Andy J. Minn

Genes that mediate breast cancer metastasis to lung

By means of in vivo selection, transcriptomic analysis, functional verification and clinical validation, here we identify a set of genes that marks and mediates breast cancer metastasis to the lungs. Some of these genes serve dual functions, providing growth advantages both in the primary tumour and in the lung microenvironment. Others contribute to aggressive growth selectively in the lung. Many encode extracellular proteins and are of previously unknown relevance to cancer metastasis.

CD28 costimulation can promote T cell survival by enhancing the expression of Bcl-xL

T cell activation through the TCR can result in either cell proliferation or cell death. The role of costimulatory receptors in regulating T cell survival has not been defined. Here, we present data demonstrating that CD28 costimulation enhances the in vitro survival of activated T cells. One mechanism for this enhancement is the ability of CD28 costimulation to augment the production of IL-2, which acts as an extrinsic survival factor for T cells. In addition, CD28 costimulation augments the intrinsic ability of T cells to resist apoptosis. Although CD28 signal transduction had no effect on Bcl-2 expression, CD28 costimulation was found to augment the expression of Bcl-XL substantially. Transfection experiments demonstrated that this level of Bcl-XL could prevent T cell death in response to TCR cross-linking, Fas cross-linking, or IL-2 withdrawal. These data suggest that an important role of CD28 costimulation is to augment T cell survival during antigen activation.

Genes that mediate breast cancer metastasis to the brain

The molecular basis for breast cancer metastasis to the brain is largely unknown. Brain relapse typically occurs years after the removal of a breast tumour, suggesting that disseminated cancer cells must acquire specialized functions to take over this organ. Here we show that breast cancer metastasis to the brain involves mediators of extravasation through non-fenestrated capillaries, complemented by specific enhancers of blood–brain barrier crossing and brain colonization. We isolated cells that preferentially infiltrate the brain from patients with advanced disease. Gene expression analysis of these cells and of clinical samples, coupled with functional analysis, identified the cyclooxygenase COX2 (also known as PTGS2), the epidermal growth factor receptor (EGFR) ligand HBEGF, and the α2,6-sialyltransferase ST6GALNAC5 as mediators of cancer cell passage through the blood–brain barrier. EGFR ligands and COX2 were previously linked to breast cancer infiltration of the lungs, but not the bones or liver, suggesting a sharing of these mediators in cerebral and pulmonary metastases. In contrast, ST6GALNAC5 specifically mediates brain metastasis. Normally restricted to the brain, the expression of ST6GALNAC5 in breast cancer cells enhances their adhesion to brain endothelial cells and their passage through the blood–brain barrier. This co-option of a brain sialyltransferase highlights the role of cell-surface glycosylation in organ-specific metastatic interactions.

Tumour exosome integrins determine organotropic metastasis.

Ever since Stephen Paget’s 1889 hypothesis, metastatic organotropism has remained one of cancer’s greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.

Radiation and dual checkpoint blockade activate non-redundant immune mechanisms in cancer

Immune checkpoint inhibitors result in impressive clinical responses, but optimal results will require combination with each other and other therapies. This raises fundamental questions about mechanisms of non-redundancy and resistance. Here we report major tumour regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation, and reproduced this effect in mouse models. Although combined treatment improved responses in irradiated and unirradiated tumours, resistance was common. Unbiased analyses of mice revealed that resistance was due to upregulation of PD-L1 on melanoma cells and associated with T-cell exhaustion. Accordingly, optimal response in melanoma and other cancer types requires radiation, anti-CTLA4 and anti-PD-L1/PD-1. Anti-CTLA4 predominantly inhibits T-regulatory cells (Treg cells), thereby increasing the CD8 T-cell to Treg (CD8/Treg) ratio. Radiation enhances the diversity of the T-cell receptor (TCR) repertoire of intratumoral T cells. Together, anti-CTLA4 promotes expansion of T cells, while radiation shapes the TCR repertoire of the expanded peripheral clones. Addition of PD-L1 blockade reverses T-cell exhaustion to mitigate depression in the CD8/Treg ratio and further encourages oligoclonal T-cell expansion. Similarly to results from mice, patients on our clinical trial with melanoma showing high PD-L1 did not respond to radiation plus anti-CTLA4, demonstrated persistent T-cell exhaustion, and rapidly progressed. Thus, PD-L1 on melanoma cells allows tumours to escape anti-CTLA4-based therapy, and the combination of radiation, anti-CTLA4 and anti-PD-L1 promotes response and immunity through distinct mechanisms.

Lung metastasis genes couple breast tumor size and metastatic spread

The association between large tumor size and metastatic risk in a majority of clinical cancers has led to questions as to whether these observations are causally related or whether one is simply a marker for the other. This is partly due to an uncertainty about how metastasis-promoting gene expression changes can arise in primary tumors. We investigated this question through the analysis of a previously defined “lung metastasis gene-expression signature” (LMS) that mediates experimental breast cancer metastasis selectively to the lung and is expressed by primary human breast cancer with a high risk for developing lung metastasis. Experimentally, we demonstrate that the LMS promotes primary tumor growth that enriches for LMS+ cells, and it allows for intravasation after reaching a critical tumor size. Clinically, this corresponds to LMS+ tumors being larger at diagnosis compared with LMS− tumors and to a marked rise in the incidence of metastasis after LMS+ tumors reach 2 cm. Patients with LMS-expressing primary tumors selectively fail in the lung compared with the bone or other visceral sites and have a worse overall survival. The mechanistic linkage between metastasis gene expression, accelerated tumor growth, and likelihood of metastatic recurrence provided by the LMS may help to explain observations of prognostic gene signatures in primary cancer and how tumor growth can both lead to metastasis and be a marker for cells destined to metastasize.

Raf kinase inhibitory protein suppresses a metastasis signalling cascade involving LIN28 and let-7

Raf kinase inhibitory protein (RKIP) negatively regulates the MAP kinase (MAPK), G protein‐coupled receptor kinase‐2, and NF‐κB signalling cascades. RKIP has been implicated as a metastasis suppressor for prostate cancer, but the mechanism is not known. Here, we show that RKIP inhibits invasion by metastatic breast cancer cells and represses breast tumour cell intravasation and bone metastasis in an orthotopic murine model. The mechanism involves inhibition of MAPK, leading to decreased transcription of LIN28 by Myc. Suppression of LIN28 enables enhanced let‐7 processing in breast cancer cells. Elevated let‐7 expression inhibits HMGA2, a chromatin remodelling protein that activates pro‐invasive and pro‐metastatic genes, including Snail. LIN28 depletion and let‐7 expression suppress bone metastasis, and LIN28 restores bone metastasis in mice bearing RKIP‐expressing breast tumour cells. These results indicate that RKIP suppresses invasion and metastasis in part through a signalling cascade involving MAPK, Myc, LIN28, let‐7, and downstream let‐7 targets. RKIP regulation of two pluripotent stem cell genes, Myc and LIN28, highlights the importance of RKIP as a key metastasis suppressor and potential therapeutic agent.

Identification of a novel regulatory domain in Bcl‐xL and Bcl‐2

Bcl‐xL, a member of the Bcl‐2 family, can inhibit many forms of programed cell death. The three‐dimensional structure of Bcl‐xL identified a 60 amino acid loop lacking defined structure. Although amino acid sequence within this region is not conserved among Bcl‐2 family members, structural modeling suggested that Bcl‐2 also contains a large unstructured region. Compared with the full‐length protein, loop deletion mutants of Bcl‐xL and Bcl‐2 displayed an enhanced ability to inhibit apoptosis. Despite enhanced function, the deletion mutants did not have significant alterations in the ability to bind pro‐apoptotic proteins such as Bax. The loop deletion mutant of Bcl‐2 also displayed a qualitative difference in its ability to inhibit apoptosis. Full‐length Bcl‐2 was unable to prevent anti‐IgM‐induced cell death of the immature B cell line WEHI‐231. In contrast, the Bcl‐2 deletion mutant protected WEHI‐231 cells from death. Substantial differences were observed in the ability of WEHI‐231 cells to phosphorylate the deletion mutant of Bcl‐2 compared with full‐length Bcl‐2. Bcl‐2 phosphorylation was found to be dependent on the presence of an intact loop domain. These results suggest that the loop domain in Bcl‐xL and Bcl‐2 can suppress the anti‐apoptotic function of these genes and may be a target for regulatory post‐translational modifications.

Exosome Transfer from Stromal to Breast Cancer Cells Regulates Therapy Resistance Pathways

Stromal communication with cancer cells can influence treatment response. We show that stromal and breast cancer (BrCa) cells utilize paracrine and juxtacrine signaling to drive chemotherapy and radiation resistance. Upon heterotypic interaction, exosomes are transferred from stromal to BrCa cells. RNA within exosomes, which are largely noncoding transcripts and transposable elements, stimulates the pattern recognition receptor RIG-I to activate STAT1-dependent antiviral signaling. In parallel, stromal cells also activate NOTCH3 on BrCa cells. The paracrine antiviral and juxtacrine NOTCH3 pathways converge as STAT1 facilitates transcriptional responses to NOTCH3 and expands therapy-resistant tumor-initiating cells. Primary human and/or mouse BrCa analysis support the role of antiviral/NOTCH3 pathways in NOTCH signaling and stroma-mediated resistance, which is abrogated by combination therapy with gamma secretase inhibitors. Thus, stromal cells orchestrate an intricate crosstalk with BrCa cells by utilizing exosomes to instigate antiviral signaling. This expands BrCa subpopulations adept at resisting therapy and reinitiating tumor growth.

An interferon-related gene signature for DNA damage resistance is a predictive marker for chemotherapy and radiation for breast cancer

Individualization of cancer management requires prognostic markers and therapy-predictive markers. Prognostic markers assess risk of disease progression independent of therapy, whereas therapy-predictive markers identify patients whose disease is sensitive or resistant to treatment. We show that an experimentally derived IFN-related DNA damage resistance signature (IRDS) is associated with resistance to chemotherapy and/or radiation across different cancer cell lines. The IRDS genes STAT1, ISG15, and IFIT1 all mediate experimental resistance. Clinical analyses reveal that IRDS(+) and IRDS(−) states exist among common human cancers. In breast cancer, a seven–gene-pair classifier predicts for efficacy of adjuvant chemotherapy and for local-regional control after radiation. By providing information on treatment sensitivity or resistance, the IRDS improves outcome prediction when combined with standard markers, risk groups, or other genomic classifiers.