The Role of Lipolysis Stimulated Lipoprotein Receptor in Breast Cancer and Directing Breast Cancer Cell Behavior

The claudin-low molecular subtype of breast cancer is of particular interest for clinically the majority of these tumors are poor prognosis, triple negative, invasive ductal carcinomas. Claudin-low tumors are characterized by cancer stem cell-like features and low expression of cell junction and adhesion proteins. Herein, we sought to define the role of lipolysis stimulated lipoprotein receptor (LSR) in breast cancer and cancer cell behavior as LSR was recently correlated with tumor-initiating features. We show that LSR was expressed in epithelium, endothelium, and stromal cells within the healthy breast tissue, as well as in tumor epithelium. In primary breast tumor bioposies, LSR expression was significantly correlated with invasive ductal carcinomas compared to invasive lobular carcinomas, as well as ERα positive tumors and breast cancer cell lines. LSR levels were significantly reduced in claudin-low breast cancer cell lines and functional studies illustrated that re-introduction of LSR into a claudin-low cell line suppressed the EMT phenotype and reduced individual cell migration. However, our data suggest that LSR may promote collective cell migration. Re-introduction of LSR in claudin-low breast cancer cell lines reestablished tight junction protein expression and correlated with transepithelial electrical resistance, thereby reverting claudin-low lines to other intrinsic molecular subtypes. Moreover, overexpression of LSR altered gene expression of pathways involved in transformation and tumorigenesis as well as enhanced proliferation and survival in anchorage independent conditions, highlighting that reestablishment of LSR signaling promotes aggressive/tumor initiating cell behaviors. Collectively, these data highlight a direct role for LSR in driving aggressive breast cancer behavior

Nuclear Localized LSR: A Novel Regulator of Breast Cancer Behavior and Tumorigenesis

Lipolysis Stimulated Lipoprotein Receptor (LSR) has been found in the plasma membrane and is believed to function in lipoprotein endocytosis and tight junctions. Given the impact of cellular metabolism and junction signaling pathways on tumor phenotypes and patient outcome, it is important to understand how LSR cellular localization mediates its functions. We conducted localization studies, evaluated DNA binding, and examined the effects of nuclear LSR in cells, xenografts, and clinical specimens. We found LSR within the membrane, cytoplasm, and the nucleus of breast cancer cells representing multiple intrinsic subtypes. Chromatin immunoprecipitation (ChIP) showed direct binding of LSR to DNA, and sequence analysis identified putative functional motifs and post-translational modifications of the LSR protein. While neither overexpression of transcript variants, nor pharmacological manipulation of post-translational modification significantly altered localization, inhibition of nuclear export enhanced nuclear localization, suggesting a mechanism for nuclear retention. Co-immunoprecipitation and proximal ligation assays indicated LSR-pericentrin interactions, presenting potential mechanisms for nuclear-localized LSR. The clinical significance of LSR was evaluated using data from over 1,100 primary breast tumors, which showed high LSR levels in basal-like tumors and tumors from African-Americans. In tumors histosections, nuclear localization was significantly associated with poor outcomes. Finally, in vivo xenograft studies revealed that basal-like breast cancer cells that over-express LSR exhibited both membrane and nuclear localization, and developed tumors with 100% penetrance, while control cells lacking LSR developed no tumors. These results show that nuclear LSR alters gene expression and may promote aggressive cancer phenotypes

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Challenging the roles of CD44 and lipolysis stimulated lipoprotein receptor in conveying Clostridium perfringens iota toxin cytotoxicity in breast cance

Nuclear Localized LSR: A Novel Regulator of Breast Cancer Behavior and Tumorigenesis

Lipolysis Stimulated Lipoprotein Receptor (LSR) has been found in the plasma membrane and is believed to function in lipoprotein endocytosis and tight junctions. Given the impact of cellular metabolism and junction signaling pathways on tumor phenotypes and patient outcome, it is important to understand how LSR cellular localization mediates its functions. We conducted localization studies, evaluated DNA binding, and examined the effects of nuclear LSR in cells, xenografts, and clinical specimens. We found LSR within the membrane, cytoplasm, and the nucleus of breast cancer cells representing multiple intrinsic subtypes. Chromatin immunoprecipitation (ChIP) showed direct binding of LSR to DNA, and sequence analysis identified putative functional motifs and post-translational modifications of the LSR protein. While neither overexpression of transcript variants, nor pharmacological manipulation of post-translational modification significantly altered localization, inhibition of nuclear export enhanced nuclear localization, suggesting a mechanism for nuclear retention. Co-immunoprecipitation and proximal ligation assays indicated LSR-pericentrin interactions, presenting potential mechanisms for nuclear-localized LSR. The clinical significance of LSR was evaluated using data from over 1,100 primary breast tumors, which showed high LSR levels in basal-like tumors and tumors from African-Americans. In tumors histosections, nuclear localization was significantly associated with poor outcomes. Finally, in vivo xenograft studies revealed that basal-like breast cancer cells that over-express LSR exhibited both membrane and nuclear localization, and developed tumors with 100% penetrance, while control cells lacking LSR developed no tumors. These results show that nuclear LSR alters gene expression and may promote aggressive cancer phenotypes

Re-introduction of LSR alters tight junction and breast cancer related gene expression.

<p>Hs578t cells were stably transfected with either a control plasmid (pCMV), or a plasmid containing the full-length gene for LSR variant 1 (LSR+). Quantitative real-time PCR analysis was performed for genes associated and with tight junctions and adhesion proteins (A). Data show representative scatterplots and corresponding list of genes significantly altered by expression of LSR. (B) Transepithelial Electrical Resistance in breast epithelial cells and breast cancer cells +/− LSR expression. Cells were plated on transwell inserts and grown to confluence. Two days post formation of a high-density monolayer TER was measured directly in culture medium. Cell monolayers were directly lysed on the transwell insert following final TER measurement and subjected to western analysis using an LSR specific antibody. Tubulin was used as a loading control. Date represent a minimum of three independent experiments, each measured in triplicate. (C) Quantitative real-time PCR analysis was performed for genes associated and with deregulation in cancer. Data show representative scatterplots and corresponding list of genes significantly altered by expression of LSR. (D) Representative western blots highlighting protein level changes of a panel of genes found to be differentially expressed in the arrays.</p

LSR protein expression in breast biopsies and correlation with clinical variables.

<p>(A) Representative images of breast cancer biopsy tissue arrays subjected to immunohistochemical analysis using a LSR specific antibody or corresponding negative control. Scale bar = 200 uM. Intensity of LSR expression in correlation with (B) breast cancer subtype IDC  =  invasive ductal carcinoma, ILC  =  invasive lobular carcinoma, (C) tumor invasion into sentinel lymph node and/or distant metastasis, (C) ERα status in tumor biopsy, and (D) ERα status in ERα positive breast cancer cell lines (MCF7, ZR75-1, and T47D) and ERα negative breast cancer cell lines (MDA-MD-231, SUM159, Hs578t). Data represent mean relative intensity +/− SE. *<i>P<</i>0.05, **<i>P<</i>0.01. A total of 248 patient samples were analyzed.</p

LSR enhances cell survival in non-adherent culture conditions.

<p>Hs578t cells were stably transfected with either a control plasmid (pCMV), or a plasmid containing the full-length gene for LSR variant 1 (LSR+). (A) Soft agar assays. Cells were plated on soft agar coated wells, grown for seven days, then stained with nitrobluetetrazolium before counting. The entire dish was analyzed and colonies larger than 50 um in diameter were counted. Data represent mean colonies counted per well ± SE of three separate experiments; *<i>P<</i>0.001. Bottom panels are representative images at 10X. (B) Sphere forming efficiency. Cells were plated in DMEM +10 ng/ml EGF +20 ng/ml FGF +2% B27 in ultralow attachment dishes for seven days then spheres counted and imaged. Data represent mean +/− SD of three independent experiments at the indicated cell plating densities. Bottom panels are representative phase images of anchorage-independent, single-cell derived spheres from LSR+ and control pCMV cells after seven days of growth. Scale bar, 50 <i>u</i>m.</p

LSR expression in breast cancer molecular subtypes.

<p>Representative breast cancer cell lines were grown to 80% confluence, lysates were isolated and analyzed via western analysis using a LSR specific antibody; α-tubulin was used as a loading control. (A, B) Representative western blot and corresponding intensity measured via ImageJ. (C) Analysis based on molecular subtype of cell lines. Data represent mean relative intensity +/− SE. **<i>P<</i>0.01.</p

LSR protein expression in breast tissue and epithelial cells.

<p>(A) Representative images of normal breast tissue arrays subjected to immunohistochemical analysis using a LSR specific antibody or corresponding negative control. Scale bar = 200 uM. (B) Representative images of breast epithelial cells subjected to immunocytofluorescence using LSR and ZO-1 specific antibodies (DNA stained with DAPI). Control images are primary breast epithelial cells simultaneously subjected to all steps with the exception of the addition of the primary antibody. Images were obtained at 20X.</p

Paralemmin-1 is over-expressed in estrogen-receptor positive breast cancers

Background Paralemmin-1 is a phosphoprotein lipid-anchored to the cytoplasmic face of membranes where it functions in membrane dynamics, maintenance of cell shape, and process formation. Expression of paralemmin-1 and its major splice variant (Δ exon 8) as well as the extent of posttranslational modifications are tissue- and development-specific. Paralemmin-1 expression in normal breast and breast cancer tissue has not been described previously. Results Paralemmin-1 mRNA and protein expression was evaluated in ten breast cell lines, 26 primary tumors, and 10 reduction mammoplasty (RM) tissues using real time RT-PCR. Paralemmin-1 splice variants were assessed in tumor and RM tissues using a series of primers and RT-PCR. Paralemmin-1 protein expression was examined in cell lines using Western Blots and in 31 ductal carcinomas in situ, 65 infiltrating ductal carcinomas, and 40 RM tissues using immunohistochemistry. Paralemmin-1 mRNA levels were higher in breast cancers than in RM tissue and estrogen receptor (ER)-positive tumors had higher transcript levels than ER-negative tumors. The Δ exon 8 splice variant was detected more frequently in tumor than in RM tissues. Protein expression was consistent with mRNA results showing higher paralemmin-1 expression in ER-positive tumors. Conclusions The differential expression of paralemmin-1 in a subset of breast cancers suggests the existence of variation in membrane dynamics that may be exploited to improve diagnosis or provide a therapeutic target