EGCG, alone or combined with temsirolimus, inhibits tumor growth of sensitive and resistant HER2+ orthoxenopatients.

<p><b>(a)</b> Mice bearing HER2-PDX and resistant HER2-PDX (HER2-PDXR) were treated with control (C), EGCG (30 mg/kg, 3 days a week), temsirolimus (10 mg/kg, 1 day a week) or the combination (EGCG plus temsirolimus) for 21 days. Dots are mean of each experimental group and bars, SE. * (p ≤ 0.05), ** (p ≤ 0.01) and *** (p ≤ 0.001). <b>(b)</b> Apoptosis, by TUNEL fluorescent assay, was perfomed in control (C), EGCG (E), temsirolimus (T) and combination (T+E) treated as in (A) tumors. Tumors were collected at the end of the experiment and fixed in formalin. Pictures are representative of two samples of each group. <b>(c)</b> Body weight of the mice treated as in (A). Data are expressed as body weight at the end of the experiment and boxes show the 25th to 75th percentiles, whereas whiskers extend to the 5th and 95th percentiles.</p

EGCG, alone or combined with pertuzumab, inhibits tumor growth of sensitive and resistant HER2+ orthoxenopatients.

<p><b>(a)</b> Mice bearing HER2-PDX and resistant HER2-PDX (HER2-PDXR) were treated with control (C), EGCG (30 mg/kg, 3 days a week), pertuzumab (30 mg/kg, 1 day a week) or the combination (EGCG plus pertuzumab) for 24 days. Dots are mean of each experimental group and bars, SE. * (p ≤ 0.05), ** (p ≤ 0.01) and *** (p ≤ 0.001). <b>(b)</b> Apoptosis, by TUNEL fluorescent assay, was perfomed in control (C), EGCG (E), pertuzumab (P) and combination (P+E) treated as in (A) tumors. Tumors were collected at the end of the experiment and fixed in paraffin. Pictures are representative of two samples of each group. <b>(c)</b> Body weight of the mice treated as in (A). Data are expressed as body weight at the end of the experiment and boxes show the 25th to 75th percentiles, whereas whiskers extend to the 5th and 95th percentiles.</p

Pertuzumab plus trastuzumab combination improves effects in SK and SKLTR.

<p>Cells were treated with trastuzumab (20 μM), pertuzumab (5 μg/ml) and the combination of both for 5 days. Results were determined using an MTT assay and are expressed as the percentage of cell proliferation inhibition from three independent experiments performed in triplicate. Columns represent % of cell proliferation inhibition after trastuzumab or pertuzumab exposure and bars SE. * (p ≤ 0.05), ** (p ≤ 0.01) and *** (p ≤ 0.001) indicate levels of statistically significant difference compared with effect of the same drug in SKBr3 cells or compared with drugs administered alone (dashed line).</p

FASN inhibitors improve pertuzumab and temsirolimus activity in parental and resistant cells.

<p><b>(a)</b> Cells were treated with pertuzumab (5 μg/ml) combined with EGCG (60 μM) or G28UCM (5 μM) for 5 days. Results were determined using an MTT assay and are expressed as ratio of inhibition of cell proliferation induced for each treatment alone versus inhibition induced for co- treatment from three independent experiments performed in triplicate. Dashed lines represent the effect of each drug alone, ratio 1. <b>(b)</b> Cells were treated with temsirolimus (0.05, 0.1, 0.5 and 1 μM) combined with EGCG (60 μM) or G28UCM (5 μM) for 2 days. Results were determined using an MTT assay and are expressed as ratio of inhibition of cell proliferation induced for each treatment alone versus inhibition induced for co- treatment from three independent experiments performed in triplicate and with several temsirolimus concentrations. Dashed lines represent the effect of each drug alone, ratio 1. * (p ≤ 0.05), ** (p ≤ 0.01) and *** (p ≤ 0.001) indicate levels of statistically significant difference compared with drugs administered alone.</p

Lymphangioleiomyomatosis Biomarkers Linked to Lung Metastatic Potential and Cell Stemness

<div><p>Lymphangioleiomyomatosis (LAM) is a rare lung-metastasizing neoplasm caused by the proliferation of smooth muscle-like cells that commonly carry loss-of-function mutations in either the tuberous sclerosis complex 1 or 2 (<i>TSC1</i> or <i>TSC2</i>) genes. While allosteric inhibition of the mechanistic target of rapamycin (mTOR) has shown substantial clinical benefit, complementary therapies are required to improve response and/or to treat specific patients. However, there is a lack of LAM biomarkers that could potentially be used to monitor the disease and to develop other targeted therapies. We hypothesized that the mediators of cancer metastasis to lung, particularly in breast cancer, also play a relevant role in LAM. Analyses across independent breast cancer datasets revealed associations between low <i>TSC1/2</i> expression, altered mTOR complex 1 (mTORC1) pathway signaling, and metastasis to lung. Subsequently, immunohistochemical analyses of 23 LAM lesions revealed positivity in all cases for the lung metastasis mediators fascin 1 (FSCN1) and inhibitor of DNA binding 1 (ID1). Moreover, assessment of breast cancer stem or luminal progenitor cell biomarkers showed positivity in most LAM tissue for the aldehyde dehydrogenase 1 (ALDH1), integrin-ß3 (ITGB3/CD61), and/or the sex-determining region Y-box 9 (SOX9) proteins. The immunohistochemical analyses also provided evidence of heterogeneity between and within LAM cases. The analysis of <i>Tsc2</i>-deficient cells revealed relative over-expression of FSCN1 and ID1; however, <i>Tsc2</i>-deficient cells did not show higher sensitivity to ID1-based cancer inhibitors. Collectively, the results of this study reveal novel LAM biomarkers linked to breast cancer metastasis to lung and to cell stemness, which in turn might guide the assessment of additional or complementary therapeutic opportunities for LAM.</p></div

Positivity for breast cancer stemness biomarkers in LAM tissue.

<p>Representative positive results for ALDH1, CD61 and SOX9 in two LAM cases. Arrows mark magnified fields shown in the insets.</p

Positivity for breast cancer lung metastasis mediators in LAM tissue.

<p>(A) Representative results for FSCN1 and ID1 in two LAM cases. Arrows mark magnified fields shown in the insets. (B) Representative immunohistochemistry results for CD34 and FSCN1 in LAM tissue. Positivity for FSCN1 is greater and not limited to endothelial cells.</p

Intra-tissue and inter-case heterogeneity in the staining of canonical and novel LAM biomarkers.

<p>(A) Left panels, details of immunohistochemical results for canonical and novel biomarkers (FSNC1 and ID1) in a given LAM lesion, revealing expression heterogeneity. Right panels, evidence of heterogeneity based on the staining of SMA and FSCN1, SMA and ID1, and SMA and HMB-45 in three independent lesions/cases. (B) Top panel, heterogeneity for pS6 staining in a characteristic LAM cystic structure. The arrows mark two different tissue regions (a, b) that are magnified (bottom panels) for the immunohistochemical results of pS6 and the novel biomarkers. (C) Double immunofluorescence staining results also show intra-tissue heterogeneity for the novel biomarkers. (D) In large LAM lesions, pS6 is mostly apparent at the front.</p

Expression of mTOR pathway components and breast cancer metastasis to lung.

<p>(A) Kaplan-Meier lung metastasis-free survival (LMFS) and bone metastasis-free survival (BMFS) curves based on categorization of <i>TSC2</i> expression. The <i>P</i> values of the Cox proportional-hazards regression analysis are shown. (B) Tumor sample and gene expression clustering, and correlations of <i>TSC1/2</i> and genes from the lung metastasis signature, in the seminal breast cancer dataset [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132546#pone.0132546.ref027" target="_blank">27</a>]. (C) GSEA results for Cox regression values of the mTOR pathway gene set and LMFS or BMFS. (D) GSEA results for Cox regression values of metabolic pathway gene sets and LMFS or BMFS. (E) Tumor sample and gene expression clustering, and correlations between <i>TSC1/2</i> and genes from lung metastasis signature, in the TCGA dataset [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132546#pone.0132546.ref070" target="_blank">70</a>]. (F) GSEA results for the expression difference of the lung metastasis signature between MCF7 cells transduced with control or <i>TSC2</i>-target shRNAs. The left top panel shows the results for absolute expression differences, and the middle and bottom panels show the results for real differences of the up-regulated and down-regulated subsets of the signature, respectively. The right panels show the Western blot results for tuberin, pS6 and control loading, TUBA.</p

Immunohistochemical characterization of biomarkers in normal breast and lung tissue.

<p>(A) Representative hematoxylin-eosin and immunohistochemical staining in normal breast tissue. The observed patterns of positivity were those expected with the exception of ALDH1, which could have showed positivity in the basal and luminal cell layers of the acini; nonetheless, this can only be observed at the growing end and branching of the ducts [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132546#pone.0132546.ref048" target="_blank">48</a>], which may be represented by the image shown in the right panel. Expression of ALDH1, CD61, FSCN1 and SOX9 was also seen in spindle-like cells surrounding the terminal extra-lobular ducts as well as in similar cells of the loose specialized intra-lobular stroma (arrows in insets). The results of CD61 are detailed for the basal cell layer in differentiated acini (a) and for spindle-like intra-lobular cells (b). The arrows mark magnified fields. (B) Representative hematoxylin-eosin and immunohistochemical staining in normal lung tissue. ALDH1 and FSCN1 mark the alveolar endothelium, and ALDH1 also marks the basal and luminal layers of the bronchioles. CD61, ID1 and SOX9 are not expressed in differentiated alveoli, and CD61 and SOX9 show positivity in the luminal and/or basal layers of the bronchioles.</p