Galam Khan

The angiogenic factor midkine is aberrantly expressed in NF1 -deficient Schwann cells and is a mitogen for neurofibroma-derived cells

Loss of the tumor suppressor gene NF1 in neurofibromatosis type 1 (NF1) contributes to the development of a variety of tumors, including malignant peripheral nerve sheath tumors (MPNST) and benign neurofibromas. Of the different cell types found in neurofibromas, Schwann cells usually provide between 40 and 80%, and are thought to be critical for tumor growth. Here we describe the identification of growth factors that are upregulated in NF1−/− mouse Schwann cells and are potential regulators of angiogenesis and cell growth. Basic fibroblast growth factor (FGF-2), platelet-derived growth factor (PDGF) and midkine (MK) were found to be induced by loss of neurofibromin and MK was further characterized. MK was induced in human neurofibromas, schwannomas, and various nervous system tumors associated with NF1 or NF2; midkine showed an expression pattern overlapping but distinct from its homolog pleiotrophin (PTN). Immunohistochemistry revealed expression of MK in S-100 positive Schwann cells of dermal and plexiform neurofibromas, and in endothelial cells of tumor blood vessels, but not in normal blood vessels. Furthermore, MK demonstrated potent mitogenic activity for human systemic and brain endothelial cells in vitro and stimulated proliferation and soft agar colony formation of human MPNST derived S100 positive cells and fibroblastoid cells derived from an NF1 neurofibroma. The data support a possible central role for MK as a mediator of angiogenesis and neurofibroma growth in NF1.

High birth weight increases mammary tumorigenesis in rats.

Epidemiological studies have investigated whether a high birth weight is associated with increased breast cancer risk, but the results remain inconclusive. This study was designed to determine whether high birth weight increases later susceptibility to carcinogen‐induced mammary tumorigenesis in an animal model and to determine mechanisms mediating this association. Pregnant female Sprague Dawley rats were fed either a control or a high‐fat diet during the extent of gestation. Maternal exposure to the high‐fat diet increased pregnancy leptin levels and offsprings birth weight, but had no effect on pregnancy estradiol or insulin‐like growth factor 1 levels. Changes in the offsprings mammary gland morphology and protein expression were assessed. The mammary epithelial tree of the high‐birth‐weight offspring was denser, contained more terminal end buds and exhibited higher number of proliferating cells. Further, their mammary glands expressed lower levels of ER‐α, but higher levels of activated MAPK. No alterations in apoptosis were noted. High‐birth‐weight rats developed 7,12‐dimethylbenz[a]anthracene‐induced mammary tumors significantly earlier, and tumors grew larger than in the controls. The tumors in this group expressed higher levels of leptin receptor and activated Akt, and contained fewer apoptotic cells than those in the controls. Our results indicate that high birth weight is related to shortened latency to develop mammary tumors—perhaps reflecting an increase in activated MAPK levels and increased tumor growth—perhaps caused by a lower apoptotic response due to higher leptin receptor and activated Akt levels in the tumors.

In utero alcohol exposure increases mammary tumorigenesis in rats

Findings in humans and animal models suggest that in utero hormonal and dietary exposures increase later breast cancer risk. Since alcohol intake by adult women consistently increases their breast cancer risk, we wondered whether maternal alcohol consumption during pregnancy increases female offsprings mammary tumorigenesis. In our study, pregnant female rats were pair-fed isocaloric diets containing either 0 (control), 16 or 25 g alcohol kg−1 feed between days 7 and 19 of gestation. These alcohol exposures generate blood alcohol levels that correspond to low and moderate alcohol consumption and are lower than those that induce foetal alcohol syndrome. Serum oestradiol levels were elevated in pregnant rats exposed to alcohol (P<0.003). When adult, female offspring of alcohol-exposed dams developed significantly more 7,12-dimethylbenz[a]anthracene -induced mammary tumours, compared to the controls (tumour multiplicity; mean±s.e.m., controls: 2.0±0.3, 16 g alcohol: 2.7±0.4 and 25 g alcohol: 3.7±0.4; P<0.006). In addition, the mammary epithelial tree of the alcohol-exposed offspring was denser (P<0.004) and contained more structures that are susceptible for the initiation of breast cancer (P<0.001). Immunohistochemical assessment indicated that the mammary glands of 22-week-old in utero alcohol-exposed rats contained elevated levels of oestrogen receptor-α (P<0.04) that is consistent with the changes in mammary gland morphology. In summary, maternal alcohol intake during pregnancy increases female offsprings mammary tumorigenesis, perhaps by programming the foetal mammary gland to exhibit persistent alterations in morphology and gene expression. It remains to be determined whether an increase in pregnancy oestradiol levels mediated alcohols effects on offsprings mammary tumorigenesis.

Gene Signaling Pathways Mediating the Opposite Effects of Prepubertal Low-Fat and High-Fat n-3 Polyunsaturated Fatty Acid Diets on Mammary Cancer Risk

In rats, prepubertal exposure to low-fat diet containing n-3 polyunsaturated fatty acids (PUFA) reduces mammary cell proliferation, increases apoptosis, and lowers risk of mammary tumors in adulthood, whereas prepubertal high-fat n-3 PUFA exposure has opposite effects. To identify signaling pathways mediating these effects, we performed gene microarray analyses and determined protein levels of genes related to mammary epithelial cell proliferation. Nursing female rats and rat pups were fed low-fat (16% energy from fat) or high-fat (39% energy from fat) n-3 or n-6 PUFA diets between postnatal days 5 and 24. cDNA gene expression microarrays were used to identify global changes in the mammary glands of 50-day-old rats. Differences in gene expression were confirmed by real-time quantitative PCR, and immunohistochemistry was used to assess changes in the peroxisome proliferator–activated receptor γ and cyclin D1 levels. DNA damage was determined by 8-hydroxy-2′-deoxyguanosine assay. Expressions of the antioxidant genes thioredoxin, heme oxygenase, NADP-dependent isocitrate dehydrogenase, and metallothionein III, as well as peroxisome proliferator–activated receptor γ protein, were increased in the mammary glands of 50-day-old rats prepubertally fed the low-fat n-3 PUFA diet. Prepubertal exposure to the high-fat n-3 PUFA diet increased DNA damage and cyclin D1 protein and reduced expression of BRCA1 and cardiotrophin-1. Reduction in mammary tumorigenesis among rats prepubertally fed a low-fat n-3 PUFA diet was associated with an up-regulation of antioxidant genes, whereas the increase in mammary tumorigenesis in the high-fat n-3 PUFA fed rats was linked to up-regulation of genes that induce cell proliferation and down-regulation of genes that repair DNA damage and induce apoptosis.

Therapeutic efficacy of G207 in a novel peripheral nerve sheath tumor model.

Nerve involvement poses a significant obstacle for the management of peripheral nervous system tumors, and nerve injury provides a frequent source of postoperative morbidity. The lack of suitable animal models for peripheral nerve tumors has impeded the development of alternative nerve-sparing therapies. To evaluate the effect of a multimutated replication-competent herpes simplex virus (G207) on the growth of peripheral nerve tumors and on nerve function, we developed a novel peripheral nerve sheath tumor model. Human neuroblastoma-derived cells injected into murine sciatic nerve consistently caused tumor development within the nerve sheath after 2 weeks followed by increasingly severe impairment of nerve function. Tumor treatment by a single intratumoral injection of G207 resulted in significant reduction of functional impairment, inhibition of tumor growth and prolonged survival. Direct injection of G207 viral particles into the healthy nerve sheath caused no obvious neurologic sequelae, whereas injections of wild-type virus resulted in uniform lethality. The results indicate that viral therapy might be considered as a safe alternative to surgical removal of tumors with peripheral nerve involvement.

Maternal dietary exposure to fiber during pregnancy and mammary tumorigenesis among rat offspring

Maternal diet during pregnancy has been proposed to modify female offsprings later susceptibility to develop breast cancer; however, most of the dietary factors identified thus far have led to increased risk. To identify dietary factors that might reduce offsprings breast cancer risk, pregnant rat dams were fed diets containing 6% fiber originating either from cellulose (control), or oat, whole wheat or defatted flax flour. At birth, dams were switched to the AIN93 semi‐purified diet. Mammary tumor incidence and multiplicity, induced by administering the offspring 5 mg 7,12‐dimethylbenz[a]anthracene (DMBA) at the age of 50 days, was reduced in the whole wheat flour‐exposed offspring and increased in the defatted flax‐exposed offspring. To identify the mechanisms mediating the effects of in utero dietary exposures, changes in mammary gland morphology and gene expression were assessed before puberty onset (3 weeks of age) and at the time rats are most susceptible to malignant transformation (8 weeks of age). The number of terminal end buds (TEBs), i.e., the targets of malignant transformation, was reduced in the mammary glands of whole wheat‐ and oat flour‐exposed offspring, as compared to the controls. Further, the number of apoptotic epithelial cells (based on ISOL assay) was elevated in the whole wheat flour offspring, but no changes in cell proliferation (PCNA), estrogen receptor α (ER‐α) or cyclin D1 mRNA or protein levels were seen. The mRNA and/or protein levels of BRCA1 and p53 were significantly increased in the mammary glands of whole wheat flour offspring. Further, the levels of 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG), a marker of DNA damage, were significantly reduced in these rats, suggesting that maternal dietary exposure to whole wheat during pregnancy may reduce offsprings breast cancer risk by improving DNA damage repair mechanisms.

Effects of maternal dietary exposure to cadmium during pregnancy on mammary cancer risk among female offspring

Background: Since heavy metal cadmium is an endocrine disrupting chemical, we investigated whether maternal exposure to cadmium during the pregnancy alters mammary tumorigenesis among female offspring. Methods: From gestation day 10 to day 19, pregnant rat dams were fed modified American Institute of Nutrition (AIN93G) diet containing 39% energy from fat (baseline diet), or the baseline diet containing moderate (75 μg/kg of feed) or high (150 μg/kg) cadmium levels. Some dams were injected with 10 μg 17β-estradiol (E2) daily between gestation days 10 and 19. Results: Rats exposed to a moderate cadmium dose in utero were heavier and exhibited accelerated puberty onset. Both moderate and high cadmium dose led to increased circulating testosterone levels and reduced the expression of androgen receptor in the mammary gland. The moderate cadmium dose mimicked the effects of in utero E2 exposure on mammary gland morphology and increased both the number of terminal end buds and pre-malignant hyperplastic alveolar nodules (HANs), but in contrast to the E2, it did not increase 7, 12-dimethylbenz (a) anthracene-induced mammary tumorigenesis. Conclusions: The effects of in utero cadmium exposure were dependent on the dose given to pregnant dams: Moderate, but not high, cadmium dose mimicked some of the effects seen in the in utero E2 exposed rats, such as increased HANs in the mammary gland.

TDP-43 protein variants as biomarkers in amyotrophic lateral sclerosis

BackgroundTDP-43 aggregates accumulate in individuals affected by amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases, representing potential diagnostic and therapeutic targets. Using an atomic force microscopy based biopanning protocol developed in our lab, we previously isolated 23 TDP-43 reactive antibody fragments with preference for human ALS brain tissue relative to frontotemporal dementia, a related neurodegeneration, and healthy samples from phage-displayed single chain antibody fragment (scFv) libraries. Here we further characterize the binding specificity of these different scFvs and identify which ones have promise for detecting ALS biomarkers in human brain tissue and plasma samples.ResultsWe developed a sensitive capture ELISA for detection of different disease related TDP-43 variants using the scFvs identified from the ALS biopanning. We show that a wide variety of disease selective TDP-43 variants are present in ALS as the scFvs show different reactivity profiles amongst the ALS cases. When assaying individual human brain tissue cases, three scFvs (ALS-TDP6, ALS-TDP10 and ALS-TDP14) reacted with all the ALS cases and 12 others reacted with the majority of the ALS cases, and none of the scFvs reacted with any control samples. When assaying individual human plasma samples, 9 different scFvs reacted with all the sporadic ALS samples and again none of them reacted with any control samples. These 9 different scFvs had different patterns of reactivity with plasma samples obtained from chromosome 9 open reading frame 72 (c9orf72) cases indicating that these familial ALS genetic variants may display different TDP-43 pathology than sporadic ALS cases.ConclusionsThese results indicated that a range of disease specific TDP-43 variants are generated in ALS patients with different variants being generated in sporadic and familial cases. We show that a small panel of scFvs recognizing different TDP-43 variants can generate a neuropathological and plasma biomarker profile with potential to distinguish different TDP-43 pathologies.

Novel atomic force microscopy based biopanning for isolation of morphology specific reagents against TDP-43 variants in amyotrophic lateral sclerosis

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinsons disease and beta-amyloid and tau in Alzheimers disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases.

Prepubertal exposure to cow's milk reduces susceptibility to carcinogen-induced mammary tumorigenesis in rats.

Cows milk contains high levels of estrogens, progesterone and insulin‐like growth factor 1 (IGF‐1), all of which are associated with breast cancer. We investigated whether prepubertal milk exposure affects mammary gland development and carcinogenesis in rats. Sprague–Dawley rats were given either whole milk or tap water to drink from postnatal day (PND) 14 to PND 35, and thereafter normal tap water. Mammary tumorigenesis was induced by administering 7,12‐dimethylbenz[a]anthracene on PND 50. Milk exposure increased circulating E2 levels on PND 25 by 10‐fold (p < 0.001) and accelerated vaginal opening, which marks puberty onset, by 2.5 days (p < 0.001). However, rats exposed to milk before puberty exhibited reduced carcinogen‐induced mammary carcinogenesis; that is, their tumor latency was longer (p < 0.03) and incidence was lower (p < 0.05) than in the controls. On PND 25 and 50, mammary glands of the milk‐exposed rats had significantly less terminal end buds (TEBs) than the tap water‐exposed controls (p < 0.019). ER‐α protein levels were elevated in the TEBs and lobules of milk rats, compared to rats given tap water (p < 0.019), but no changes in cyclin D1 expression, cell proliferation or apoptosis were seen. IGF‐1 mRNA levels were reduced on PND 50 in the mammary glands of rats exposed to milk at puberty. Our results suggest that drinking milk before puberty reduces later risk of developing mammary cancer in rats. This might be mediated by a reduction in the number of TEBs and lower expression of IGF‐1 mRNA in the mammary glands of milk‐exposed animals.