Three-dimensional growth sensitizes breast cancer cells to treatment with ferroptosis-promoting drugs.

Drugs causing ferroptosis, iron-mediated cell death, represent promising tools for cancer treatment. While exploring the effect of these drugs on breast cancer (BC), we found that a ferroptosis-inducing drug erastin dramatically inhibits tumorigenicity of human BC cells in mice but when used at a concentration known to effectively kill other cell types only modestly reduces such growth in 2D monolayer culture. BCs grow in vivo as 3D masses, and we found that ferroptosis inducers erastin and sulfasalazine inhibit growth of multiple human BC cell lines in 3D culture significantly stronger than in 2D culture. To understand the mechanism of this differential effect, we found that ferroptosis inducers upregulate mRNAs encoding multiple direct and indirect autophagy stimulators, such as ATG16L2, ATG9A, ATG4D, GABARAP, SQSTM/p62, SEC23A and BAX, in tumor cells growing in 2D but not in 3D culture. Furthermore, these drugs promoted autophagy of tumor cells growing in a 2D but not in a 3D manner. We observed that pharmacological inhibition of autophagy-stimulating protein kinase ULK1 or RNA interference-mediated knockdown of autophagy mediator ATG12 significantly sensitized tumor cells to erastin treatment in 2D culture. We also found that ferroptosis-promoting treatments upregulate heme oxygenase-1 (HO-1) in BC cells. HO-1 increases cellular free iron pool and can potentially promote ferroptosis. Indeed, we observed that HO-1 knockdown by RNA interference reversed the effect of ferroptosis inducers on BC cell 3D growth. Hence, the effect of these drugs on such growth is mediated by HO-1. In summary, autophagy triggered by ferroptosis-promoting drugs reduces their ability to kill BC growing in a 2D manner. This protection mechanism is inhibited in BC cells growing as a 3D mass, and ferroptosis-promoting drugs kill such cells more effectively. Moreover, this death is mediated by HO-1. Thus, ferroptosis induction represents a promising strategy for blocking 3D BC growth.

ErbB2/Her2-dependent downregulation of a cell death-promoting protein BLNK in breast cancer cells is required for 3D breast tumor growth.

A significant proportion of breast cancers are driven by ErbB2/Her2 oncoprotein that they overexpress. These malignancies are typically treated with various ErbB2-targeted drugs, but many such cancers develop resistance to these agents and become incurable. Conceivably, treatment of ErbB2-positive cancers could be facilitated by use of agents blocking oncogenic signaling mechanisms downstream of ErbB2. However, current understanding of these mechanisms is limited. The ability of solid tumor cells to resist anoikis, cell death triggered by cell detachment from the extracellular matrix (ECM), is thought to be critical for 3D tumor growth. In an effort to understand the mechanisms of ErbB2-driven breast cancer cell anoikis resistance we found that detachment of non-malignant breast epithelial cells from the ECM upregulates a cell death-promoting tumor suppressor adapter protein BLNK and that ErbB2 blocks this upregulation by reducing tumor cell levels of transcription factor IRF6. We further observed that trastuzumab, a therapeutic anti-ErbB2 antibody, upregulates BLNK in human trastuzumab-sensitive but not trastuzumab-resistant ErbB2-positive breast cancer cells. Moreover, we established that BLNK promotes anoikis by activating p38 MAP kinase and that ErbB2-dependent BLNK downregulation blocks breast cancer cell anoikis. In search for pharmacological approaches allowing to upregulate BLNK in tumor cells we found that clinically approved proteasome inhibitor bortezomib upregulates IRF6 and BLNK in human breast cancer cells and inhibits their 3D growth in a BLNK-dependent manner. In addition, we found that BLNK upregulation in human ErbB2-positive breast cancer cells blocks their ability to form tumors in mice. Furthermore, we used publicly available data on mRNA levels in multiple breast cancers to demonstrate that increased BLNK mRNA levels correlate with increased relapse-free survival in a cohort of approximately 400 patients with ErbB2-positive breast cancer. In summary, we discovered a novel mechanism of ErbB2-driven 3D breast tumor growth mediated by ErbB2-dependent BLNK downregulation.

Killer Cell Immunoglobulin-Like Receptor Haplotype B Modulates Susceptibility to EBV-Associated Classic Hodgkin Lymphoma.

Tumor cells of classic Hodgkin lymphoma (cHL) are derived from antigen presenting B cells that are infected by Epstein Barr virus (EBV) in ~30% of patients. Polymorphic Killer cell immunoglobulin-like receptors (KIRs) expressed on NK cells interact with human leukocyte antigen (HLA) class I and play a key role in immune surveillance against virally infected cells and tumor cells. We investigated the effect of KIR types on cHL susceptibility overall (n=211) and in EBV-stratified subgroups using the Dutch GoNL cohort as controls (n=498). The frequency of the KIR haplotype B subgroup was significantly different between EBV+ and EBV- cHL patients (62% vs. 77%, p=0.04) and this difference was more pronounced in nodular sclerosis (NS) cHL (49% vs. 79%, p=0.0003). The frequency of KIR haplotype B subgroup was significantly lower in EBV+ NS cHL compared to controls (49% vs. 67%, p=0.01). Analyses of known KIR - HLA interaction pairs revealed lower carrier frequencies of KIR2DS2 - HLA-C1 (29% vs. 46%, p=0.03) and KIR2DL2 - HLA-C1 (29% vs. 45%, p=0.04) in EBV+ NS cHL patients compared to controls. Carriers of the KIR haplotype B subgroup are less likely to develop EBV+ NS cHL, probably because of a more efficient control over EBV-infected B cells.

HLA Expression in Relation to HLA Type in Classic Hodgkin Lymphoma Patients.

Several human leukocyte antigen (HLA) alleles are strongly associated with susceptibility to classic Hodgkin lymphoma (cHL), also in subgroups stratified for presence of the Epstein-Barr virus (EBV). We tested the hypothesis that the pressure on cHL tumour cells to lose HLA expression is associated with HLA susceptibility alleles. A meta-analysis was carried out to identify consistent protective and risk HLA alleles in a combined cohort of 839 cHL patients from the Netherlands and the United Kingdom. Tumour cell HLA expression was studied in 338 cHL cases from these two cohorts and correlated to the presence of specific susceptibility HLA alleles. Carriers of the HLA-DRB1*07 protective allele frequently lost HLA class II expression in cHL overall. Patients carrying the HLA-DRB1*15/16 (DR2) risk allele retained HLA class II expression in EBV- cHL and patients with the HLA-B*37 risk allele retained HLA class I expression more frequently than non-carriers in EBV+ cHL. The other susceptibility alleles showed no significant differences in expression. Thus, HLA expression by tumour cells is associated with a subset of the protective and risk alleles. This strongly suggests that HLA associations in cHL are related to peptide binding capacities of specific HLA alleles.

Interaction between ERAP Alleles and HLA Class I Types Support a Role of Antigen Presentation in Hodgkin Lymphoma Development.

Genetic variants in the HLA region are the strongest risk factors for developing Hodgkin lymphoma (HL), suggesting an important role for antigen presentation. This is supported by another HL-associated genomic region which contains the loci of two enzymes that process endogenous proteins to peptides to be presented by HLA class I, i.e., endoplasmic reticulum aminopeptidase 1 (ERAP1) and ERAP2. We hypothesized that ERAP and HLA class I type interact in HL susceptibility, as shown previously for several autoimmune diseases. We detected ERAP1 and ERAP2 expression in tumor cells and cells in the microenvironment in primary HL tissue samples. Seven ERAP SNPs and ERAP1 haplotypes showed strong associations with RNA and protein levels of ERAP1 and ERAP2 in LCLs and HL cell lines. Analysis of HLA class I types, ERAP SNPs and ERAP haplotypes by direct genotyping or imputation from genome-wide association data in 390 HL patients revealed significant interactions between HLA-A11, rs27038 and the rs27038 associated ERAP haplotype, as well as between HLA-Cw2 and rs26618. In conclusion, our results show that ERAP and HLA class I interact in genetic susceptibility to HL, providing further evidence that antigen presentation is an important process in HL susceptibility and pathogenesis.

MORC2 regulates C/EBPα-mediated cell differentiation via sumoylation.

The expression and activity of CCAAT/enhancer-binding protein α (C/EBPα) are involved in sumoylation modification, which is critical to divert normal cells from differentiation to proliferation. However, the role and underlying mechanism of C/EBPα in cancer is poorly understood. Human MORC2 (microrchidia family CW-type zinc-finger 2), is a member of the MORC proteins family containing a CW-type zinc-finger domain. Here, we found that MORC2 interacted with TE-III domain of C/EBPα, and the overexpression of MORC2 promoted wild-type C/EBPα sumoylation and its subsequent degradation, which didn't significantly observe in mutant C/EBPα-K161R. Furthermore, the overexpression of MORC2 inhibited C/EBPα-mediated C2C12 cell differentiation to maintain cell cycle progression. Moreover, the striking correlation between the decreased C/EBPα expression and the increased MORC2 expression was also observed in the poor differentiation status of gastric cancer tissues. Most notably, the high expression of MORC2 is correlated  with an aggressive phenotype of clinical gastric cancer and shorter overall survival of patients. Taken together, our findings demonstrated that MORC2 expression regulated C/EBPα-mediated the axis of differentiation/proliferation via sumoylation modification, and affected its protein stability, causing cell proliferation and tumorigenesis.