The NFIB-ERO1A axis promotes breast cancer metastatic colonization of disseminated tumour cells.

Metastasis is the main cause of deaths related to solid cancers. Active transcriptional programmes are known to regulate the metastatic cascade but the molecular determinants of metastatic colonization remain elusive. Using an inducible piggyBac (PB) transposon mutagenesis screen, we have shown that overexpression of the transcription factor nuclear factor IB (NFIB) alone is sufficient to enhance primary mammary tumour growth and lung metastatic colonization. Mechanistically and functionally, NFIB directly increases expression of the oxidoreductase ERO1A, which enhances HIF1α-VEGFA-mediated angiogenesis and colonization, the last and fatal step of the metastatic cascade. NFIB is thus clinically relevant: it is preferentially expressed in the poor-prognostic group of basal-like breast cancers, and high expression of the NFIB/ERO1A/VEGFA pathway correlates with reduced breast cancer patient survival.

Serpin E2 promotes breast cancer metastasis by remodeling the tumor matrix and polarizing tumor associated macrophages.

The extracellular serine protease inhibitor serpinE2 is overexpressed in breast cancer and has been shown to foster metastatic spread. Here, we investigated the hypothesis that serpinE2 creates tumor-promoting conditions in the tumor microenvironment (TME) by affecting extracellular matrix remodeling. Using two different breast cancer models, we show that blocking serpinE2, either by knock-down (KD) in tumor cells or in response to a serpinE2 binding antibody, decreases metastatic dissemination from primary tumors to the lungs. We demonstrate that in response to serpinE2 KD or antibody treatment there are dramatic changes in the TME. Multiphoton intravital imaging revealed deposition of a dense extracellular collagen I matrix encapsulating serpinE2 KD or antibody-treated tumors. This is accompanied by a reduction in the population of tumor-promoting macrophages, as well as a decrease in chemokine ligand 2, which is known to affect macrophage abundance and polarization. In addition, TIMP-1 secretion is increased, which may directly inhibit matrix metalloproteases critical for collagen degradation in the tumor. In summary, our findings suggest that serpinE2 is required in the extracellular milieu of tumors where it acts in multiple ways to regulate tumor matrix deposition, thereby controlling tumor cell dissemination.

Interleukin-33 Expression Indicates a Favorable Prognosis in Malignant Salivary Gland Tumors.

The cytokine interleukin-33 (IL-33) is abundantly expressed in epithelial barrier tissues such as salivary glands. Here, we characterized nuclear IL-33 protein expression by immunohistochemistry in benign and malignant salivary gland tumors and associated it with disease outcome. Most benign salivary gland tumors expressed IL-33, and all Warthin's tumors showed strong and consistent IL-33 expression in the basally oriented cells of their bilayered epithelium. In the malignant group of neoplasms, nuclear IL-33 expression was limited to specific tumor entities-for example, to epithelial-myopepithelial carcinomas (n = 9/11), acinic cell carcinomas (n = 13/27), and oncocytic carcinomas (n = 2/2). IL-33 expression in the combined group of malignant salivary gland neoplasms was significantly associated with favorable histological parameters, lack of metastasis, and longer overall survival, compared with IL-33-negative tumors. We conclude that IL-33 expression is a novel prognostic marker for malignant salivary gland tumors with potential use in clinical diagnostics.

Cessation of CCL2 inhibition accelerates breast cancer metastasis by promoting angiogenesis.

Secretion of C-C chemokine ligand 2 (CCL2) by mammary tumours recruits CCR2-expressing inflammatory monocytes to primary tumours and metastatic sites, and CCL2 neutralization in mice inhibits metastasis by retaining monocytes in the bone marrow. Here we report a paradoxical effect of CCL2 in four syngeneic mouse models of metastatic breast cancer. Surprisingly, interruption of CCL2 inhibition leads to an overshoot of metastases and accelerates death. This is the result of monocyte release from the bone marrow and enhancement of cancer cell mobilization from the primary tumour, as well as blood vessel formation and increased proliferation of metastatic cells in the lungs in an interleukin (IL)-6- and vascular endothelial growth factor (VEGF)-A-dependent manner. Notably, inhibition of CCL2 and IL-6 markedly reduced metastases and increased survival of the animals. CCL2 has been implicated in various neoplasias and adopted as a therapeutic target. However, our results call for caution when considering anti-CCL2 agents as monotherapy in metastatic disease and highlight the tumour microenvironment as a critical determinant of successful anti-metastatic therapy.

If you don't look, you won't see: intravital multiphoton imaging of primary and metastatic breast cancer.

A fundamental hallmark of cancer is progression to metastasis and the growth of breast cancer metastases in lung, bone, liver and/or brain causes fatal complications. Unfortunately, the cellular and biochemical mechanisms of the metastatic process remain ill-defined. Recent application of intravital multiphoton microscopy (MP-IVM) to image fluorescently labeled cells in mouse models of cancer has allowed dynamic observation of this multi-step process at the cellular and subcellular levels. In this article, we discuss the use of MP-IVM in studies of breast cancer metastasis, as well as surgical techniques for exposing tumors prior to imaging. We also describe a versatile multiphoton microscope for imaging tumor-stroma interactions.

Xenografts of highly resistant leukemia recapitulate the clonal composition of the leukemogenic compartment.

Clonal evolution of the leukemogenic compartment may contribute to alter the therapeutic response in acute lymphoblastic leukemia (ALL). Using xenotransplantation of primary leukemia cells, we evaluated the phenotypic and genetic composition of de novo resistant very high risk precursor B-cell ALL, a subgroup defined by the persistence of minimal residual disease despite intensive chemotherapy. Analysis of copy number alterations (CNAs) showed that the xenografted leukemia, even when reconstituted from 100 cells, remained highly related to the diagnostic sample, with minor changes in CNAs, mostly deletions, emerging in most cases in the first passage into mice. At the single-cell level, the pattern of monoallelic and biallelic deletions of the CDKN2A locus revealed distinct leukemia subpopulations, which were reproducibly tracked in xenografts. In most very high risk ALL cases, the predominant diagnostic clones were reconstituted in xenografts, as shown by multiplex polymerase chain reaction analysis of immunoglobulin and T-cell receptor loci. In other cases, the pattern in CNAs and immunoglobulin and T-cell receptor rearrangement was less concordant in xenografts, suggesting the outgrowth of subclones. These results unequivocally demonstrate the existence of clonally closely related but distinct subsets of leukemia initiating cells in ALL, which has important implications for drug development and preclinical disease modeling.

Induction of autophagy-dependent necroptosis is required for childhood acute lymphoblastic leukemia cells to overcome glucocorticoid resistance.

In vivo resistance to first-line chemotherapy, including to glucocorticoids, is a strong predictor of poor outcome in children with acute lymphoblastic leukemia (ALL). Modulation of cell death regulators represents an attractive strategy for subverting such drug resistance. Here we report complete resensitization of multidrug-resistant childhood ALL cells to glucocorticoids and other cytotoxic agents with subcytotoxic concentrations of obatoclax, a putative antagonist of BCL-2 family members. The reversal of glucocorticoid resistance occurred through rapid activation of autophagy-dependent necroptosis, which bypassed the block in mitochondrial apoptosis. This effect was associated with dissociation of the autophagy inducer beclin-1 from the antiapoptotic BCL-2 family member myeloid cell leukemia sequence 1 (MCL-1) and with a marked decrease in mammalian target of rapamycin (mTOR) activity. Consistent with a protective role for mTOR in glucocorticoid resistance in childhood ALL, combination of rapamycin with the glucocorticoid dexamethasone triggered autophagy-dependent cell death, with characteristic features of necroptosis. Execution of cell death, but not induction of autophagy, was strictly dependent on expression of receptor-interacting protein (RIP-1) kinase and cylindromatosis (turban tumor syndrome) (CYLD), two key regulators of necroptosis. Accordingly, both inhibition of RIP-1 and interference with CYLD restored glucocorticoid resistance completely. Together with evidence for a chemosensitizing activity of obatoclax in vivo, our data provide a compelling rationale for clinical translation of this pharmacological approach into treatments for patients with refractory ALL.

Cell cycle alteration and decreased cell proliferation in the hippocampal dentate gyrus and in the neocortical germinal matrix of fetuses with Down syndrome and in Ts65Dn mice.

Down syndrome (DS), the leading genetic cause of mental retardation, is characterized by reduced number of cortical neurons and brain size. The occurrence of these defects starting from early life stages points at altered developmental neurogenesis as their major determinant. The goal of our study was to obtain comparative evidence for impaired neurogenesis in the hippocampal dentate gyrus (DG) of DS fetuses and Ts65Dn mice, an animal model for DS. Cell proliferation in human fetuses was evaluated with Ki-67 (a marker of cells in S + G(2) + M phases of cell cycle) and cyclin A (a marker of cells in S phase) immunohistochemistry. We found that in the DG of DS fetuses the number of proliferating cells was notably reduced when compared with controls. A similar reduction was observed in the germinal matrix of the lateral ventricle. In both structures, DS fetuses showed a reduced ratio between cyclin A- and Ki-67-positive cells when compared with controls, indicating that they had a reduced number of cycling cells in S phase. In the DG of P2 Ts65Dn mice cell proliferation, assessed 2 h after an injection of bromodeoxyuridine (BrdU), was notably reduced, similarly to DS fetuses. After 28 days, Ts65Dn mice had still less BrdU-positive cells than controls. Phenotypic analysis of the surviving cells showed that Ts65Dn mice had a percent number of cells with astrocytic phenotype larger than controls. Using phospho-histone H3 immunohistochemistry we found that both DS fetuses and P2 Ts65Dn mice had a higher number of proliferating cells in G(2) and a smaller number of cells in M phase of cell cycle. Results provide novel evidence for proliferation impairment in the hippocampal DG of the DS fetal brain, comparable to that of the P2 mouse model, and suggest that cell cycle alterations may be critical determinants of the reduced proliferation potency.

Low-dose arsenic trioxide sensitizes glucocorticoid-resistant acute lymphoblastic leukemia cells to dexamethasone via an Akt-dependent pathway.

Incorporation of apoptosis-inducing agents into current therapeutic regimens is an attractive strategy to improve treatment for drug-resistant leukemia. We tested the potential of arsenic trioxide (ATO) to restore the response to dexamethasone in glucocorticoid (GC)-resistant acute lymphoblastic leukemia (ALL). Low-dose ATO markedly increased in vitro GC sensitivity of ALL cells from T-cell and precursor B-cell ALL patients with poor in vivo response to prednisone. In GC-resistant cell lines, this effect was mediated, at least in part, by inhibition of Akt and affecting downstream Akt targets such as Bad, a proapoptotic Bcl-2 family member, and the X-linked inhibitor of apoptosis protein (XIAP). Combination of ATO and dexamethasone resulted in increased Bad and rapid down-regulation of XIAP, while levels of the antiapoptotic regulator Mcl-1 remained unchanged. Expression of dominant-active Akt, reduction of Bad expression by RNA interference, or overexpression of XIAP abrogated the sensitizing effect of ATO. The inhibitory effect of XIAP overexpression was reduced when the Akt phosphorylation site was mutated (XIAP-S87A). These data suggest that the combination of ATO and glucocorticoids could be advantageous in GC-resistant ALL and reveal additional targets for the evaluation of new antileukemic agents.