Lnc-DC promotes estrogen independent growth and tamoxifen resistance in breast cancer

Selective estrogen receptor modulators (SERMs) such as tamoxifen have proven to be effective in the treatment of estrogen receptor (ER) positive breast cancer. However, a major obstacle for such endocrine therapy is estrogen independent growth, leading to resistance, and the underlying mechanism is not fully understood. The purpose of this study was to determine whether long non-coding RNAs (lncRNAs) are involved in regulation of estrogen independent growth and tamoxifen resistance in ER positive breast cancer. Using a CRISPR/Cas9-based SAM (synergistic activation mediator) library against a focus group of lncRNAs, we identify Lnc-DC as a candidate lncRNA. Further analysis suggests that Lnc-DC is able to reduce tamoxifen-induced apoptosis by upregulation of anti-apoptotic genes such as Bcl2 and Bcl-xL. Furthermore, Lnc-DC activates STAT3 by phosphorylation (pSTAT3Y705), and the activated STAT3 subsequently induces expression of cytokines which in turn activate STAT3, forming an autocrine loop. Clinically, upregulation of Lnc-DC is associated with poor prognosis. In particular, analysis of a tamoxifen-treated patient cohort indicates that Lnc-DC expression can predict the response to tamoxifen. Together, this study demonstrates a previously uncharacterized function of Lnc-DC/STAT3/cytokine axis in estrogen independent growth and tamoxifen resistance, and Lnc-DC may serve as a potential predictor for tamoxifen response.

Comprehensive Analysis of VEGFR2 Expression in HPV-Positive and -Negative OPSCC Reveals Differing VEGFR2 Expression Patterns

VEGF signaling regulated by the vascular endothelial growth factor receptor 2 (VEGFR2) plays a decisive role in tumor angiogenesis, initiation and progression in several tumors including HNSCC. However, the impact of HPV-status on the expression of VEGFR2 in OPSCC has not yet been investigated, although HPV oncoproteins E6 and E7 induce VEGF-expression. In a series of 56 OPSCC with known HPV-status, VEGFR2 expression patterns were analyzed both in blood vessels from tumor-free and tumor-containing regions and within tumor cells by immunohistochemistry using densitometry. Differences in subcellular colocalization of VEGFR2 with endothelial, tumor and stem cell markers were determined by double-immunofluorescence imaging. Immunohistochemical results were correlated with clinicopathological data. HPV-infection induces significant downregulation of VEGFR2 in cancer cells compared to HPV-negative tumor cells (p = 0.012). However, with respect to blood vessel supply, the intensity of VEGFR2 staining differed only in HPV-positive OPSCC and was upregulated in the blood vessels of tumor-containing regions (p < 0.0001). These results may suggest different routes of VEGFR2 signaling depending on the HPV-status of the OPSCC. While in HPV-positive OPSCC, VEGFR2 might be associated with increased angiogenesis, in HPV-negative tumors, an autocrine loop might regulate tumor cell survival and invasion.

Molecular Dissection of Pro-Fibrotic IL11 Signaling in Cardiac and Pulmonary Fibroblasts

In fibroblasts, TGFβ1 stimulates IL11 upregulation that leads to an autocrine loop of IL11-dependent pro-fibrotic protein translation. The signaling pathways downstream of IL11, which acts via IL6ST, are contentious with both STAT3 and ERK implicated. Here we dissect IL11 signaling in fibroblasts and study IL11-dependent protein synthesis pathways in the context of approved anti-fibrotic drug mechanisms of action. We show that IL11-induced ERK activation drives fibrogenesis and while STAT3 phosphorylation (pSTAT3) is also seen, this appears unrelated to fibroblast activation. Ironically, recombinant human IL11, which has been used extensively in mouse experiments to infer STAT3 activity downstream of IL11, increases pSTAT3 in Il11ra1 null mouse fibroblasts. Unexpectedly, inhibition of STAT3 was found to induce severe proteotoxic ER stress, generalized fibroblast dysfunction and cell death. In contrast, inhibition of ERK prevented fibroblast activation in the absence of ER stress. IL11 stimulated an axis of ERK/mTOR/P70RSK protein translation and its selectivity for Collagen 1 synthesis was ascribed to an EPRS-regulated, ribosome stalling mechanism. Surprisingly, the anti-fibrotic drug nintedanib caused dose-dependent ER stress and lesser pSTAT3 expression. Pirfenidone had no effect on ER stress whereas anti-IL11 specifically inhibited the ERK/mTOR axis while reducing ER stress. These studies define the translation-specific signaling pathways downstream of IL11, intersect immune and metabolic signaling and reveal unappreciated effects of nintedanib.

LRP-1 Commands Two Parallel Signalling Mechanisms to Support Constitutive Tumour Cell Motility in the Absence of Blood Supply

Tumour cells often face the stress of ischemic (nutrient paucity and hypoxia) environment and must act self-sufficient to migrate toward the nearest blood supply or die. The mechanism that supports the constitutive motility of tumour cells under stress is poorly understood. We and others have previously shown that the low-density lipoprotein receptor-related protein 1 (LRP-1) plays a critical role in tumour cell migration and invasion in vitro and tumour formation in mice. Herein we show that depletion of LRP-1 completely abolishes the self-supported and serum-independent tumour cell motility. More intriguingly, we demonstrate that LRP-1 commands the full tumour cell motility by connecting with two independent cell surface signalling pathways. First, LRP-1 mediates secreted Hsp90α signalling via the Hsp90α > LRP-1 receptor autocrine loop for a half of tumour cell motility. Second, LRP1 stabilizes constitutively activated EGFR signalling that contributes the other half of tumour cell motility. Only combined inhibitions of the secreted Hsp90α autocrine and the EGFR signalling reproduces the effect of LRP1 down-regulation on constitutive tumour cell motility. This study reveals a novel mechanism of how tumour cells migrate in the absence of blood support.

HMGA1, Moonlighting Protein Function, and Cellular Real Estate: Location, Location, Location!

The gene encoding the High Mobility Group A1 (HMGA1) chromatin remodeling protein is upregulated in diverse cancers where high levels portend adverse clinical outcomes. Until recently, HMGA1 was assumed to be a nuclear protein exerting its role in cancer by transcriptionally modulating gene expression and downstream signaling pathways. However, the discovery of an extracellular HMGA1-RAGE autocrine loop in invasive triple-negative breast cancer (TNBC) cell lines implicates HMGA1 as a “moonlighting protein” with different functions depending upon cellular location. Here, we review the role of HMGA1, not only as a chromatin regulator in cancer and stem cells, but also as a potential secreted factor that drives tumor progression. Prior work found that HMGA1 is secreted from TNBC cell lines where it signals through the receptor for advanced glycation end products (RAGE) to foster phenotypes involved in tumor invasion and metastatic progression. Studies in primary TNBC tumors also suggest that HMGA1 secretion associates with distant metastasis in TNBC. Given the therapeutic potential to target extracellular proteins, further work to confirm this role in other contexts is warranted. Indeed, crosstalk between nuclear and secreted HMGA1 could change our understanding of tumor development and reveal novel therapeutic opportunities relevant to diverse human cancers overexpressing HMGA1.

The calcium-binding protein S100B reduces IL6 production in malignant melanoma via inhibition of RSK cellular signaling

S100B is frequently elevated in malignant melanoma. A regulatory mechanism was uncovered here in which elevated S100B lowers mRNA and secreted protein levels of interleukin-6 (IL6) and inhibits an autocrine loop whereby IL6 activates STAT3 signaling. Our results showed that S100B affects IL6 expression transcriptionally. S100B was shown to form a calcium-dependent protein complex with the p90 ribosomal S6 kinase (RSK), which in turn sequesters RSK into the cytoplasm. Consistently, S100B inhibition was found to restore phosphorylation of a nuclear located RSK substrate, CREB, which is a potent transcription factor for IL6 expression. Thus, elevated S100B reduces IL6-STAT3 signaling via RSK signaling pathway in malignant melanoma. Indeed, the elevated S100B levels in malignant melanoma cell lines correspond to low levels of IL6 and p-STAT3.

A Self-Limiting Circuit Regulates Mammary Cap Cell Plasticity Through TGF-beta Signaling

The organization and maintenance of complex tissues requires emergent properties driven by self-organizing and self-limiting cell-cell interactions. We examined these interactions in the murine mammary gland. Luminal and myoepithelial subpopulations of the postnatal mammary gland arise from unipotent progenitors, but the destiny of cap cells, which enclose terminal end buds (TEB) in pubertal mice, remains controversial. Using a transgenic strain (Tg11.5kb-GFP) that specifically marks cap cells, we found ~50% of these cells undergo divisions perpendicular to the TEB surface, suggesting they might contribute to the underlying luminal cell population. To address their stemness potential we developed a lineage tracing mouse driven from the s-SHIP (11.5 kb) promoter. Induction of tdTomato (tdTom) from this promoter in vivo demonstrated that all cap cell progeny are myoepithelial, with no conversion to luminal lineage. Organoid cultures also exhibited unipotency. However, isolated cap cells cultured as mammospheres generated mixed luminal/myoepithelial spheres. Moreover, ablation of luminal cells in vivo using diphtheria toxin triggered repopulation by progeny of tdTom+ cap cells. A signaling inhibitor screen identified the TGFb pathway as a potential regulator of multipotency. TGFbR inhibitors or gene deletion blocked conversion to the luminal lineage, consistent with an autocrine loop in which cap cells secrete TGFb to activate the receptor and promote luminal transdifferentiation. Ductal tree regeneration in vivo from isolated cap cells was much more efficient when they were pre-treated with inhibitor, consistent with more cells retaining cap cell potential prior to transplantation. Notably, in vitro transdifferentiation of cap cells was blocked by co-culture with luminal cells. Overall, these data reveal a self-limiting cell circuit through which mammary luminal cells suppress cap cell conversion to the luminal lineage.

High Mobility Group Box 1 (HMGB1) Induces Toll-Like Receptor 4-Mediated Production of the Immunosuppressive Protein Galectin-9 in Human Cancer Cells

High mobility group box 1 (HMGB1) is a non-histone protein which is predominantly localised in the cell nucleus. However, stressed, dying, injured or dead cells can release this protein into the extracellular matrix passively. In addition, HMGB1 release was observed in cancer and immune cells where this process can be triggered by various endogenous as well as exogenous stimuli. Importantly, released HMGB1 acts as a so-called “danger signal” and could impact on the ability of cancer cells to escape host immune surveillance. However, the molecular mechanisms underlying the functional role of HMGB1 in determining the capability of human cancer cells to evade immune attack remain unclear. Here we report that the involvement of HMGB1 in anti-cancer immune evasion is determined by Toll-like receptor (TLR) 4, which recognises HMGB1 as a ligand. We found that HGMB1 induces TLR4-mediated production of transforming growth factor beta type 1 (TGF-β), displaying autocrine/paracrine activities. TGF-β induces production of the immunosuppressive protein galectin-9 in cancer cells. In TLR4-positive cancer cells, HMGB1 triggers the formation of an autocrine loop which induces galectin-9 expression. In malignant cells lacking TLR4, the same effect could be triggered by HMGB1 indirectly through TLR4-expressing myeloid cells present in the tumour microenvironment (e. g. tumour-associated macrophages).

Rewiring of lactate-IL-1β auto-regulatory loop with Clock-Bmal1: A feed-forward circuit in glioma

De-synchronized circadian rhythm in tumors is coincident with aberrant inflammation and dysregulated metabolism. As their inter-relationship in cancer etiology is largely unknown, we investigated the link between the three in glioma. Tumor metabolite lactate- mediated increase in pro-inflammatory cytokine IL-1β was concomitant with elevated levels of core circadian regulators Clock and Bmal1. siRNA mediated knockdown of Bmal1 and Clock decreased (i) LDHA and IL-1β levels and (ii) release of lactate and pro-inflammatory cytokines. Lactate mediated deacetylation of Bmal1 and its interaction with Clock, regulate IL-1β levels and vice versa. Site-directed mutagenesis and luciferase reporter assay indicated the functionality of E-box sites on LDHA and IL-1β promoters. ChIP-re-ChIP revealed that lactate-IL-1β crosstalk positively affects co-recruitment of Clock-Bmal1 to these E-box sites. Clock-Bmal1 enrichment was accompanied by decreased H3K9me3, and increased H3K9ac and RNA pol II occupancy. Lactate-IL-1β-Clock (LIC) loop positively regulated expression of genes associated with cell cycle, DNA damage and cytoskeletal organization involved in glioma progression. TCGA data analysis suggested the presence of lactate- IL-1β-crosstalk in other cancers. The responsiveness of stomach and cervical cancer cells to lactate inhibition followed the same trend exhibited by glioma cells. In addition, components of LIC loop were found to be correlated with (i) patient survival, (ii) clinically actionable genes, and (iii) anti-cancer drug sensitivity. Our findings provide evidence for a potential cancer-specific axis wiring of IL-1β and LDHA through Clock -Bmal1, the outcome of which is to fuel an IL-1β-lactate autocrine loop that drives pro-inflammatory and oncogenic signals.

Molecular dissection of pro-fibrotic signaling identifies the mechanism underlying IL11-driven fibrosis gene translation, reveals non-specific effects of STAT3 and suggests a new mechanism of action for nintedanib

In fibroblasts, TGFβ1 stimulates IL11 upregulation that leads to an autocrine loop of IL11-dependent pro-fibrotic protein translation. The signalling pathways downstream of IL11 are contentious and both STAT3 and ERK have been implicated. Here we show that TGFβ1- or IL11- induced ERK activation is consistently associated with fibrogenesis whereas STAT3 phosphorylation (pSTAT3) is unrelated to fibroblast activation. Surprisingly, recombinant human IL11, which has been used extensively in mouse experiments to infer STAT3 activity downstream of IL11, non-specifically increases pSTAT3 in Il11ra1 null mouse fibroblasts. Pharmacologic inhibition of STAT3 prevents TGFβ1-induced fibrogenesis but this effect was found to reflect fibroblast dysfunction due to severe proteotoxic ER stress. In contrast, inhibition of MEK/ERK prevented fibrosis in the absence of ER stress. TGFβ1-stimulated ERK/mTOR/P70RSK-driven protein translation was IL11-dependent and selectivity for pro-fibrotic protein synthesis was ascribed to an EPRS-related mechanism. In TGFβ1-stimulated fibroblasts, the anti-fibrotic drug nintedanib caused dose-dependent ER stress, reduced pSTAT/pERK and inhibited pro-fibrotic protein translation, similarly to generic STAT3 inhibitors or ER stressors. Pirfenidone, while anti-fibrotic, had no effect on ER stress whereas anti-IL11 inhibited the ERK/mTOR axis while reducing ER stress. These studies discount a specific role for STAT3 in pro-fibrotic signaling, suggest a novel mechanism of action for nintedanib and prioritise further the IL11 pathway as a therapeutic target for fibrosis.