Increased Expression of Interleukin-1 Receptor Characterizes Anti-estrogen-Resistant ALDH+ Breast Cancer Stem Cells.

Estrogen-receptor-positive breast tumors are treated with anti-estrogen (AE) therapies but frequently develop resistance. Cancer stem cells (CSCs) with high aldehyde dehydrogenase activity (ALDH+ cells) are enriched following AE treatment. Here, we show that the interleukin-1ß (IL-1ß) signaling pathway is activated in ALDH+ cells, and data from single cells reveals that AE treatment selects for IL-1 receptor (IL1R1)-expressing ALDH+ cells. Importantly, CSC activity is reduced by an IL1R1 inhibitor in AE-resistant models. Moreover, IL1R1 expression is increased in the tumors of patients treated with AE therapy and predicts treatment failure. Single-cell gene expression analysis revealed that at least two subpopulations exist within the ALDH+ population, one proliferative and one quiescent. Following AE therapy the quiescent population is expanded, which suggests CSC dormancy as an adaptive strategy that facilitates treatment resistance. Targeting of ALDH+IL1R1+ cells merits testing as a strategy to combat AE resistance in patients with residual disease.

Targeting STAT3 signaling using stabilised sulforaphane (SFX-01) inhibits endocrine resistant stem-like cells in ER-positive breast cancer.

Estrogen receptor (ER) positive breast cancer is frequently sensitive to endocrine therapy. Multiple mechanisms of endocrine therapy resistance have been identified, including cancer stem-like cell (CSC) activity. Here we investigate SFX-01, a stabilised formulation of sulforaphane (SFN), for its effects on breast CSC activity in ER+ preclinical models. SFX-01 reduced mammosphere formation efficiency (MFE) of ER+ primary and metastatic patient samples. Both tamoxifen and fulvestrant increased MFE and aldehyde dehydrogenase (ALDH) activity of patient-derived xenograft (PDX) tumors, which was reversed by combination with SFX-01. SFX-01 significantly reduced tumor-initiating cell frequency in secondary transplants and reduced the formation of spontaneous lung micrometastases by PDX tumors in mice. Mechanistically, we establish that both tamoxifen and fulvestrant induce STAT3 phosphorylation. SFX-01 suppressed phospho-STAT3 and SFN directly bound STAT3 in patient and PDX samples. Analysis of ALDH+ cells from endocrine-resistant patient samples revealed activation of STAT3 target genes MUC1 and OSMR, which were inhibited by SFX-01 in patient samples. Increased expression of these genes after 3 months' endocrine treatment of ER+ patients (n = 68) predicted poor prognosis. Our data establish the importance of STAT3 signaling in CSC-mediated resistance to endocrine therapy and the potential of SFX-01 for improving clinical outcomes in ER+ breast cancer.

Microenvironmental IL1ß promotes breast cancer metastatic colonisation in the bone via activation of Wnt signalling.

Dissemination of tumour cells to the bone marrow is an early event in breast cancer, however cells may lie dormant for many years before bone metastases develop. Treatment for bone metastases is not curative, therefore new adjuvant therapies which prevent the colonisation of disseminated cells into metastatic lesions are required. There is evidence that cancer stem cells (CSCs) within breast tumours are capable of metastasis, but the mechanism by which these colonise bone is unknown. Here, we establish that bone marrow-derived IL1ß stimulates breast cancer cell colonisation in the bone by inducing intracellular NFkB and CREB signalling in breast cancer cells, leading to autocrine Wnt signalling and CSC colony formation. Importantly, we show that inhibition of this pathway prevents both CSC colony formation in the bone environment, and bone metastasis. These findings establish that targeting IL1ß-NFKB/CREB-Wnt signalling should be considered for adjuvant therapy to prevent breast cancer bone metastasis.

PAK4 regulates stemness and progression in endocrine resistant ER-positive metastatic breast cancer.

Despite the effectiveness of endocrine therapies to treat estrogen receptor-positive (ER+) breast tumours, two thirds of patients will eventually relapse due to de novo or acquired resistance to these agents. Cancer Stem-like Cells (CSCs), a rare cell population within the tumour, accumulate after anti-estrogen treatments and are likely to contribute to their failure. Here we studied the role of p21-activated kinase 4 (PAK4) as a promising target to overcome endocrine resistance and disease progression in ER + breast cancers. PAK4 predicts for resistance to tamoxifen and poor prognosis in 2 independent cohorts of ER + tumours. We observed that PAK4 strongly correlates with CSC activity in metastatic patient-derived samples irrespective of breast cancer subtype. However, PAK4-driven mammosphere-forming CSC activity increases alongside progression only in ER + metastatic samples. PAK4 activity increases in ER + models of acquired resistance to endocrine therapies. Targeting PAK4 with either CRT PAKi, a small molecule inhibitor of PAK4, or with specific siRNAs abrogates CSC activity/self-renewal in clinical samples and endocrine-resistant cells. Together, our findings establish that PAK4 regulates stemness during disease progression and that its inhibition reverses endocrine resistance in ER + breast cancers.

Acquired Resistance of ER-Positive Breast Cancer to Endocrine Treatment Confers an Adaptive Sensitivity to TRAIL through Posttranslational Downregulation of c-FLIP.

Purpose: One third of ER-positive breast cancer patients who initially respond to endocrine therapy become resistant to treatment. Such treatment failure is associated with poor prognosis and remains an area of unmet clinical need. Here, we identify a specific posttranslational modification that occurs during endocrine resistance and which results in tumor susceptibility to the apoptosis-inducer TRAIL. This potentially offers a novel stratified approach to targeting endocrine-resistant breast cancer.Experimental Design: Cell line and primary-derived xenograft models of endocrine resistance were investigated for susceptibility to TRAIL. Tumor viability, cancer stem cell (CSC) viability (tumorspheres), tumor growth kinetics, and metastatic burden were assessed. Western blots for the TRAIL-pathway inhibitor, c-FLIP, and upstream regulators were performed. Results were confirmed in primary culture of 26 endocrine-resistant and endocrine-naïve breast tumors.Results: Breast cancer cell lines with acquired resistance to tamoxifen (TAMR) or faslodex were more sensitive to TRAIL than their endocrine-sensitive controls. Moreover, TRAIL eliminated CSC-like activity in TAMR cells, resulting in prolonged remission of xenografts in vivo In primary culture, TRAIL significantly depleted CSCs in 85% endocrine-resistant, compared with 8% endocrine-naïve, tumors, whereas systemic administration of TRAIL in endocrine-resistant patient-derived xenografts reduced tumor growth, CSC-like activity, and metastases. Acquired TRAIL sensitivity correlated with a reduction in intracellular levels of c-FLIP, and an increase in Jnk-mediated phosphorylation of E3-ligase, ITCH, which degrades c-FLIP.Conclusions: These results identify a novel mechanism of acquired vulnerability to an extrinsic cell death stimulus, in endocrine-resistant breast cancers, which has both therapeutic and prognostic potential. Clin Cancer Res; 24(10); 2452-63. ©2018 AACR.

Structural and lipid-binding characterization of human annexin A13a reveals strong differences with its long A13b isoform.

Annexin A13 is the founder member of the vertebrate family of annexins, which are comprised of a tetrad of unique conserved domains responsible for calcium-dependent binding to membranes. Its expression is restricted to epithelial intestinal and kidney cells. Alternative splicing in the N-terminal region generates two isoforms, A13a and A13b, differing in a deletion of 41 residues in the former. We have confirmed the expression of both isoforms in human colon adenocarcinoma cells at the mRNA and protein levels. We have cloned, expressed, and purified human annexin A13a for the first time to analyze its structural characteristics. Its secondary structure and thermal stability differs greatly from the A13b isoform. The only tryptophan residue (Trp186) is buried in the protein core in the absence of calcium but is exposed to the solvent after calcium binding even though circular dichroism spectra are quite similar. Non-myristoylated annexin A13a binds in a calcium-dependent manner to acidic phospholipids but not to neutral or raft-like liposomes. Calcium requirements for binding to phosphatidylserine are around 6-fold lower than those required by the A13b isoform. This fact could account for the different subcellular localization of both annexins as binding to basolateral membranes seems to be calcium-dependent and myristoylation-independent.

Patient-derived Mammosphere and Xenograft Tumour Initiation Correlates with Progression to Metastasis.

Breast cancer specific mortality results from tumour cell dissemination and metastatic colonisation. Identification of the cells and processes responsible for metastasis will enable better prevention and control of metastatic disease, thus reducing relapse and mortality. To better understand these processes, we prospectively collected 307 patient-derived breast cancer samples (n = 195 early breast cancers (EBC) and n = 112 metastatic samples (MBC)). We assessed colony-forming activity in vitro by growing isolated cells in both primary (formation) and secondary (self-renewal) mammosphere culture, and tumour initiating activity in vivo through subcutaneous transplantation of fragments or cells into mice. Metastatic samples formed primary mammosphere colonies significantly more frequently than early breast cancers and had significantly higher primary mammosphere colony formation efficiency (0.9 % vs. 0.6 %; p < 0.0001). Tumour initiation in vivo was significantly higher in metastatic than early breast cancer samples (63 % vs. 38 %, p = 0.04). Of 144 breast cancer samples implanted in vivo, we established 20 stable patient-derived xenograft (PDX) models at passage 2 or greater. Lung metastases were detected in mice from 14 PDX models. Mammosphere colony formation in vitro significantly correlated with the ability of a tumour to metastasise to the lungs in vivo (p = 0.05), but not with subcutaneous tumour initiation. In summary, the breast cancer stem cell activities of colony formation and tumour initiation are increased in metastatic compared to early samples, and predict metastasis in vivo. These results suggest that breast stem cell activity will predict for poor outcome tumours, and therapy targeting this activity will improve outcomes for patients with metastatic disease.

Anti-estrogen Resistance in Human Breast Tumors Is Driven by JAG1-NOTCH4-Dependent Cancer Stem Cell Activity.

Breast cancers (BCs) typically express estrogen receptors (ERs) but frequently exhibit de novo or acquired resistance to hormonal therapies. Here, we show that short-term treatment with the anti-estrogens tamoxifen or fulvestrant decrease cell proliferation but increase BC stem cell (BCSC) activity through JAG1-NOTCH4 receptor activation both in patient-derived samples and xenograft (PDX) tumors. In support of this mechanism, we demonstrate that high ALDH1 predicts resistance in women treated with tamoxifen and that a NOTCH4/HES/HEY gene signature predicts for a poor response/prognosis in 2 ER+ patient cohorts. Targeting of NOTCH4 reverses the increase in Notch and BCSC activity induced by anti-estrogens. Importantly, in PDX tumors with acquired tamoxifen resistance, NOTCH4 inhibition reduced BCSC activity. Thus, we establish that BCSC and NOTCH4 activities predict both de novo and acquired tamoxifen resistance and that combining endocrine therapy with targeting JAG1-NOTCH4 overcomes resistance in human breast cancers.

P-cadherin signals through the laminin receptor α6ß4 integrin to induce stem cell and invasive properties in basal-like breast cancer cells.

P-cadherin is a classical cell-cell adhesion molecule that, in contrast to E-cadherin, has a positive role in breast cancer progression, being considered a poor prognostic factor in this disease. In previous reports, we have shown that this protein induces cancer stem cell and invasive properties to basal-like breast cancer cells. Here, we clarify the downstream signaling pathways that are triggered by P-cadherin to mediate these effects. We demonstrated that P-cadherin inhibition led to a significant decreased adhesion of cancer cells to the basement membrane substrate laminin, as well as to a major reduction in the expression of the laminin receptor α6ß4 integrin. Remarkably, the expression of this heterodimer was required for the invasive capacity and increased mammosphere forming efficiency induced by P-cadherin expression. Moreover, we showed that P-cadherin transcriptionally up-regulates the α6 integrin subunit expression and directly interacts with the ß4 integrin subunit. We still showed that P-cadherin downstream signaling, in response to laminin, involves the activation of focal adhesion (FAK), Src and AKT kinases. The association between the expression of P-cadherin, α6ß4 heterodimer and the active FAK and Src phosphorylated forms was validated in vivo. Our data establish that there is a crosstalk between P-cadherin and the laminin receptor α6ß4 integrin signaling pathway, which link has never been previously described. The activation of this heterodimer explains the stem cell and invasive properties induced by P-cadherin to breast cancer cells, pointing to a new molecular mechanism that may be targeted to counteract the effects induced by this adhesion molecule.

4F2hc-silencing impairs tumorigenicity of HeLa cells via modulation of galectin-3 and ß-catenin signaling, and MMP-2 expression.

4F2hc is a type-II glycoprotein whose covalent-bound association with one of several described light chains yields a heterodimer mainly involved in large neutral amino acid transport. Likewise, it is well known that the heavy chain interacts with ß-integrins mediating integrin-dependent events such as survival, proliferation, migration and even transformation. 4F2hc is a ubiquitous protein whose overexpression has been related to tumor development and progression. Stable silencing of 4F2hc in HeLa cells using an artificial miRNA impairs in vivo tumorigenicity and leads to an ineffective proliferation response to mitogens. 4F2hc colocalizes with ß1-integrins and CD147, but this interaction does not occur in lipid rafts in HeLa cells. Moreover, silenced cells present defects in integrin- (FAK, Akt and ERK1/2) and hypoxia-dependent signaling, and reduced expression/activity of MMP-2. These alterations seem to be dependent on the inappropriate formation of CD147/4F2hc/ß1-integrin heterocomplexes on the cell surface, arising when CD147 cannot interact with 4F2hc. Although extracellular galectin-3 accumulates due to the decrease in MMP-2 activity, galectin-3 signaling events are blocked due to an impaired interaction with 4F2hc, inducing an increased degradation of ß-catenin. Furthermore, cell motility is compromised after protein silencing, suggesting that 4F2hc is related to tumor invasion by facilitating cell motility. Therefore, here we propose a molecular mechanism by which 4F2hc participates in tumor progression, favoring first steps of epithelial-mesenchymal transition by inhibition of ß-catenin proteasomal degradation through Akt/GSK-3ß signaling and enabling cell motility.

Resistance to butyrate impairs bile acid-induced apoptosis in human colon adenocarcinoma cells via up-regulation of Bcl-2 and inactivation of Bax.

A critical risk factor in colorectal carcinogenesis and tumor therapy is the resistance to the apoptotic effects of different compounds from the intestinal lumen, among them butyrate (main regulator of colonic epithelium homeostasis). Insensitivity to butyrate-induced apoptosis yields resistance to other agents, as bile acids or chemotherapy drugs, allowing the selective growth of malignant cell subpopulations. Here we analyze bile acid-induced apoptosis in a butyrate-resistant human colon adenocarcinoma cell line (BCS-TC2.BR2) to determine the mechanisms that underlay the resistance to these agents in comparison with their parental butyrate-sensitive BCS-TC2 cells. This study demonstrates that DCA and CDCA still induce apoptosis in butyrate-resistant cells through increased ROS production by activation of membrane-associated enzymes and subsequent triggering of the intrinsic mitochondrial apoptotic pathway. Although this mechanism is similar to that described in butyrate-sensitive cells, cell viability is significantly higher in resistant cells. Moreover, butyrate-resistant cells show higher Bcl-2 levels that confer resistance to bile acid-induced apoptosis sequestering Bax and avoiding Bax-dependent pore formation in the mitochondria. We have confirmed that this resistance is reverted using the Bcl-2 inhibitor ABT-263, thus demonstrating that the lower sensitivity of butyrate-resistant cells to the apoptotic effects of bile acids is mainly due to increased Bcl-2 levels.

Histone deacetylase inhibitors upregulate MMP11 gene expression through Sp1/Smad complexes in human colon adenocarcinoma cells.

MMP-11 (stromelysin-3) is a matrix metalloproteinase associated with tumor progression and poor prognosis. Its expression was initially described exclusively in stromal cells surrounding tumors, but more recently it has also been detected in macrophages and hepatocarcinoma cells. Here we show MMP-11 expression in human epithelial colon adenocarcinoma cell lines (Caco-2, HT-29 and BCS-TC2). Treatment of BCS-TC2 cells with butyrate and trichostatin A (TSA) (histone deacetylase inhibitors) increases MMP11 promoter activity and protein expression. Using electrophoretic mobility shift assay (EMSA) and supershift assays, we demonstrate for the first time that Sp1 is able to bind to the GC-boxes within the MMP11 proximal promoter region; this binding has been confirmed by chromatin immunoprecipitation. Sp1 is involved in MMP11 basal expression and it is essential for the upregulation of transcription by histone deacetylase inhibitors as deduced from mutant constructs lacking the Sp1 sites and by inhibition of its binding to the promoter with mithramycin. This regulation requires the formation of Sp1/Smad2 heterocomplexes, which is stimulated by an increase in the acetylation status of Smad after butyrate or TSA treatments. We have also found that ERK1/2-mitogen-activated protein kinase (MAPK), but not p38-MAPK or JNK, is involved in the upregulation of MMP11 by HDAC inhibitors.

Deoxycholic and chenodeoxycholic bile acids induce apoptosis via oxidative stress in human colon adenocarcinoma cells.

The continuous exposure of the colonic epithelium to high concentrations of bile acids may exert cytotoxic effects and has been related to pathogenesis of colon cancer. A better knowledge of the mechanisms by which bile acids induce toxicity is still required and may be useful for the development of new therapeutic strategies. We have studied the effect of deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) treatments in BCS-TC2 human colon adenocarcinoma cells. Both bile acids promote cell death, being this effect higher for CDCA. Apoptosis is detected after 30 min-2 h of treatment, as observed by cell detachment, loss of membrane asymmetry, internucleosomal DNA degradation, appearance of mitochondrial transition permeability (MPT), and caspase and Bax activation. At longer treatment times, apoptosis is followed in vitro by secondary necrosis due to impaired mitochondrial activity and ATP depletion. Bile acid-induced apoptosis is a result of oxidative stress with increased ROS generation mainly by activation of plasma membrane enzymes, such as NAD(P)H oxidases and, to a lower extent, PLA2. These effects lead to a loss of mitochondrial potential and release of pro-apoptotic factors to the cytosol, which is confirmed by activation of caspase-9 and -3, but not caspase-8. This initial apoptotic steps promote cleavage of Bcl-2, allowing Bax activation and formation of additional pores in the mitochondrial membrane that amplify the apoptotic signal.

Structural characterization and unfolding mechanism of human 4F2hc ectodomain.

4F2hc (CD98hc) is a multifunctional type II membrane glycoprotein involved in several functions as amino acid transport, cell fusion, ß1-integrin-signaling and transformation. 4F2hc ectodomain has been crystallized and its three-dimensional structure determined. We have carried out a spectroscopical/structural characterization of the recombinant ectodomain in order to obtain information on its dynamic structure in solution and on its ability to form homodimers by itself in the absence of the transmembrane helix and of the potential interactions with the plasma membrane. Analytical ultracentrifugation and crosslinking experiments showed that the ectodomain is monomeric in solution. The secondary structure determined by far-UV circular dichroism (CD) spectroscopy (around 30% α-helix and 20% ß-sheets, 12% antiparallel and 8% parallel) reveals a compact and thermally stable structure with a high melting temperature (57-59°C). Tryptophan residues are mainly buried and immobilized in the hydrophobic core of the protein as suggested by near-UV CD spectrum, the position of the Trp maximum fluorescence emission (323nm) and from the acrylamide quenching constant (2.6M(-1)). Urea unfolding equilibrium has been studied by far-UV CD and fluorescence spectroscopy to gain information on the folding/unfolding process of the ectodomain. The analyses suggest the existence of two intermediate states as reported for other TIM barrel-containing proteins rather than an independent unfolding of each domain [A, (ßα)(8) barrel; C, antiparallel ß(8) sandwich]. Folding seems to be directed by the initial formation of hydrophobic clusters within the first strands of the ß-barrel of domain A followed by additional hydrophobic interactions in domain C.

Kinetic analysis of butyrate transport in human colon adenocarcinoma cells reveals two different carrier-mediated mechanisms.

Butyrate has antitumorigenic effects on colon cancer cells, inhibits cell growth and promotes differentiation and apoptosis. These effects depend on its intracellular concentration, which is regulated by its transport. We have analysed butyrate uptake kinetics in human colon adenocarcinoma cells sensitive to the apoptotic effects of butyrate (BCS-TC2, Caco-2 and HT-29), in butyrate-resistant cells (BCS-TC2.BR2) and in normal colonic cells (FHC). The properties of transport were analysed with structural analogues, specific inhibitors and different bicarbonate and sodium concentrations. Two carrier-mediated mechanisms were detected: a low-affinity/high-capacity (K(m)=109+/-16 mM in BCS-TC2 cells) anion exchanger and a high-affinity/low-capacity (K(m)=17.9+/-4.0 microM in BCS-TC2 cells) proton-monocarboxylate co-transporter that was energy-dependent and activated via PKCdelta (protein kinase Cdelta). All adenocarcinoma cells analysed express MCT (monocarboxylate transporter) 1, MCT4, ancillary protein CD147 and AE2 (anion exchanger 2). Silencing experiments show that MCT1, whose expression increases with butyrate treatment in butyrate-sensitive cells, plays a key role in high-affinity transport. Low-affinity uptake was mediated by a butyrate/bicarbonate antiporter along with a possible contribution of AE2 and MCT4. Butyrate treatment increased uptake in a time- and dose-dependent manner in butyrate-sensitive but not in butyrate-resistant cells. The two butyrate-uptake activities in human colon adenocarcinoma cells enable butyrate transport at different physiological conditions to maintain cell functionality. The high-affinity/low-capacity transport functions under low butyrate concentrations and may be relevant for the survival of carcinoma cells in tumour regions with low glucose and butyrate availability as well as for the normal physiology of colonocytes.