Distinctive Left-Sided Distribution of Adrenergic-Derived Cells in the Adult Mouse Heart

Adrenaline and noradrenaline are produced within the heart from neuronal and non-neuronal sources. These adrenergic hormones have profound effects on cardiovascular development and function, yet relatively little information is available about the specific tissue distribution of adrenergic cells within the adult heart. The purpose of the present study was to define the anatomical localization of cells derived from an adrenergic lineage within the adult heart. To accomplish this, we performed genetic fate-mapping experiments where mice with the cre-recombinase (Cre) gene inserted into the phenylethanolamine-n-methyltransferase (Pnmt) locus were cross-mated with homozygous Rosa26 reporter (R26R) mice. Because Pnmt serves as a marker gene for adrenergic cells, offspring from these matings express the β-galactosidase (βGAL) reporter gene in cells of an adrenergic lineage. βGAL expression was found throughout the adult mouse heart, but was predominantly (89%) located in the left atrium (LA) and ventricle (LV) (p<0.001 compared to RA and RV), where many of these cells appeared to have cardiomyocyte-like morphological and structural characteristics. The staining pattern in the LA was diffuse, but the LV free wall displayed intermittent non-random staining that extended from the apex to the base of the heart, including heavy staining of the anterior papillary muscle along its perimeter. Three-dimensional computer-aided reconstruction of XGAL+ staining revealed distribution throughout the LA and LV, with specific finger-like projections apparent near the mid and apical regions of the LV free wall. These data indicate that adrenergic-derived cells display distinctive left-sided distribution patterns in the adult mouse heart

Metabotropic glutamate receptor-1 contributes to progression in triple negative breast cancer.

TNBC is an aggressive breast cancer subtype that does not express hormone receptors (estrogen and progesterone receptors, ER and PR) or amplified human epidermal growth factor receptor type 2 (HER2), and there currently exist no targeted therapies effective against it. Consequently, finding new molecular targets in triple negative breast cancer (TNBC) is critical to improving patient outcomes. Previously, we have detected the expression of metabotropic glutamate receptor-1 (gene: GRM1; protein: mGluR1) in TNBC and observed that targeting glutamatergic signaling inhibits TNBC growth both in vitro and in vivo. In this study, we explored how mGluR1 contributes to TNBC progression, using the isogenic MCF10 progression series, which models breast carcinogenesis from nontransformed epithelium to malignant basal-like breast cancer. We observed that mGluR1 is expressed in human breast cancer and that in MCF10A cells, which model nontransformed mammary epithelium, but not in MCF10AT1 cells, which model atypical ductal hyperplasia, mGluR1 overexpression results in increased proliferation, anchorage-independent growth, and invasiveness. In contrast, mGluR1 knockdown results in a decrease in these activities in malignant MCF10CA1d cells. Similarly, pharmacologic inhibition of glutamatergic signaling in MCF10CA1d cells results in a decrease in proliferation and anchorage-independent growth. Finally, transduction of MCF10AT1 cells, which express c-Ha-ras, using a lentiviral construct expressing GRM1 results in transformation to carcinoma in 90% of resultant xenografts. We conclude that mGluR1 cooperates with other factors in hyperplastic mammary epithelium to contribute to TNBC progression and therefore propose that glutamatergic signaling represents a promising new molecular target for TNBC therapy

Expression of <i>GRM1</i> and mGluR1 in the MCF10 progression series.

<p><b>A. <i>GRM1</i> mRNA expression.</b> RNA lysates collected from the progression series cells were synthesized to cDNA and qRT-PCR analysis performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081126#s2" target="_blank">Materials and Methods</a>. Relative fold change <i>GRM1</i> expression of MCF10AT1, MCF10DCIS.com, and MCF10CA1d are compared to MCF10A cells. <b>B. mGluR1 protein expression.</b> Protein lysates were immunoprecipitated with mGluR1 antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081126#s2" target="_blank">Materials and Methods</a>. Premalignant MCF10AT1 and malignant MCF10DCIS.com, and MCF10CA1d express increased levels of <i>GRM1</i> and mGluR1 compared to parental MCF10A cells, with MCF10DCIS.com expressing the highest levels.</p

mGluR1 facilitates anchorage-independent growth.

<p><b>A and B. MCF10AT1 cells.</b> Cells were transduced with either with pLenti-<i>GRM1</i>, pLenti-<i>LacZ</i>, sh<i>GRM1</i>, or NS control vector and stably selected before plating in soft agar. <i>GRM1</i> overexpression significantly increased number of colonies on soft agar compared to the control <i>LacZ</i> cells (<b>A</b>), an effect reversed by sh<i>GRM1</i> (<b>B</b>). <b>C. MCF10CA1d cells.</b> Silencing <i>GRM1</i> significantly decreased colony formation compared to the non-silencing (NS) vector control in MCF10CA1d cells. Finally, colony formation is inhibited by BAY in both MCF10DCIS.com cells (<b>D</b>) and MCF10CA1d cells (<b>E</b>). For all assays 1×10⁵ cells were suspended in 0.4% agarose and colonies formed on the soft agar were counted after three weeks on Gel Count machine. Results are recorded as the mean ± SEM of three experiments, where *p<0.05 compared to control. <b>F. Live cell imaging of 4 day MAME cultures of MCF10AT1-</b><b><i>LacZ</i></b><b> and MCF10AT1-</b><b><i>GRM1</i></b><b> cells illustrate increases in proliferative and invasive phenotype in cells expressing </b><b><i>GRM1</i></b><b>.</b> 15,000 cells were seeded per coverslip on reconstituted basement membrane. Differential Interference Contrast images from 16 contiguous fields were obtained with a Zeiss LSM-510 META confocal microscope. Tiled images allow one to see the relative sizes of the structures formed over a 4-day period. Bar, 100 µm. <b>Legend</b>: <i>LacZ</i> = pLenti-<i>LacZ</i>; <i>GRM1</i> = pLenti-<i>GRM1</i>.</p

mGluR1 is active in a TNBC cell line.

<p>BT549 cells were stimulated in glutamate-free media containing GlutaMAX™ (Life Technologies, Grand Island, NY) with mGluR1 agonist, L-quisqualate (10 µM). Cells were harvested and the fold-increase in phosphorylated ERK1/2 was assayed by Western blot. Pretreatment with mGluR1 antagonist LY367385 for 30 minutes markedly reduced pERK1/2 induction by L-quisqualate (right side of gel). <b>Bottom gel:</b> Same blot stripped and re-probed with ERK antibody for normalization. Experiments were repeated two times with similar results.</p

Increasing mGluR1 activity has no effect on measures of oncogenesis in MCF10A cells.

<p><b>A. <i>GRM1</i> expression.</b> RNA lysates from stable transfectants of <i>GRM1</i>-MCF10AT1 or control <i>LACZ</i> cells were isolated and subjected to qPCR as described in Materials/Subjects and Methods. <i>GRM1</i> is expressed at a level approximately 1.8×10⁴ times higher in GRM1-transduced MCF10A cells than in <i>LacZ</i> control infected cells. <b>B. mGluR1 expression.</b> Protein lysate (30 µg) was loaded in each lane. The membrane was stripped and reprobed for tubulin antibody to show equal loadings. (<b><i>LacZ</i></b> = pLenti-LacZ; <b><i>GRM1</i></b> = pLenti-<i>GRM1</i>). <b>C. Effect of mGluR1 overexpression on proliferation.</b> Lentivirus-driven mGluR1 expression had no effect on the proliferation of MCF10AT1 cells compared to the control as measured by MTT assay. For MTT assay, 1×10⁴ cells were plated and cell proliferation was analyzed on day 3. <b>D. Effect of mGluR1 expression on MCF10A migration.</b> <i>GRM1</i> overexpression in MCF10AT1 cells had no effect on MCF10A migration compared to the control <i>LacZ-</i>transduced cells. <b>E. Effect of mGluR1 expression on anchorage-independent colony formation.</b> MCF10A cells were transduced with either with pLenti-<i>GRM1</i> or pLenti-<i>LacZ</i> and plated in soft agar as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081126#s2" target="_blank">Materials and Methods</a>. <i>GRM1</i> overexpression had no effect on the number of colonies on soft agar compared to the control <i>LacZ</i> cells. All experimenbts were performed in triplicate and results are recorded as the mean ± SEM of three experiments, where *p<0.05 compared to control.</p

mGluR1 activity regulates cell proliferation.

<p><b>A. Proliferation.</b> mGluR1 overexpression significantly increased proliferation of MCF10AT1 cells compared to the control as measured by MTT assay. For MTT assay, 1×10⁴ cells were plated and cell proliferation was analyzed on day 3. <b>B. Cell counts.</b> Cell counting by Coulter counter also show that mGluR1 overexpression significantly increased proliferation of MCF10AT1 cells compared to the control LACZ cells. <b>C. Pharmacological inhibition of mGluR1 inhibits TNBC cell proliferation.</b> Proliferation, as measured by cell counts of MCF10CA1d cells was significantly inhibited by glutamatergic signaling inhibitor, BAY. <b>D and E. Effect of silencing mGluR1.</b> Silencing <i>GRM1</i> significantly decreased cell growth compared to the NS vector control in <i>GRM1-</i> MCF10AT1 cells (<b>D</b>) and in malignant MCF10CA1d cells (<b>E</b>). For B, <b>C, D</b>, and <b>E</b>, growth assays, 5×10⁴ cells were plated in 6-well tissue culture plates and cell numbers counted by Coulter counter on day 3 for B, D, E growth assays and for C growth assay cells were counted on day 6 after plating. Results are recorded as mean ± SEM of replicate experiments, where <b>*</b>p<0.05 compared to control. <b>Legend</b>: NS = pLenti vector containing nonsilencing control; sh<i>GRM1</i> = pLenti vector containing shRNA against <i>GRM1</i>; <i>LacZ</i> = pLenti-<i>LacZ</i>; <i>GRM1</i> = pLenti-<i>GRM1</i>.</p

mGluR1 transforms MCF10AT1 cells.

<p>MCF10AT1 cells, wild type or transduced with either <i>LacZ</i> or <i>GRM1</i>, were implanted into both flanks of athymic nude mice and allowed to grow for 8 weeks, after which MCF10AT1 lesions were harvested. <b>A. Representative wild type MCF10AT1 lesion</b> with both papillary enfolding (green arrow) and cribiform foci (red arrow). <b>B. </b><b><i>LacZ</i></b><b> control MCF10AT1 lesion.</b> The morphology indicates a hyperplastic lesion with both papillary (green arrow) and initial cribiforming present (red arrow). <b>C. mGluR1-overexpressing MCF10AT1 lesion.</b> The morphology indicates invasive cancer. These figures (<b>A</b> through <b>C</b>) are representative of 10 lesions analyzed for each group (magnification: 100×). <b>D. </b><b><i>GRM1</i></b><b> overexpression results in malignant transformation of MCF10AT1 cells.</b> Standard H&E sections of tumors were examined by a trained observer blinded to experimental group, and MCF10AT1 xenografts assessed for the presence and number of foci of carcinoma.</p

mGluR1 expression and silencing. <i>GRM1</i> overexpression results in mGluR1 protein overexpression in MCF10AT1 cells.

<p><b>A. <i>GRM1</i> expression.</b> RNA lysates from stable transfectants of <i>GRM1</i>-MCF10AT1 or control <i>LACZ</i> cells were isolated and subjected to QPCR as described in Materials/Subjects and Methods. <i>GRM1</i> was expressed at a level 1.6×10⁵ times higher in stable transformed GRM1-MCF10AT1 cells than in <i>LacZ</i> control infected cells. <b>B. mGluR1 expression.</b> Protein lysate (30 µg) was loaded in each lane. The membrane was stripped and reprobed for tubulin antibody to show equal loadings. (<b><i>LacZ</i></b> = pLenti-LacZ; <b><i>GRM1</i></b> = pLenti-<i>GRM1</i>). <b>C, D. </b><b><i>GRM1</i></b><b> silencing.</b> RNA lysates from MCF10DCIS.com and MCF10CA1d cells with Lentivirus-driven expression of sh<i>GRM1</i> or NS vector were subjected to qPCR analysis. <i>GRM1</i> expression was significantly silenced by 0.5 and 0.25 fold in sh<i>GRM1</i> transduced MCF10DCIS.com and MCF10CA1d cells respectively, compared to the NS control infected cells (<b>C</b>). GRM1-MCF10AT1 cells were silenced with either sh<i>GRM1</i> or NS control vector and <i>GRM1</i> mRNA message was analyzed using QPCR. <i>GRM1</i> is significantly silenced by 0.48 fold in <i>GRM1-</i>MCF10AT1 cells compared to the NS control infected cells (<b>D</b>). <b>E. mGluR1 silencing.</b> Protein lysate (50 µg) was loaded in each lane. mGluR1 is also silenced by sh<i>GRM1</i> in <i>GRM1</i>-MCF10AT1 cells compared to the NS control cells. qPCR was performed in triplicate and results are recorded as the mean ± SEM of three experiments, where *p<0.05 compared to control. (<b>For C, D, and E: NS</b> = pLenti vector containing nonsilencing control: <b>sh</b><b><i>GRM1</i></b> = pLenti vector containing shRNA against <i>GRM1</i>.)</p

mGluR1 regulates TNBC migration and invasion.

<p>MCF10AT1 or MCF10CA1d cells were transduced either with pLenti-<i>GRM1</i>, pLenti-<i>LacZ</i>, sh<i>GRM1</i>, or NS control. 2.5×10⁵ cells were plated on polycarbonate containing cell inserts and migration or invasion towards chemoattractant (10% serum) was measured after 24 hours. For invasion assays, polycarbonate membranes were coated with reconstituted basement membrane. <b>A. mGluR1 stimulates MCF10AT1 migration.</b> <i>GRM1</i> overexpression in MCF10AT1 cells significantly increased migration of the cells compared to the control <i>LacZ</i> cells, which was reversed (<b>B</b>) by silencing with sh<i>GRM1</i>. <b>C. MCF10AT1 invasion.</b> GRM1 overexpression significantly stimulated the invasion of MCF10AT1 cells through polycarbonate membranes coated with reconstituted basement membrane. <b>D. MCF10CA1d invasion.</b> <i>shGRM1</i> knockdown inhibited the invasion of malignant MCF10CA1d cells compared to the NS transduced cells. Results are recorded as the mean ± SEM of three experiments, where *p<0.05 compared to control. <b>Legend</b>: NS = nonsilencing control; sh<i>GRM1</i> = shRNA against <i>GRM1</i>; <i>LacZ</i> = pLenti-<i>LacZ</i>; <i>GRM1</i> = pLenti-<i>GRM1</i>.</p