Steven Van Laere

Inflammatory breast cancer (IBC) is an aggressive breast cancer subtype characterized by tumor emboli, lymphovascular invasion (LVI), and early dissemination. Herein, we establish adaptive stress response (ASR) as a biologic feature linking stress adaptation to tumor emboli survival, lymphatic dissemination, therapeutic response, and disparities. Using a previously defined 226 ASR-related genes, complementary preclinical models of tumor emboli and lymphatic circulating cell clusters, and independent patient cohorts, we identified ASR genes enriched for XIAP-NFκB, oxidative stress response, inflammatory, and immune pathways. CXCL8 emerged as one of the most highly upregulated transcripts in tumor emboli and was shared across both models; however, CXCL8 , IL6 , and PTGS2 were downregulated in lymphatic circulating cell clusters and LVI-positive triple-negative IBC patients, suggesting dynamic remodeling of inflammatory signaling during dissemination. CYP4B1 was associated with ER status, LVI, and therapeutic response across multiple cohorts, implicating metabolic stress adaptation in dissemination. IL6 and PTGS2 were elevated in self-reported Black patients with triple-negative IBC compared to White patients. Pharmacologic inhibition of XIAP-NFκB and oxidative stress pathways suppressed tumor emboli formation. Collectively, these findings identify ASR signaling as a framework linking tumor emboli survival, dissemination, and therapeutic vulnerability in IBC.

Recent studies have shown that heart failure (HF) and left ventricular (LV) dysfunction are associated with enhanced tumor growth. However, whether treating LV dysfunction mitigates its impact on cancer progression remains unknown. We hypothesized that HF treatments would attenuate tumor growth in a rodent model of post-myocardial infarction (MI)-induced LV dysfunction, and that different pharmacological agents (carvedilol, enalapril, and empagliflozin) might exert distinct effects on tumor progression. MI was induced in female BALB/c mice, leading to LV dysfunction. Mice with LV ejection fraction < 40% two weeks after MI received daily vehicle or one of three HF treatments by gavage. Two weeks later, 2 × 105 4T1 metastatic breast cancer cells were injected, with a three-week follow-up. The effects of HF treatments on cancer progression were assessed by analyzing: (i) tumor size in vivo over 22 days, (ii) lung metastasis development and (iii) gene expression levels in tumor tissue by RNA sequencing. Enalapril reversed several MI-induced transcriptomic changes in tumor tissue. Empagliflozin reduced primary tumor growth, while carvedilol reduced metastatic clusters. These effects were absent in control mice without MI, and neither drug directly affected cultured 4T1 cells. Transcriptomic analysis revealed treatment-specific inflammatory pathway regulation. Notably, carvedilol and empagliflozin restored MI-suppressed IFN-γ expression in tumors, accompanied by increased STAT1 expression in 4T1 cells. Specific HF treatments can mitigate cancer growth in a mouse model of MI-induced LV dysfunction, with outcomes varying by treatment. These benefits occurred only with LV dysfunction, suggesting they result from changes in HF pathophysiology rather than direct drug effects on tumor cells. Restoration of immune signaling may contribute to these effects.

Inflammatory breast cancer (IBC) tumors are characterized by diffuse clusters of cells found in dermal tissue and lymphatic vessels, known as tumor emboli. Thus, IBC needs a novel treatment because it is the most aggressive breast cancer subtype. We hypothesized that the interaction between tumor emboli and the tumor immune microenvironment (TiME) fosters survival signaling, leading to the aggressiveness of the IBC. In this study, ex vivo tumor emboli were generated from patient-derived cell lines cultured in a lymphatic-like environment, which was compared to 2D monolayer cultures, revealing upregulation of TNFR signaling networks, CXCL8, and immune cell chemotaxis genes. Spatial immunophenotyping of IBC patient tumors demonstrated high levels of CD163+ tumor-associated macrophages (TAMs). Furthermore, intravital imaging of CX3cr1 ^GFP mice confirmed macrophage movement toward tumor cell clusters. Finally, targeting macrophage-associated TNF-α-signaling using Birinapant, a SMAC mimetic, inhibited the tumor emboli phenotype in vivo, identifying Birinapant as a potential therapeutic agent to disrupt this signaling axis.

Therapeutic resistance presents a significant hurdle in combating inflammatory breast cancer (IBC), adding to the complexity of its management. To investigate these mechanisms, we conducted a comprehensive analysis using transcriptomic and proteomic profiling in a preclinical model alone with correlates of treatment response in IBC patients. This included SUM149 cell lines derived from treatment-naïve patients, along with acquired drug resistance (rSUM149) and others in a state of resistance reversal (rrSUM149), aiming to uncover drug resistance networks. We identified specific ribosomal proteins associated with acquiring resistance. These correlated with elevated levels of molecular markers such as pERK, CDK1, XIAP, and SOD2. While resistance reversal in rrSUM149 cells largely normalized the expression profile, VIPER analysis revealed persistent alterations in ribosomal process-related proteins (AGO2, Exportin 1, RPL5), suggesting their continued involvement in drug resistance. Moreover, genes linked to ribosomal processes were significantly enriched (P < 0.001) among overexpressed genes in IBC patients (n = 87) who exhibited a pathological complete response (pCR) to neoadjuvant chemotherapy. Given the common hyperactivation of MAPK in IBC tumors, including rSUM149, we evaluated Merestinib, a multikinase inhibitor in clinical trials. It effectively targeted pERK and peIF4E pathways, suppressed downstream targets, induced cell death in drug-resistant rSUM149 cells, and showed synergistic effects with another tyrosine kinase inhibitor (Lapatinib) in parental cells. This underscores its significant impact on protein synthesis signaling, crucial for combating translational dependence in cancer cells. In summary, our study elucidates adaptive changes in IBC cells in response to therapy and treatment pauses, guiding precision medicine approaches for this challenging cancer type.

Supplementary Figure S3. Top 20 master regulators that were differentially expressed in Cas9-p15 control and MELK knockout (KO) MDA-NB-231 cells.

Supplementary Materials and Methods

Clinicopathologic characteristics of patients in the World IBC Consortium dataset whose data were used for our analysis (n = 314)

Figure S1. Inhibitory effect of treatment with MELK-In-17 on the expression of epithelial-to-mesenchymal transition markers in murine 4T1 triple-negative breast cancer cells.

Clinical relevance of MELK in breast cancer and expression of MELK in breast cancer cell lines. A,MELK mRNA levels in HR−HER2+, HR+HER2−, HR+HER2+, and TNBC tumors in the World Consortium IBC dataset, analyzed by multiple comparison using a general linear model. Data are presented as mean ± SD. – represents median, X represents mean. B, IHC images (200× magnification) showing MELK protein expression in normal epithelial tissue and luminal, HER2+, and TNBC breast tumors. Correlation between MELK mRNA levels (<7 or ≥7) and OS, PFS, and DMFS in patients in the overall cohort (C) and among those with TNBC (D), determined by the Kaplan–Meier method. MELK mRNA levels (E) and protein levels (F) in TNBC and non-TNBC cells. Inset, correlation between MELK mRNA and protein levels in TNBC cell lines. In F, α-tubulin was used as a loading control. MELK mRNA levels were determined using quantitative reverse transcriptase PCR.