Subhash C. Juneja

BubR1 insufficiency causes early onset of aging-associated phenotypes and infertility in mice.

Faithful segregation of replicated chromosomes is essential for maintenance of genetic stability and seems to be monitored by several mitotic checkpoints. Various components of these checkpoints have been identified in mammals, but their physiological relevance is largely unknown. Here we show that mutant mice with low levels of the spindle assembly checkpoint protein BubR1 develop progressive aneuploidy along with a variety of progeroid features, including short lifespan, cachectic dwarfism, lordokyphosis, cataracts, loss of subcutaneous fat and impaired wound healing. Graded reduction of BubR1 expression in mouse embryonic fibroblasts causes increased aneuploidy and senescence. Male and female mutant mice have defects in meiotic chromosome segregation and are infertile. Natural aging of wild-type mice is marked by decreased expression of BubR1 in multiple tissues, including testis and ovary. These results suggest a role for BubR1 in regulating aging and infertility.

Genetically obese MMTV-TGF-α/Lepob Lepob female mice do not develop mammary tumors

Elevated body weight is a risk factor for postmenopausal breast cancer and is associated with increased incidence of spontaneous and chemically induced mammary tumors (MTs) in rodents. In this study, genetically obese LepobLepob female mice that overexpress human TGF-α (transforming growth factor-alpha) were used to assess the role of body weight on oncogene-induced MT development in comparison to lean counterparts. MMTV (mouse mammary tumor virus)-TGF-α and Lep strain mice were crossed to produce TGF-α/Lep+Lep+ (homozygous lean), TGF-α/Lep+Lepob (heterozygous lean) and TGF-α/LepobLepob (homozygous obese) genotypes. Body weights were determined weekly and mice palpated for the presence of MTs until 104 weeks of age. Despite their significantly higher body weight, obese TGF-α/LepobLepob mice failed to develop MTs. MTs were detected between 48 and 104 weeks of age for 26/39 TGF-α/Lep+Lepob mice and for 19/38 TGF-α/Lep+Lep+ mice between 67 and 104 weeks of age. Although MT incidence was not statistically different between the lean groups, age of MT detection tended to be younger for TGF-α/Lep+Lepob mice (p < 0.09). There were significant effects of both genotype and MTs on final body weight, that is, TGF-α/Lep+Lepob mice weighed more than homozygous lean mice, and mice with MTs weighed more than those without MTs. TGF-α/LepobLepob mice are not a good model to evaluate the effect of body weight on MT development possibly due to leptin deficiency. However, the finding that increased body weight is associated with increased oncogene-induced MT development within the normal weight range provides experimental support for the role of body weight in breast cancer.

Leptin Receptor-Deficient MMTV-TGF-α/LeprdbLepr db Female Mice Do Not Develop Oncogene-Induced Mammary Tumors

Being overweight is a risk factor for postmenopausal breast cancer and is associated with an increased incidence and shortened latency of spontaneous and chemically Induced mammary tumors in rodents. However, leptin-deficient obese Lepob Lepob female mice have reduced incidences of spontaneous and oncogene-induced mammary tumors. Of interest, leptin enhances the proliferation of human breast cancer cell lines in which leptin receptors are expressed, which suggests that leptin signaling plays a role in tumor development. We evaluated oncogene-induced mammary tumor development in obese MMTV-TGF-α/Leprdb Leprdb mice that exhibit a defect in OB-Rb, which is considered to be the major signaling isoform of the leptin receptor. Lepr and MMTV-TGF-α mice were crossed, and the offspring were genotyped for oncogene expression and the determination of Lepr status. Lean MMTV-TGF-α/Lepr+ Lepr+ (homozygous) and MMTV-TGF-α/Lepr+ Leprdb (heterozygous) mice and obese MMTV-TGF-α/Leprdb Leprdb mice were monitored until age 104 weeks. Body weights of MMTV-TGF-α/Leprdb Leprdb mice were significantly heavier than those of the lean groups. No mammary tumors were detected in MMTV-TGF-α/LeprdbLeprdb mice, whereas the incidence of mammary tumors in MMTV-TGF-α/Lepr+ Lepr+ and MMTV-TGF-α/Lepr+ Leprdb mice was 69% and 82%, respectively. Examination of mammary tissue whole mounts indicated an absence of duct formation and branching for MMTV-TGF-α/Leprdb Leprdb mice. Both age at mammary tumor detection and tumor burden (tumors/mouse and tumor weights) were similar for the lean genotypes. Serum leptin levels of MMTV-TGF-α/Leprdb Leprdb mice were 12-20-fold higher than levels of lean mice. Thus, despite elevated serum leptin levels, leptin receptor-deficient MMTV-TGF-α/Leprdb Leprdb mice do not develop mammary tumors. This study provides additional evidence that leptin and its cognate receptor may be involved in mammary tumorigenesis.

Diet-Induced Obesity and Mammary Tumor Development in MMTV-neu Female Mice

Abstract: Obesity is a risk factor for postmenopausal breast cancer and is associated with shortened latency and/or increased mammary tumor (MT) incidence in animals. Elevated body weight is usually associated with hormone-responsive tumors. In agreement with these data we previously showed that latency of hormone-responsive MTs in MMTV-TGF-α mice with diet-induced obesity was significantly shortened. Here, we used the same protocol to determine the impact of diet-induced obesity on estrogen receptor-negative MT development in MMTV-neu (strain 202) mice. Mice were fed a low-fat diet (n = 20) or a high-fat diet (n = 54) from 10 wk of age. Body weight at 19 wk of age was used to assign high-fat mice to obesity-prone, overweight, and obesity-resistant groups. Mice were euthanized due to MT size or at 85 wk of age. Final body weights of obesity-prone mice were heaviest, and those of obesity-resistant and low-fat groups were similar. Fat pad weights were heaviest in obesity-prone mice followed by overweight and obesity-resistant groups, and lightest in low-fat mice. Serum IGF-I levels were similar for low-fat and high-fat mice, whereas leptin was higher in high-fat mice (P < 0.0001). MT latency, incidence, metastasis, and burden were similar for all groups. These findings support that obesity is not a risk factor for development of estrogen-negative breast cancer.

Prevention of mammary tumorigenesis by intermittent caloric restriction: does caloric intake during refeeding modulate the response?

Chronic caloric restriction (CCR) prevents mammary tumorigenesis in rodents, but a protective effect for intermittent caloric restriction (ICR) is less well documented. We recently reported that ICR reduced mammary tumor (MT) incidence of mouse mammary tumor virus–transforming growth factor (MMTV-TGF)-α mice to a greater extent than did CCR. Here, we repeated this protocol and obtained serum and tissue samples. Ad libitum (AL) MMTV-TGF-α mice were fed AIN-93M diet. Beginning at 10 weeks of age, ICR mice received isocaloric AIN-93M-mod diet (2-fold increases in protein, fat, vitamins, and minerals) at 50% of ad libitum for 3 weeks followed by 3 weeks refeeding with AIN-93M diet. CCR mice were pair-fed AIN-93M:AIN-93M-mod (2:1) matching intakes for restriction/refeeding cycles. Mice were sacrificed for MT size, at 79 (end of 12th restriction) or at 80 (1 week after 12th refeeding) weeks of age. AL and ICR-80 mice had heavier body weights than ICR-79 and CCR mice (P < 0.0001). Cumulative food intakes of ICR and CCR mice were reduced 12% and 15% versus AL mice (P < 0.0001). However, ICR mice consumed significantly (P < 0.0001) more food than did AL mice during refeeding. MT incidence was 84%, 13%, and 27% for AL, ICR, and CCR mice, respectively. MT weight (P < 0.0011) and number (P < 0.01) were higher for AL mice compared with ICR and CCR mice. AL and ICR-80 mice had similar serum IGF-I levels, but only AL values were higher than those of ICR-79 and CCR mice (P < 0.0017). ICR mice had more MT DNA breaks compared with AL and CCR mice, suggesting enhanced apoptosis (P < 0.02). AL mice had higher mammary fat pad ObR and ObRb leptin receptor mRNA expression than did ICR and CCR mice (P < 0.001), but there was no effect on MTs. These results confirm that ICR prevents development of MTs to a greater extent than does CCR, although “overeating” during refeeding may compromise this protection.

Grb2 regulation of the actin-based cytoskeleton is required for ligand-independent EGF receptor-mediated oncogenesis

Mutations within members of the EGF/ErbB receptor family frequently release the oncogenic potential of these receptors, resulting in the activation of downstream signaling events independent of ligand regulatory constraints. We previously have demonstrated that the signal transduction events originating from S3-v-ErbB, a ligand-independent, oncogenic EGF receptor mutant, are qualitatively distinct from the ligand-dependent mitogenic signaling pathways associated with the wild-type EGF receptor. Specifically, expression of S3-v-ErbB in primary fibroblasts results in anchorage-independent growth, increased invasive potential, and the formation of a transformation-specific phosphoprotein signaling complex, all in a Ras-independent manner. Here we demonstrate the transformation-specific interaction between two components of this complex: the adaptor protein Grb2 and the cytoskeletal regulatory protein caldesmon. This interaction is mediated via both the amino-terminal SH3 and central SH2 domains of Grb2, and the amino-terminal (myosin-binding) domain of caldesmon. Expression of a dominant-negative Grb2 deletion mutant, which lacks the carboxy-terminal SH3 domain, in fibroblasts expressing S3-v-ErbB results in a reduction in phosphoprotein complex formation, the loss of anchorage-independent growth, and a reduction in invasive potential. Together, these results demonstrate a Ras-independent role for Grb2 in modulating cytoskeletal function during ligand-independent EGF receptor-mediated transformation, and provide further support for the hypothesis that ligand-independent oncogenic signaling is qualitatively distinct from ligand-dependent mitogenic signaling by the EGF receptor.

Development of infertility at young adult age in a mouse model of human Sandhoff disease

Sandhoff disease is a human lysosomal storage disease. In a knockout mouse model of Sandhoff disease, which lacks the beta-subunit of beta-hexosaminidase A (Hex A, alphabeta subunits) and B (Hex B, betabeta subunits), the mutant homozygous mice (Hexb(-/-)) are healthy until 15 weeks of age when they develop neurodegenerative symptoms. This study was designed to analyse the fertility profile of male and female Hexb(-/-) mice. Mating behaviour of Hexb(-/-) mice was assessed at different ages. The ovarian function of Hexb(-/-) females was determined by superovulation studies. The quality of spermatozoa and ova was assessed by an in vitro fertilization (IVF) procedure. Hexb(-/-) mice were fertile at a young age. Males were fertile up to the age of 69.3 +/- 6.3 days (mean +/- SD) and females were fertile up to the age of 56-63 days. Since both the Hexb (-/-) sexes showed fertility, the results indicate that Hex A and Hex B (major isozymes of beta-hexosaminidase) may not be required for sperm-ovum interactions, in contrast to the widely accepted belief. On the other hand, young adult Hexb(-/-) males showed a reduction in mating behaviour at the age of 84.8 +/- 2.2 days and an absence of mating behaviour at 94.2 +/- 2.0 days. Spermatozoa from Hexb(-/-) mice (aged 109.2 +/- 1.8 days) showed a lower IVF rate. Among Hexb (-/-) females aged 85.6 +/- 2.1 days, no mice became pregnant although they were positive for a vaginal plug when caged with fertile males. The number of ova recovered from Hexb(-/-) females (aged 111.0 +/- 3.1 days) and the IVF rate of ova were lower than those of controls. In conclusion, Hex A and Hex B may not be required for sperm-ovum interactions. Mice lacking Hex A and Hex B activities develop infertility at a young adult age in an age-dependent manner.