Intratumoral heterogeneity impacts the success or failure of anti-cancer therapies. Here, we investigated the evolution and mechanistic heterogeneity in clonal populations of cell models for estrogen receptor positive breast cancer. To this end, we established barcoded models of luminal breast cancer and rendered them resistant to commonly applied first line endocrine therapies. By isolating single clones from the resistant cell pools and characterizing replicates of individual clones we observed inter- (between cell lines) and intra-tumor (between different clones from the same cell line) heterogeneity. Molecular characterization at RNA and phospho-proteomic levels revealed private clonal activation of the unfolded protein response and respective sensitivity to inhibition of the proteasome, and potentially shared sensitivities for repression of protein kinase C. Our in vitro findings are consistent with tumor-heterogeneity that is observed in breast cancer patients thus highlighting the need to uncover heterogeneity at an individual patient level and to adjust therapies accordingly.

Ionizing radiation-induced bystander effects (RIBE) encompass a number of effects with potential for a plethora of damages in adjacent non-irradiated tissue. The cascade of molecular events is initiated in response to the exposure to ionizing radiation (IR), something that may occur during diagnostic or therapeutic medical applications. In order to better investigate these complex response mechanisms, we employed a unified framework integrating statistical microarray analysis, signal normalization, and translational bioinformatics functional analysis techniques. This approach was applied to several microarray datasets from Gene Expression Omnibus (GEO) related to RIBE. The analysis produced lists of differentially expressed genes, contrasting bystander and irradiated samples versus sham-irradiated controls. Furthermore, comparative molecular analysis through BioInfoMiner, which integrates advanced statistical enrichment and prioritization methodologies, revealed discrete biological processes, at the cellular level. For example, the negative regulation of growth, cellular response to Zn²⁺-Cd²⁺, and Wnt and NIK/NF-kappaB signaling, thus refining the description of the phenotypic landscape of RIBE. Our results provide a more solid understanding of RIBE cell-specific response patterns, especially in the case of high-LET radiations, like α-particles and carbon-ions.

Colorectal cancer (CRC) patients with microsatellite‐stable (MSS) tumors are mostly treated with chemotherapy. Clinical benefits of targeted therapies depend on mutational states and tumor location. Many tumors carry mutations in KRAS proto‐oncogene, GTPase (KRAS) or B‐Raf proto‐oncogene, serine/threonine kinase (BRAF), rendering them more resistant to therapies. We performed whole‐exome sequencing and RNA‐Sequencing of 28 tumors of the Athens Comprehensive Cancer Center CRC cohort, and molecularly characterized CRC patients based on their microsatellite instability (MSI) status, single‐nucleotide variations (SNVs)/copy number alterations (CNAs), and pathway/transcription factor activities at the individual patient level. Variants were classified using a computational score for integrative cancer variant annotation and prioritization. Complementing this with public multi‐omics datasets, we identified activation of transforming growth factor beta (TGFβ) signaling to be more strongly activated in MSS patients, whereas Janus kinase (JAK)–signal transducer and activator of transcription (STAT) and mitogen‐activated protein kinase (MAPK) molecular cascades were activated specifically in MSI tumors. We unraveled mechanisms consistently perturbed in the transcriptional and mutational circuits and identified Runt‐related transcription factors (RUNX transcription factors) as putative biomarkers in CRC, given their role in the regulation of pathways involved in tumor progression and immune evasion. Assessing the immunogenicity of CRC tumors in the context of RAS/RAF mutations and MSI/MSS status revealed a critical impact that KRAS mutations have on immunogenicity, particularly in the MSS patient subgroup, with implications for diagnosis and treatment.

Breast cancer is the most frequent malignancy and the second leading cause of cancer‐related mortality in women. Estrogen receptor‐positive (ER+) tumors are treated with endocrine therapies such as tamoxifen or aromatase inhibitors (AI), aimed at disrupting estrogen signaling. While these therapies are initially effective, resident tumor cells can develop resistance, leading to relapse. The p21‐activated kinase 1 (PAK1), a regulator of oncogenic signaling pathways, has been implicated in tamoxifen resistance. However, it remains unclear whether PAK1 also affects the response to other endocrine therapies. Here we show PAK1 activity was elevated in tamoxifen‐resistant and long‐term estrogen‐deprived MCF7 cell lines and showed enhanced responsiveness to EGF stimulation. Inhibition of PAK1 effectively reduced cell proliferation in both models, with distinct effects on PAK1 downstream substrates. In the tamoxifen resistance context, PAK1 inhibition induced activation of the pro‐apoptotic protein BAD and triggered apoptosis while proliferation‐related kinases were suppressed in the estrogen‐deprived model. Our findings position PAK1 as a mediator of resistance to endocrine therapies suggesting that targeting PAK1 may present a novel strategy to overcome endocrine therapy resistance in ER+ breast cancer.

The 3D cultures provide more insight into cell-to-cell and cell-to-matrix interactions, better mimicking the environment where stem cells reside compared to traditional 2D cultures. Although the precise molecular pathways involved in the regulation of stem and progenitor cell fate remain unknown, it is widely accepted that transcription factors play a crucial role as intrinsic regulators in these fate decisions. In this study, we carried out a microRNA screen to track the behaviour of adult stem/progenitor cells derived from human mammary epithelial cells grown in 3D cultures. We identified miR-106a-3p, which enriches the adult stem cell-like lineage and promotes the expansion of 3D cultures. Transcriptomic analysis showed that this miRNA regulates transcription factors such as REST, CBFB, NF-YA, and GATA3, thereby enhancing the maintenance of adult stem/progenitor cells in human epithelial cells. These data reveal a clear transcriptional program that governs the maintenance of adult stem/progenitor cells and controls their fate.

Intratumoral heterogeneity drastically impacts the success or failure of anti-cancer therapies. Here, we investigated the evolution and mechanistic heterogeneity in clonal populations of cell models for estrogen receptor positive breast cancer. To this end, we established barcoded models of luminal breast cancer and rendered them resistant to commonly applied first line endocrine therapies. By deconvoluting complex resistant cell pools and characterizing replicates of individual clones we observed inter- (between cell lines) and intra-tumor (between different clones from the same cell line) heterogeneity. Molecular characterization at RNA and phospho-proteomic levels revealed private clonal activation of the unfolded protein response and respective sensitivity to inhibition of the proteasome, and shared sensitivities for repression of protein kinase C. Our in vitro findings mirror the tumor-heterogeneity that is observed in breast cancer patients thus highlighting the urgent need to uncover heterogeneity at an individual patient level and to adjust therapies accordingly.