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

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

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