Continuous mowing differentially affects floral defenses in the noxious and invasive weed Solanum elaeagnifolium in its native range.

In weeds, disturbance has been found to affect life history traits and mediate trophic interactions. In urban landscapes, mowing is an important disturbance, and we previously showed that continuous mowing leads to enhanced fitness and defense traits in Solanum elaeagnifolium, Silverleaf Nightshade (SLN). However, most studies have been focused on foliar defenses, ignoring floral defenses. In this study we examined whether continuous mowing affected floral defenses in SLN using mowed and unmowed populations in South Texas, their native range. We found flowers of mowed SLN plants larger but lighter than unmowed plants. Additionally, flowers on plants that were mowed frequently were both heavier and larger. Mowed plants had higher spine density and consequently unmowed flowers had higher herbivore damage. Additionally, early instar Manduca sexta fed on mowed flower-based artificial diets showed no difference in mass than the control and unmowed; however, later instars caterpillars on unmowed diets gained significantly more mass than the mowed treatment and control. Mowed plants had higher spine density which may shed light on why unmowed flowers experienced higher herbivore damage. We found caterpillars fed on high mowing frequency diets were heavier than those on low mowing frequency diets. Collectively, we show that mowing compromises floral traits and enhances plant defenses against herbivores and should be accounted for in management.

Morphological characterization of intraspecific variation for trichome traits in tomato (Solanum lycopersicum).

Trichomes, the hairlike protuberances in plants, have been well known to act as the first line of defense against herbivores, and abiotic stresses, along with other structural defenses such as spines, thorns, and waxes. We previously reported the tremendous variation in trichome traits among different wild and cultivated Solanum species and demonstrated that trichomes types and density are traditionally miscalculated and often misnamed. However, intraspecific variation in trichome traits is poorly understood, although this has implications for stress tolerance and resistance breeding programs in economically important crop species and can also mediate ecological interactions at multiple trophic levels in their wild congeners. In this study, using tomato as a model, we characterized the trichomes from 10 commonly grown varieties using a minimal sample prep desktop scanning electron microscopy, and followed up with estimating their dimensions across the varieties and trichome types. We hypothesized that although trichome number may vary, the varieties will have similar trichome types, based on current literature. Our results show that there is significant variation for trichome number as well as dimensions of trichome types among these varieties. Furthermore, when we separately analyzed the number and dimensions of commonly found glandular and non-glandular trichomes, the results were consistent with broad assessment of trichomes, showing consistent variation among varieties, suggesting that trichome studies should not be limited to basic classification into glandular and non-glandular, and should accommodate the sub-types and their dimensions.

Questing by Tick Larvae (Acari: Ixodidae): A Review of the Influences That Affect Off-Host Survival.

Questing is a host-seeking behavior in which ticks ascend plants, extend their front legs, and wait poised for a chance to attach to a passing host. Hard ticks are ectoparasites of terrestrial vertebrates and because some species vector disease, they are among the most medically important of arthropod pests. All ixodid ticks require blood to survive and reproduce with the number of blood-hosts needed to complete their life cycle varying among species. The vast majority are three-host ticks requiring a different host for each developmental stage: larva, nymph, and adult. A few, including some of the most economically important species, are one-host ticks, that quest only in the larval stage. Questing is a rate-limiting behavior critical to tick survival and disease transmission. For the off-host larval stage, survival is highly dependent on ecological and physiological factors. Yet, off-host larval ecophysiology is often overlooked for the more obvious adult and nymphal tick-host interactions. This review summarizes the literature on ixodid larval questing with emphasis on how specific biotic and abiotic factors affect off-host survival.

Survival of off-host unfed Rhipicephalus (Boophilus) annulatus (Acari: Ixodidae) larvae in study arenas in relation to climatic factors and habitats in South Texas, USA.

The cattle fever tick, Rhipicephalus annulatus (Say), is an economically destructive arthropod because of its ability to vector bovine babesiosis. Cattle fever ticks can spend more than 90% of their life cycle as questing larvae, but the effect of climatic factors on their off-host behavior and survival is unclear. The goal of this study was to measure the effects of specific ecological factors on off-host larvae in nature. The study was conducted in a south Texas pasture over a 20-mo period, during which time larval populations were surveyed and ambient weather variables - relative humidity and temperatures - were recorded. Oviposition success and larval survival varied between cattle fever tick cohorts and was affected by relative humidity and canopied (with tree cover) versus exposed habitat. The results show that relative humidity and the interaction of relative humidity and inhabiting canopied habitats play a key role in oviposition success. Additionally, canopied habitats have a positive influence on off-host larval survival in the spring and summer.

Correction to: A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone.

The original article [1] contains three erroneous mentions of usage of a restriction enzyme-BstZ17I-in the Methods section as displayed in the following sentences.

Population Dynamics of Off-Host Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) Larvae in Response to Habitat and Seasonality in South Texas.

The cattle tick, Rhipicephalus microplus (Canestrini), is an economically destructive arthropod because of its ability to vector bovine babesiosis. It is known that cattle ticks can spend 80-90% of their lifecycle as questing larvae, yet the effect of climatic factors on their off-host behavior and survival is unclear. The goal of this study was to measure the effects of specific ecological factors on off-host questing larvae in nature. The study was conducted in a south Texas pasture over a two-year period, during which time larval populations were surveyed. Simultaneously, weather variables-precipitation, relative humidity, and ambient temperatures-were recorded. Larval survival rates varied among seasons, with the overall highest populations recorded in the spring and the lowest in the fall by a ratio of 20:1. In the winter, the larger numbers were collected from exposed habitats at a ratio of 6:1. Conversely, canopied habitats in the summer had 10-fold larger larval numbers. In the spring, exposed and canopied habitats showed no difference in tick larval survival rates. The results show that the interaction between season and habitat strongly influence off-host questing tick survival. Relative humidity was a key weather variable.

A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone.

BACKGROUND: Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided adaptive immune systems are found in prokaryotes to defend cells from foreign DNA. CRISPR Cas9 systems have been modified and employed as genome editing tools in wide ranging organisms. Here, we provide a detailed protocol to truncate genes in mammalian cells using CRISPR Cas9 editing. We describe custom donor vector construction using Gibson assembly with the commonly utilized pcDNA3 vector as the backbone. RESULTS: We describe a step-by-step method to truncate genes of interest in mammalian cell lines using custom-made donor vectors. Our method employs 2 guide RNAs, mutant Cas9D10A nickase (Cas9 = CRISPR associated sequence 9), and a custom-made donor vector for homologous recombination to precisely truncate a gene of interest with a selectable neomycin resistance cassette (NPTII: Neomycin Phosphotransferase II). We provide a detailed protocol on how to design and construct a custom donor vector using Gibson assembly (and the commonly utilized pcDNA3 vector as the backbone) allowing researchers to obtain specific gene modifications of interest (gene truncation, gene deletion, epitope tagging or knock-in mutation). Selection of mutants in mammalian cell lines with G418 (Geneticin) combined with several screening methods: western blot analysis, polymerase chain reaction, and Sanger sequencing resulted in streamlined mutant isolation. Proof of principle experiments were done in several mammalian cell lines. CONCLUSIONS: Here we describe a detailed protocol to employ CRISPR Cas9 genome editing to truncate genes of interest using the commonly employed expression vector pcDNA3 as the backbone for the donor vector. Providing a detailed protocol for custom donor vector design and construction will enable researchers to develop unique genome editing tools. To date, detailed protocols for CRISPR Cas9 custom donor vector construction are limited (Lee et al. in Sci Rep 5:8572, 2015; Ma et al. in Sci Rep 4:4489, 2014). Custom donor vectors are commercially available, but can be expensive. Our goal is to share this protocol to aid researchers in performing genetic investigations that require custom donor vectors for specialized applications (specific gene truncations, knock-in mutations, and epitope tagging applications).

Cattle fever tick, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae): Potential control on pastures by the application of urea fertilizer.

The southern cattle fever tick, Rhipicephalus (Boophilus) microplus, spends as much as 80-90% of its life cycle as a larva questing for a host. Standard control methods are limited to on-host applications, leaving a need for methods directed at the pasture infesting stages. Reports from Brazil indicate that pasture fertilization can reduce tick numbers. Granular urea was tested using standard pesticide efficacy methods in both the laboratory and field trials to determine if there was a significant impact on adult reproduction and larval survival. Under the conditions of this present study, there was no detectable effect on either female adults or larval stages. Ammonification in the soil may be a key factor limiting the impact of fertilizer treatments.

Lack of interaction between ErbB2 and insulin receptor substrate signaling in breast cancer.

BACKGROUND: ErbB2 Receptor Tyrosine Kinase 2 (ErbB2, HER2/Neu) is amplified in breast cancer and associated with poor prognosis. Growing evidence suggests interplay between ErbB2 and insulin-like growth factor (IGF) signaling. For example, ErbB2 inhibitors can block IGF-induced signaling while, conversely, IGF1R inhibitors can inhibit ErbB2 action. ErbB receptors can bind and phosphorylate insulin receptor substrates (IRS) and this may be critical for ErbB-mediated anti-estrogen resistance in breast cancer. Herein, we examined crosstalk between ErbB2 and IRSs using cancer cell lines and transgenic mouse models. METHODS: MMTV-ErbB2 and MMTV-IRS2 transgenic mice were crossed to create hemizygous MMTV-ErbB2/MMTV-IRS2 bigenic mice. Signaling crosstalk between ErbB2 and IRSs was examined in vitro by knockdown or overexpression followed by western blot analysis for downstream signaling intermediates and growth assays. RESULTS: A cross between MMTV-ErbB2 and MMTV-IRS2 mice demonstrated no enhancement of ErbB2 mediated mammary tumorigenesis or metastasis by elevated IRS2. Substantiating this, overexpression or knockdown of IRS1 or IRS2 in MMTV-ErbB2 mammary cancer cell lines had little effect upon ErbB2 signaling. Similar results were obtained in human mammary epithelial cells (MCF10A) and breast cancer cell lines. CONCLUSION: Despite previous evidence suggesting that ErbB receptors can bind and activate IRSs, our findings indicate that ErbB2 does not cooperate with the IRS pathway in these models to promote mammary tumorigenesis.

Targeted DNA Methylation Screen in the Mouse Mammary Genome Reveals a Parity-Induced Hypermethylation of Igf1r That Persists Long after Parturition.

The most effective natural prevention against breast cancer is an early first full-term pregnancy. Understanding how the protective effect is elicited will inform the development of new prevention strategies. To better understand the role of epigenetics in long-term protection, we investigated parity-induced DNA methylation in the mammary gland. FVB mice were bred or remained nulliparous and mammary glands harvested immediately after involution (early) or 6.5 months following involution (late), allowing identification of both transient and persistent changes. Targeted DNA methylation (109 Mb of Ensemble regulatory features) analysis was performed using the SureSelectXT Mouse Methyl-seq assay and massively parallel sequencing. Two hundred sixty-nine genes were hypermethylated and 128 hypomethylated persistently at both the early and late time points. Pathway analysis of the persistently differentially methylated genes revealed Igf1r to be central to one of the top identified signaling networks, and Igf1r itself was one of the most significantly hypermethylated genes. Hypermethylation of Igf1r in the parous mammary gland was associated with a reduction of Igf1r mRNA expression. These data suggest that the IGF pathway is regulated at multiple levels during pregnancy and that its modification might be critical in the protective role of pregnancy. This supports the approach of lowering IGF action for prevention of breast cancer, a concept that is currently being tested clinically.

Analysis of high fat diet induced genes during mammary gland development: identifying role players in poor prognosis of breast cancer.

BACKGROUND: Epidemiological studies have shown that consumption of a high-fat diet (HFD) increases the risk of developing breast cancer (BC). Studies in rodents have shown HFD causes changes in the genetic programming of the maturing mammary gland (MG) increasing the susceptibility of developing the disease. Less is known about how HFD induced genes impact BC development. HFD exposure two weeks before conception to six weeks of age was previously shown to dramatically change MG gene expression in 10 week old mice. Therefore, we investigated these differentially expressed HFD-induced genes for their expression in BC using the NKI 295 breast tumor dataset. RESULTS: To examine the potential role of HFD induced genes in BC, we first investigated whether these HFD-induced genes in mouse MGs were differentially expressed in different types of human BC. Of the 28 HFD induced genes that were differentially expressed between BC subtypes in the NKI set, 79% were significantly higher in basal-like BC. Next, we analyzed whether HFD induced genes were associated with BC prognosis utilizing gene expression and survival data for each HFD induced gene from the NKI data and constructed Kaplan Meier survival plots. Significantly, 93% of the prognosis associated genes (13/14) were associated with poor prognosis (P = 0.002). Kaplan Meier analysis with 249 non-basal-like BC found that all but one of the genes examined were still significantly associated with poor prognosis. Furthermore, gene set enrichment analysis (GSEA) with HFD microarray data revealed that invasive BC genes where enriched in HFD samples that also had lost expression of luminal genes. CONCLUSIONS: HFD exposed mouse MGs maintain differential expression of genes that are found highly expressed in basal-like breast cancer. These HFD-induced genes associate with poor survival in numerous BC subtypes, making them more likely to directly impact prognosis. Furthermore, HFD exposure leads to a loss in the expression of luminal genes and a gain in expression of mesenchymal and BC invasion genes in MGs. Collectively, our study suggests that HFD exposure during development induces genes associated with poor prognosis, thus identifying how HFD diet may regulate BC development.

Bismuth nitrate-induced novel nitration of estradiol: an entry to new anticancer agents.

Direct nitration of estradiol was carried out using metal nitrates on solid surfaces under mild condition, and a combination of bismuth nitrate pentahydrate impregnated KSF clay was found to be the best reagent to synthesize 2- and 4-nitroestradiol effectively. Furthermore, various basic side chains were introduced, through O-linker at C-3, to these nitroestradiols. The ability of these derivatives to cause cytotoxicity in Estrogen Receptor (ER)-positive and ER-negative breast cancer cell lines, as well as cancer cell lines of other origins, was examined. Qualitative structure activity relationship (SAR) has also been studied. We found that a basic side chain containing either a piperidine or morpholine ring, when conjugated to 2-nitroestradiol, was particularly effective at causing cytotoxicity in each of the cancer cell lines examined. Surprisingly, this effective cytotoxicity was even seen in ER-negative breast cancer cells.

Prepubertal exposure to elevated manganese results in estradiol regulated mammary gland ductal differentiation and hyperplasia in female rats.

Evidence suggests that environmental substances regulating estrogenic pathways during puberty may be detrimental to the developing mammary gland (MG). Manganese (Mn) is a trace mineral required for normal physiological processes. Prepubertal exposure to Mn induces precocious puberty in rats, an event associated with early elevations in puberty-related hormones, including estradiol (E2). However, until now the effect of Mn-induced precocious MG development has not been determined. Therefore, we assessed the ability of prepubertal Mn exposure to advance normal MG development and alter E2 driven pathways involved in tumorigenesis. Sprague Dawley female rats were gavaged daily with either 10 mg/kg manganese chloride (MnCl2) or saline (control) from postnatal day (PND) 12 through PND 30. Blood and MGs were collected on PNDs 30 and 120. Compared to controls, serum E2 levels on PND 30 were elevated (p < 0.05) in the Mn-treated group. Mn exposure significantly increased differentiated MG terminal ductal structures and the percentage of MG epithelial cells that stained positive for the proliferative marker, Ki67, at PND 30 (p < 0.001) and PND 120 (p < 0.001). Levels of Mn (ppm) were not elevated in these MGs. Mn-treated animals (40%) exhibited reactive stroma and intra-luminal focal hyperplasia in hemotoxylin and eosin stained MGs at PND 120. Furthermore, Mn exposure resulted in elevated protein expression levels of estrogen receptor α, activator protein 2α, phosphorylated (p)-Akt, and p53 in MGs on PND 120, but not on PND 30. Collectively, these data show that exposure to a supplemental dose of Mn causes accelerated pubertal MG growth which can progress to adult hyperplasia; thus, providing evidence that early life Mn exposure may increase susceptibility to breast cancer.

New Frontiers for the NFIL3 bZIP Transcription Factor in Cancer, Metabolism and Beyond.

The bZIP transcription factor NFIL3 (Nuclear factor Interleukin 3 regulated, also known as E4 binding protein 4, E4BP4) regulates diverse biological processes from circadian rhythm to cellular viability. Recently, a host of novel roles have been identified for NFIL3 in immunological signal transduction, cancer, aging and metabolism. Elucidating the signaling pathways that are impacted by NFIL3 and the regulatory mechanisms that it targets, inhibits or activates will be critical for developing a clearer picture of its physiological roles in disease and normal processes. This review will discuss the recent advances and emerging issues regarding NFIL3-mediated transcriptional regulation of CEBPß and FOXO1 activated genes and signal transduction.

Prepubertal exposure to arsenic(III) suppresses circulating insulin-like growth factor-1 (IGF-1) delaying sexual maturation in female rats.

Arsenic (As) is a prevalent environmental toxin readily accessible for human consumption and has been identified as an endocrine disruptor. However, it is not known what impact As has on female sexual maturation. Therefore, in the present study, we investigated the effects of prepubertal exposure on mammary gland development and pubertal onset in female rats. Results showed that prepubertal exposure to 10 mg/kg of arsenite (As(III)) delayed vaginal opening (VO) and prepubertal mammary gland maturation. We determined that As accumulates in the liver, disrupts hepatocyte function and suppresses serum levels of the puberty related hormone insulin-like growth factor 1 (IGF-1) in prepubertal animals. Overall, this is the first study to show that prepubertal exposure to As(III) acts peripherally to suppress circulating levels of IGF-1 resulting in delayed sexual maturation. Furthermore, this study identifies a critical window of increased susceptibility to As(III) that may have a lasting impact on female reproductive function.

A moderate elevation of circulating levels of IGF-I does not alter ErbB2 induced mammary tumorigenesis.

BACKGROUND: Epidemiological evidence suggests that moderately elevated levels of circulating insulin-like growth factor-I (IGF-I) are associated with increased risk of breast cancer in women. How circulating IGF-I may promote breast cancer incidence is unknown, however, increased IGF-I signaling is linked to trastuzumab resistance in ErbB2 positive breast cancer. Few models have directly examined the effect of moderately high levels of circulating IGF-I on breast cancer initiation and progression. The purpose of this study was to assess the ability of circulating IGF-I to independently initiate mammary tumorigenesis and/or accelerate the progression of ErbB2 mediated mammary tumor growth. METHODS: We crossed heterozygous TTR-IGF-I mice with heterozygous MMTV-ErbB2 mice to generate 4 different genotypes: TTR-IGF-I/MMTV-ErbB2 (bigenic), TTR-IGF-I only, MMTV-ErbB2 only, and wild type (wt). Virgin females were palpated twice a week and harvested when tumors reached 1000 mm(3). For study of normal development, blood and tissue were harvested at 4, 6 and 9 weeks of age in TTR-IGF-I and wt mice. RESULTS: TTR-IGF-I and TTR-IGF-I/ErbB2 bigenic mice showed a moderate 35% increase in circulating total IGF-I compared to ErbB2 and wt control mice. Elevation of circulating IGF-I had no effect upon pubertal mammary gland development. The transgenic increase in IGF-I alone wasn't sufficient to initiate mammary tumorigenesis. Elevated circulating IGF-I had no effect upon ErbB2-induced mammary tumorigenesis or metastasis, with median time to tumor formation being 30 wks and 33 wks in TTR-IGF-I/ErbB2 bigenic and ErbB2 mice respectively (p = 0.65). Levels of IGF-I in lysates from ErbB2/TTR-IGF-I tumors compared to ErbB2 was elevated in a similar manner to the circulating IGF-I, however, there was no effect on the rate of tumor growth (p = 0.23). There were no morphological differences in tumor type (solid adenocarcinomas) between bigenic and ErbB2 mammary glands. CONCLUSION: Using the first transgenic animal model to elevate circulating levels of IGF-I to those comparable to women at increased risk of breast cancer, we showed that moderately high levels of systemic IGF-I have no effect on pubertal mammary gland development, initiating mammary tumorigenesis or promoting ErbB2 driven mammary carcinogenesis. Our work suggests that ErbB2-induced mammary tumorigenesis is independent of the normal variation in circulating levels of IGF-I.

Parity-induced decrease in systemic growth hormone alters mammary gland signaling: a potential role in pregnancy protection from breast cancer.

Early full-term pregnancy is an effective natural protection against breast cancer in both humans and experimental rodents. The protective effect of an early pregnancy is, in part, linked to changes in circulating hormones that are involved in both normal breast development and breast cancer. For example, a reduction in circulating growth hormone (GH) has been shown to protect rats from carcinogen-induced mammary tumors. We examined the ability of a full-term pregnancy to alter the endocrine GH/insulin-like growth factor-I (IGF-I) axis and how this change affected normal mammary gland function in two commonly used rat models (Sprague-Dawley and Wistar Furth). Circulating GH and IGF-I were measured in blood drawn every 30 minutes from parous and age-matched virgin female rats. Mean serum GH levels were significantly decreased (P < 0.01) in parous compared with age-matched virgin rats for both strains. Changes in GH levels were independent of estrous cycle, indicated by a significant (P < 0.05) reduction in circulating levels of GH during estrus and diestrus in both parous strains. Despite the decrease in circulating GH, pituitary GH mRNA levels were unaltered in parous rats. Circulating IGF-I and hepatic IGF-I mRNA were also unaltered by parity in either rat strain. Immunoblot analysis of mammary glands showed decreases in phosphorylation of signal transducer and activator of transcription 5A and Janus-activated kinase 2, suggesting reduced action of GH in the mammary gland. Therefore, although the parity reduction in circulating GH does not affect circulating IGF-I levels, it is possible that reduced GH acts directly at the mammary gland and may play a role in pregnancy protection from breast cancer.

The type I insulin-like growth factor receptor pathway: a key player in cancer therapeutic resistance.

The insulin-like growth factor (IGF) ligands stimulate cellular proliferation and survival by activating the type I insulin-like growth factor receptor (IGF-IR). As a result, the IGF signaling system is implicated in a number of cancers, including those of the breast, prostate, and lung. In addition to mitogenic and anti-apoptotic roles that may directly influence tumor development, IGF-IR also appears to be a critical determinant of response to numerous cancer therapies. This review describes the role of the IGF-IR pathway in mediating resistance to both general cytotoxic therapies, such as radiation and chemotherapy, and targeted therapies, such as tamoxifen and trastuzumab. It concludes with a description of approaches to target IGF-IR and argues that inhibition of IGF signaling, in conjunction with standard therapies, may enhance the response of cancer cells to multiple modalities.

Altered mammary gland development in the p53+/m mouse, a model of accelerated aging.

The tumor suppressor p53 is important for inhibiting the development of breast carcinomas. However, little is known about the effects of increased p53 activity on mammary gland development. Therefore, the effect of p53 dosage on mammary gland development was examined by utilizing the p53+/m mouse, a p53 mutant which exhibits increased wild-type p53 activity, increased tumor resistance, a shortened longevity, and a variety of accelerated aging phenotypes. Here we report that p53+/m virgin mice exhibit a defect in mammary gland ductal morphogenesis. Transplants of mammary epithelium into p53+/m recipient mice demonstrate decreased outgrowth of wild-type and p53+/m donor epithelium, suggesting systemic or stromal alterations in the p53+/m mouse. Supporting these data, p53+/m mice display decreased levels of serum IGF-1 and reduced IGF-1 signaling in virgin glands. The induction of pregnancy or treatment of p53+/m mice with estrogen, progesterone, estrogen and progesterone in combination, or IGF-1 stimulates ductal outgrowth, rescuing the p53+/m mammary phenotype. Serial mammary epithelium transplants demonstrate that p53+/m epithelium exhibits decreased transplant capabilities, suggesting early stem cell exhaustion. These data indicate that appropriate levels of p53 activity are important in regulating mammary gland ductal morphogenesis, in part through regulation of the IGF-1 pathway.

Oncogenic transformation by the signaling adaptor proteins insulin receptor substrate (IRS)-1 and IRS-2.

Insulin receptor substrates (IRSs) are adaptor proteins that link signaling from upstream activators to multiple downstream effectors to modulate normal growth, metabolism, survival, and differentiation. Recent cell culture studies have shown that IRSs can interact with, and are functionally required for, the transforming ability of many oncogenes. Consistent with this, IRSs are elevated and hyperactive in many human tumors. IRSs respond to many extracellular signals that are critical for mammary gland development, and we have shown that IRSs disrupt normal mammary acini formation in vitro, and cause mammary tumorigenesis and metastasis in vivo. In this review we will discuss the role of IRSs in both transformation and cancer progression.