting that endometrial tumours progress by a different route where TGFbetaIIR mutations are less favourable.

The transmembrane molecule kekkon 1 acts in a feedback loop to negatively regulate the activity of the Drosophila EGF receptor during oogenesis.

We have identified the Drosophila transmembrane molecule kekkon 1 (kek1) as an inhibitor of the epidermal growth factor receptor (EGFR) and demonstrate that it acts in a negative feedback loop to modulate the activity of the EGFR tyrosine kinase. During oogenesis, kek1 is expressed in response to the Gurken/EGFR signaling pathway, and loss of kek1 activity is associated with an increase in EGFR signaling. Consistent with our loss-of-function studies, we demonstrate that ectopic overexpression of kek1 mimics a loss of EGFR activity. We show that the extracellular and transmembrane domains of Kek1 can inhibit and physically associate with the EGFR, suggesting potential models for this inhibitory mechanism.

Signal transduction pathways activated and required for mammary carcinogenesis in response to specific oncogenes.

We have assessed five signal transduction pathways to determine the role each might play in the malignant transformation of mammary epithelium initiated by neu, heregulin/NDF, TGFalpha, v-Ha-ras and c-myc in transgenic mice. The study involves a molecular and pharmacologic assessment of Erk/MAP kinase, Jnk/SAP kinase, PI 3-kinase, protein kinase C, and the Src-related kinases Lck and Fyn. Our results indicate that oncogenes capable of transforming mammary gland epithelium activate and require specific signal transduction pathways. For example, mammary tumors initiated by neu, v-Ha-ras and c-myc have high levels of active Erk/MAP kinase and their anchorage independent growth is strongly inhibited by PD098059, an inhibitor of Mek/ MAP kinase kinase. By contrast, Erk/MAP kinase activity is weak in tumors initiated by TFGalpha and heregulin/NDF and the corresponding cell lines are not growth inhibited by PD098059. Similarly, PI 3-kinase is strongly activated in neu, TGFalpha and heregulin/NDF initiated tumor cell lines, but not in c-myc or v-Ha-ras initiated tumor cell lines. The anchorage independent growth of all these tumor cell lines are, however, inhibited by the specific PI 3-kinase inhibitor LY294001. Further illustrating this oncogene-based specificity, PP1, a specific inhibitor of the Src-like kinases, Lck and Fyn, blocks anchorage-independent cell growth only in the TGFalpha initiated mammary tumor cell line. Taken together with additional observations, we conclude that certain oncogenes reliably require the recruitment/activation of specific signal transduction pathways. Such specific relationships between the initiating oncogene and a required pathway may reflect a direct activating effect or the parallel activation of a pathway that is a necessary oncogenic collaborator for transformation in the mammary gland. The work points to a molecular basis for targeting therapy when an initiating oncogene can be implicated; for example, because of amplification, increased expression, genetic alteration, or heritable characteristics.

Role for Bcl-xL in the regulation of apoptosis by EGF and TGF beta 1 in c-myc overexpressing mammary epithelial cells.

We previously showed that TGF alpha synergizes with c-myc in mammary tumorigenesis through inhibition of Myc-induced apoptosis. We therefore examined the effects of growth factors on apoptosis induction in several cell lines from MMTV-myc mammary tumors. When EGF was withdrawn or TGF beta 1 was added, cells became apoptotic after 15 h (by ELISA and morphology). Northern and Western analysis revealed high levels of Bax and p53, and low or undetectable levels of Bcl-2 and Bcl-xS under all treatment condition. In contrast, Bcl-xL expression was highest in the presence of EGF or TGF alpha, with a significant reduction upon removal of EGF or exposure to TGF beta. In mouse mammary tumors, the relative Bcl-xL/Bax ratio was higher in TGF alpha/Myc double transgenics than in Myc single transgenics, in agreement with the in vitro data. Our results suggest a role for Bcl-xL in the regulation of apoptosis by EGF and TGF beta in mammary epithelial cells.

Cooperation of TGF alpha and c-Myc in mouse mammary tumorigenesis: coordinated stimulation of growth and suppression of apoptosis.

We have previously shown that TGF alpha and c-Myc interact in a strong, synergistic fashion to induce mammary gland tumors in double transgenic mice. Here we show this interaction can be explained, at least in part, by a cooperative growth stimulus by the two proteins, and by TGF alpha-mediated inhibition of c-Myc-induced apoptosis. We initially compared rapidly progressing mammary tumors from double transgenic mice to long latency tumors from single transgenic mice and observed a striking difference in the occurrence of apoptosis among the three groups. Tumors exhibiting apoptosis were derived exclusively from mice that expressed the c-myc transgene in the absence of the TGF alpha transgene, indicating that TGF alpha might protect c-Myc-overexpressing cells from programmed cell death. Cell lines were derived from single and double transgenic mammary tumors to examine further the mechanism underlying the cooperative interaction between the two gene products. In accordance with our in vivo data, apoptosis was only detected when the c-myc transgene was expressed without the TGF alpha transgene. Furthermore, exogenous addition of TGF alpha inhibited apoptosis in cells overexpressing c-Myc alone. In addition, tumor-derived cells that overexpressed both TGF alpha and c-Myc exhibited faster growth rates in vitro and in vivo and were less sensitive to the inhibitory effects of TGF beta in vitro compared to cell lines expressing only one of the transgenes. Based on our findings we propose that TGF alpha acts both as a proliferative and a survival factor for c-Myc-expressing tumor cells. Our results indicate that TGF alpha and c-Myc cooperate in tumorigenesis via a dual mechanism: TGF alpha can inhibit c-Myc-induced apoptosis and both proteins provide a growth stimulus.

Transgenic mouse models of breast cancer.

Although valuable initial information can be gathered about transformation from in vitro studies, human cancer occurs in the context of a complex interaction with its environment and must ultimately be studied in living animals. Transgenic animal models have been used to study breast transformation for a number of years and have yielded valuable information on the subject. In this paper, we will summarize results from our laboratories, and others, regarding the use of transgenic mice to study breast tumorigenesis. We will also suggest future directions for the use of transgenic models to understand, and hopefully, one day to cure the disease.

Synergistic interaction of transforming growth factor alpha and c-myc in mouse mammary and salivary gland tumorigenesis.

The c-myc oncogene is commonly amplified in breast cancer and is known to interact synergistically with transforming growth factor alpha (TGF alpha) in vitro to promote phenotypic transformation of mammary epithelial cells. In addition, both genes are under sex steroid hormone regulation in breast cancer. We have used a bitransgenic mouse approach to test the relevance of Myc-TGF alpha interaction in mammary gland tumorigenesis of virgin animals in vivo. We mated single transgenic TGF alpha and c-myc mouse strains to yield double transgenic offspring for TGF alpha and c-myc. All (20 of 20) double transgenic TGF alpha/c-myc animals developed synchronous mammary tumors at a mean age of 66 days. An unexpected finding was that tumor latency and frequency in males and virgin females were identical. Thus, two gene products that are known to be coinduced in breast cancer by the sex hormones estrogen and progesterone strongly synergize to induce synchronous mammary tumors, independent of sex. The tumors, despite being estrogen receptor positive, were readily transplanted as highly malignant s.c. cancers in ovariectomized nude mice. Although approximately one-half of single transgenic c-myc virgin females also eventually developed mammary gland tumors, these were stochastic and arose after a long latency period of 9-12 months. Single transgenic virgin TGF alpha females and males, c-myc males, and transgene-negative littermates did not develop tumors (ages up to 15 months). The salivary glands of double transgenic animals also coexpress the two transgenes and show pathological abnormalities ranging from hyperplasias to frank adenocarcinomas. In contrast, the salivary glands of single transgenic and wild-type animals showed only mild hyperplasias or metaplasias, but tumors were not observed. In situ hybridization analysis of mammary and salivary glands revealed that hyperplastic and tumorous areas colocalize with regions that overexpress both the TGF alpha and c-myc transgenes. This indicates that there is a requirement for the presence of both proteins for transformation of these glands. In summary, TGF alpha and c-Myc synergize in an extremely powerful way to cause breast and salivary gland tumorigenesis in males and virgin females without a requirement for pregnancies.

Growth factors in breast cancer: mitogenesis to transformation.

While endocrine steroid hormones have been known for many years to regulate normal and malignant mammary epithelium, recent studies have led to an appreciation of polypeptide growth factors as locally-acting autocrine and paracrine effectors. In the current article we summarize what is known about growth factor regulation and action in the normal mammary gland and about perturbations of the steroid-growth factor interplay as cancer progresses. A major theme is that oncogenic activation modulates both regulation of production and function of growth factors in the mammary gland.