PTK6/BRK is expressed in the normal mammary gland and activated at the plasma membrane in breast tumors.
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ABSTRACT:
SUBMITTER: Peng M
PROVIDER: S-EPMC4171611 | biostudies-literature | 2014 Aug
REPOSITORIES: biostudies-literature
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Project description:Basal-type triple-negative breast cancers (TNBC) are aggressive and difficult to treat relative to luminal-type breast cancers. TNBC often express abundant Met receptors and are enriched for transcriptional targets regulated by hypoxia-inducible factor-1α (HIF-1α), which independently predict cancer relapse and increased risk of metastasis. Brk/PTK6 is a critical downstream effector of Met signaling and is required for hepatocyte growth factor (HGF)-induced cell migration. Herein, we examined the regulation of Brk by HIFs in TNBC in vitro and in vivo. Brk mRNA and protein levels are upregulated strongly in vitro by hypoxia, low glucose, and reactive oxygen species. In HIF-silenced cells, Brk expression relied upon both HIF-1α and HIF-2α, which we found to regulate BRK transcription directly. HIF-1α/2α silencing in MDA-MB-231 cells diminished xenograft growth and Brk reexpression reversed this effect. These findings were pursued in vivo by crossing WAP-Brk (FVB) transgenic mice into the MET(Mut) knockin (FVB) model. In this setting, Brk expression augmented MET(Mut)-induced mammary tumor formation and metastasis. Unexpectedly, tumors arising in either MET(Mut) or WAP-Brk × MET(Mut) mice expressed abundant levels of Sik, the mouse homolog of Brk, which conferred increased tumor formation and decreased survival. Taken together, our results identify HIF-1α/2α as novel regulators of Brk expression and suggest that Brk is a key mediator of hypoxia-induced breast cancer progression. Targeting Brk expression or activity may provide an effective means to block the progression of aggressive breast cancers.
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Project description:Breast cancer (BCa) incidence increases following aberrant hormone exposure, which has been linked to direct effects on estrogen receptor (ER)+ mammary epithelium. While estrogen exposure during mammary involution has been shown to drive tumour growth via neutrophils, the potential for the ER + immune microenvironment to mediate part (in addition to mammary epithelial cells) of hormonally controlled BCa risk during normal development has not been assessed. We collected mammary tissue, lymph nodes and blood from tumour naïve mice treated with, oophorectomy, estrogen (17β estradiol) or Fulvestrant. Flow cytometry was used to examine the impact on the frequency of innate and adaptive immune cells. Oophorectomy and fulvestrant decreased the proportion of macrophages, particularly pro-tumour polarized M2 macrophages and neutrophils. Conversely, dendritic cells were increased by these therapies, as were eosinophils. Estrogen increased the proportion of M2 macrophages and to a lesser extent CD4-CD8- double negative and FoxP3+ regulatory T cells but decreased CD8 + T cells and B cells. Excluding eosinophils, these changes were restricted to the mammary tissue. This suggests that inhibiting estrogen action lowers the immune suppressive myeloid cells, increases in antigen presentation and eosinophil-mediated direct or indirect cytotoxic effects. In contrast, estrogen exposure, which drives BCa risk, increases the suppressive myeloid cells and reduces anti-tumour cytotoxic T cells. The impact of hormonal exposure on BCa risk, may in part be linked to its immune modulatory activity.
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Project description:Breast cancer risk from pesticides may be missed if effects on mammary gland are not assessed in toxicology studies required for registration. Using US EPA's registration documents, we identified pesticides that cause mammary tumors or alter development, and evaluated how those findings were considered in risk assessment. Of 28 pesticides that produced mammary tumors, EPA's risk assessment acknowledges those tumors for nine and dismisses the remaining cases. For five pesticides that alter mammary gland development, the implications for lactation and cancer risk are not assessed. Many of the mammary-active pesticides activate pathways related to endocrine disruption: altering steroid synthesis in H295R cells, activating nuclear receptors, or affecting xenobiotic metabolizing enzymes. Clearer guidelines based on breast cancer biology would strengthen assessment of mammary gland effects, including sensitive histology and hormone measures. Potential cancer risks from several common pesticides should be re-evaluated, including: malathion, triclopyr, atrazine, propylene oxide, and 3-iodo-2-propynyl butylcarbamate (IPBC).
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Project description:Connexin26 (Cx26) is the major Cx protein expressed in the human mammary gland and is up-regulated during pregnancy while remaining elevated throughout lactation. It is currently unknown if patients with loss-of-function Cx26 mutations that result in hearing loss and skin diseases have a greater susceptibility to impaired breast development. To investigate if Cx26 plays a critical role in mammary gland development and differentiation, a novel Cx26 conditional knockout mouse model was generated by crossing Cx26fl/fl mice with mice expressing Cre under the β-Lactoglobulin promoter. Conditional knockdown of Cx26 from the mammary gland resulted in a dramatic reduction in detectable gap junction plaques confirmed by a significant ∼65-70% reduction in Cx26 mRNA and protein throughout parturition and lactation. Interestingly, this reduction was accompanied by a decrease in mammary gland Cx30 gap junction plaques at parturition, while no change was observed for Cx32 or Cx43. Whole mount, histological and immunofluorescent assessment of breast tissue revealed comparatively normal lobuloalveolar development following pregnancy in the conditionally knockdown mice compared to control mice. In addition, glands from genetically-modified mice were capable of producing milk proteins that were evident in the lumen of alveoli and ducts at similar levels as controls, suggesting normal gland function. Together, our results suggest that low levels of Cx26 expression throughout pregnancy and lactation, and not the physiological surge in Cx26, is sufficient for normal gland development and function.
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Project description:IntroductionLineage tracing using inducible genetic labeling has emerged to be a powerful method for interrogating the developmental fate of cells in intact tissues. A common induction mechanism is the use of tamoxifen-dependent Cre recombinase (CreER and CreERT2), but the effects of tamoxifen at doses normally used in lineage-tracing studies on normal adult mammary gland homeostasis are not known.MethodsWe used flow cytometry and immunostaining of intact glands to determine whether varying doses of tamoxifen skew the distribution and the apoptosis and proliferation status of different types of mammary epithelial cells in vivo. We also examined how tamoxifen influences the number of progenitor and mammary repopulating units (MRUs).ResultsOur results indicate that ≥5 mg/25 g body weight of tamoxifen induces a transient increase in cell proliferation and in the number of basal cells in the adult mammary epithelium up to 7 days after tamoxifen administration. However, in the medium term (3 weeks), all doses of tamoxifen≥1 mg/25 g body weight result in a decrease in the number of basal and EpCAM+CD49b- luminal cells and a decrease in progenitor cell function. Tamoxifen at doses≥5 mg/25 g body weight induced a transient increase in caspase-3-mediated apoptotic cell death within the mammary epithelium. However, mammary epithelial cell numbers in all subpopulations were restored to their original levels by 8 weeks. No long-lasting effects of tamoxifen on MRU numbers or on pubertal ductal development were observed.ConclusionTamoxifen can skew the distribution of mammary cell types in a dose-dependent manner, and thus caution must be taken when interpreting lineage-tracing studies using high doses of tamoxifen, particularly when short-duration analyses of a quantitative nature are being performed.
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Project description:BackgroundThe Six1 homeobox gene is highly expressed in the embryonic mammary gland, continues to be expressed in early postnatal mammary development, but is lost when the mammary gland differentiates during pregnancy. However, Six1 is re-expressed in breast cancers, suggesting that its re-instatement in the adult mammary gland may contribute to breast tumorigenesis via initiating a developmental process out of context. Indeed, recent studies demonstrate that Six1 overexpression in the adult mouse mammary gland is sufficient for initiating invasive carcinomas, and that its overexpression in xenograft models of mammary cancer leads to metastasis. These data demonstrate that Six1 is causally involved in both breast tumorigenesis and metastasis, thus raising the possibility that it may be a viable therapeutic target. However, because Six1 is highly expressed in the developing mammary gland, and because it has been implicated in the expansion of mammary stem cells, targeting Six1 as an anti-cancer therapy may have unwanted side effects in the breast.ResultsWe sought to determine the role of Six1 in mammary development using two independent mouse models. To study the effect of Six1 loss in early mammary development when Six1 is normally expressed, Six1-/- embryonic mammary glands were transplanted into Rag1-/- mice. In addition, to determine whether Six1 downregulation is required during later stages of development to allow for proper differentiation, we overexpressed Six1 during adulthood using an inducible, mammary-specific transgenic mouse model. Morphogenesis of the mammary gland occurred normally in animals transplanted with Six1-/- embryonic mammary glands, likely through the redundant functions of other Six family members such as Six2 and Six4, whose expression was increased in response to Six1 loss. Surprisingly, inappropriate expression of Six1 in the adult mammary gland, when levels are normally low to absent, did not inhibit normal mammary differentiation during pregnancy or lactation.ConclusionsSix1 is not critical for normal mammary gland development, since neither loss nor inappropriate overexpression of Six1 adversely affects normal mammary gland development or function. However, as both Six2 and Six4 levels are increased in Six1-/- mammary glands, we postulate that these Six family members are functionally redundant in the gland, as is true of many homeobox gene families. This data, in conjunction with recent findings that Six1 is capable of promoting breast cancer initiation and progression, suggest that Six1 may serve as a reasonable chemotherapeutic target in a clinical setting, particularly for those women diagnosed with breast cancer in their childbearing years.
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Project description:Breast tumour kinase (Brk/PTK6) is overexpressed in up to 86% of breast cancers and is associated with poorer patient outcomes. It is considered a potential therapeutic target in breast cancer, even though the full spectrum of its kinase activity is not known. This study investigated the role of the kinase domain in promoting tumour growth and its potential in sensitising triple negative breast cancer cells to standard of care chemotherapy. Triple negative human xenograft models revealed that both kinase-inactive and wild-type Brk promoted xenograft growth. Suppression of Brk activity in cells subsequently co-treated with the chemotherapy agents doxorubicin or paclitaxel resulted in an increased cell sensitivity to these agents. In triple negative breast cancer cell lines, the inhibition of Brk kinase activity augmented the effects of doxorubicin or paclitaxel. High expression of the alternatively spliced isoform, ALT-PTK6, resulted in improved patient outcomes. Our study is the first to show a role for kinase-inactive Brk in human breast tumour xenograft growth; therefore, it is unlikely that kinase inhibition of Brk, in isolation, would halt tumour growth in vivo. Breast cancer cell responses to chemotherapy in vitro were kinase-dependent, indicating that treatment with kinase inhibitors could be a fruitful avenue for combinatorial treatment. Of particular prognostic value is the ratio of ALT-PTK6:Brk expression in predicating patient outcomes.
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Project description:Early diagnosis of mammary gland tumors is a challenging task in animals, especially in unspayed dogs. Hence, this study investigated the role of microsatellite instability (MSI), MMR gene mRNA transcript levels and SNPs of MMR genes in canine mammary gland tumors (CMT). A total of 77 microsatellite (MS) markers in 23 primary CMT were selected from four breeds of dogs. The results revealed that 11 out of 77 MS markers were unstable and showed MSI in all the tumors (at least at one locus), while the other markers were stable. Compared to the other markers, the ABC9TETRA, MEPIA, 9A5, SCNA11 and FJL25 markers showed higher frequencies of instability. All CMT demonstrated MSI, with eight tumors presenting MSI-H. The RT-qPCR results revealed significant upregulation of the mRNA levels of cMSH3, cMLH1, and cPMSI, but downregulation of cMSH2 compared to the levels in the control group. Moreover, single nucleotide polymorphisms (SNPs) were observed in the cMSH2 gene in four exons, i.e., 2, 6, 15, and 16. In conclusion, MSI, overexpression of MMR genes and SNPs in the MMR gene are associated with CMT and could be served as diagnostic biomarkers for CMT in the future.
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