COGNITION: a prospective precision oncology trial for patients with early breast cancer at high risk following neoadjuvant chemotherapy.

BACKGROUND: COGNITION (Comprehensive assessment of clinical features, genomics and further molecular markers to identify patients with early breast cancer for enrolment on marker driven trials) is a diagnostic registry trial that employs genomic and transcriptomic profiling to identify biomarkers in patients with early breast cancer with a high risk for relapse after standard neoadjuvant chemotherapy (NACT) to guide genomics-driven targeted post-neoadjuvant therapy. PATIENTS AND METHODS: At National Center for Tumor Diseases Heidelberg patients were biopsied before starting NACT, and for patients with residual tumors after NACT additional biopsy material was collected. Whole-genome/exome and transcriptome sequencing were applied on tumor and corresponding blood samples. RESULTS: In the pilot phase 255 patients were enrolled, among which 213 were assessable: thereof 48.8% were identified to be at a high risk for relapse following NACT; 86.4% of 81 patients discussed in the molecular tumor board were eligible for a targeted therapy within the interventional multiarm phase II trial COGNITION-GUIDE (Genomics-guided targeted post neoadjuvant therapy in patients with early breast cancer) starting enrolment in Q4/2022. An in-depth longitudinal analysis at baseline and in residual tumor tissue of 16 patients revealed some cases with clonal evolution but largely stable genetic alterations, suggesting restricted selective pressure of broad-acting cytotoxic neoadjuvant chemotherapies. CONCLUSIONS: While most precision oncology initiatives focus on metastatic disease, the presented concept offers the opportunity to empower novel therapy options for patients with high-risk early breast cancer in the post-neoadjuvant setting within a biomarker-driven trial and provides the basis to test the value of precision oncology in a curative setting with the overarching goal to increase cure rates.

MiR-16-5p is frequently down-regulated in astrocytic gliomas and modulates glioma cell proliferation, apoptosis and response to cytotoxic therapy.

AIMS: Aberrant expression of microRNAs (miRNAs) is frequent in various cancers including gliomas. We aimed to characterize the role of miR-16-5p as a candidate tumour suppressor miRNA in gliomas. METHODS: Real-time PCR-based approaches were used for miRNA and mRNA expression profiling of glioma and non-neoplastic brain tissues as well as glioma cell lines. Protein levels were determined by Western blotting. In vitro analyses were performed following overexpression of miR-16-5p, trichostatin A (TSA) treatment, and siRNA-mediated knock-down of HDAC3 in glioma cells. Effects of miR-16-5p on glioma cell viability, apoptosis and response to irradiation and temozolomide (TMZ) were assessed. RESULTS: Expression of miR-16-5p was reduced relative to control brain tissue in isocitrate dehydrogenase (IDH)-mutant astrocytomas of World Health Organization (WHO) grades II, III and IV, and a subset of IDH-wildtype glioblastomas WHO grade IV. MiR-16-5p expression was lower in IDH-mutant than in IDH-wildtype gliomas, and down-regulated in IDH-wildtype glioma lines. MiR-16-5p overexpression reduced expression of important cell cycle and apoptosis regulators in glioma cells, including CDK6, CDC25A, CCND3, CCNE1, WEE1, CHEK1, BCL2 and MCL1. In line, CDK6, WEE1, CHEK1, BCL2 and MCL1 transcript levels were increased in WHO grade III or IV gliomas. TSA treatment and HDAC3 knockdown in glioma cells induced miR-16-5p up-regulation and reduced expression of its targets. Moreover, miR-16-5p overexpression inhibited proliferation and induced apoptosis in various glioma cell lines and increased sensitivity of A172 glioma cells to irradiation and TMZ. CONCLUSION: Reduced expression of miR-16-5p contributes to glioma cell proliferation, survival and resistance to cytotoxic therapy.

The branched-chain amino acid transaminase 1 sustains growth of antiestrogen-resistant and ERα-negative breast cancer.

Antiestrogen-resistant and triple-negative breast tumors pose a serious clinical challenge because of limited treatment options. We assessed global gene expression changes in antiestrogen-sensitive compared with antiestrogen-resistant (two tamoxifen resistant and two fulvestrant resistant) MCF-7 breast cancer cell lines. The branched-chain amino acid transaminase 1 (BCAT1), which catalyzes the first step in the breakdown of branched-chain amino acids, was among the most upregulated transcripts in antiestrogen-resistant cells. Elevated BCAT1 expression was confirmed in relapsed tamoxifen-resistant breast tumor specimens. High intratumoral BCAT1 levels were associated with a reduced relapse-free survival in adjuvant tamoxifen-treated patients and overall survival in unselected patients. On a tissue microarray (n=1421), BCAT1 expression was detectable in 58% of unselected primary breast carcinomas and linked to a higher Ki-67 proliferation index, as well as histological grade. Interestingly, BCAT1 was predominantly expressed in estrogen receptor-α-negative/human epidermal growth factor receptor-2-positive (ERα-negative/HER-2-positive) and triple-negative breast cancers in independent patient cohorts. The inverse relationship between BCAT1 and ERα was corroborated in various breast cancer cell lines and pharmacological long-term depletion of ERα induced BCAT1 expression in vitro. Mechanistically, BCAT1 indirectly controlled expression of the cell cycle inhibitor p27Kip1 thereby affecting pRB. Correspondingly, phenotypic analyses using a lentiviral-mediated BCAT1 short hairpin RNA knockdown revealed that BCAT1 sustains proliferation in addition to migration and invasion and that its overexpression enhanced the capacity of antiestrogen-sensitive cells to grow in the presence of antiestrogens. Importantly, silencing of BCAT1 in an orthotopic triple-negative xenograft model resulted in a massive reduction of tumor volume in vivo, supporting our findings that BCAT1 is necessary for the growth of hormone-independent breast tumors.

Genomic profiling of Acute lymphoblastic leukemia in ataxia telangiectasia patients reveals tight link between ATM mutations and chromothripsis.

Recent developments in sequencing technologies led to the discovery of a novel form of genomic instability, termed chromothripsis. This catastrophic genomic event, involved in tumorigenesis, is characterized by tens to hundreds of simultaneously acquired locally clustered rearrangements on one chromosome. We hypothesized that leukemias developing in individuals with Ataxia Telangiectasia, who are born with two mutated copies of the ATM gene, an essential guardian of genome stability, would show a higher prevalence of chromothripsis due to the associated defect in DNA double-strand break repair. Using whole-genome sequencing, fluorescence in situ hybridization and RNA sequencing, we characterized the genomic landscape of Acute Lymphoblastic Leukemia (ALL) arising in patients with Ataxia Telangiectasia. We detected a high frequency of chromothriptic events in these tumors, specifically on acrocentric chromosomes, as compared with tumors from individuals with other types of DNA repair syndromes (27 cases total, 10 with Ataxia Telangiectasia). Our data suggest that the genomic landscape of Ataxia Telangiectasia ALL is clearly distinct from that of sporadic ALL. Mechanistically, short telomeres and compromised DNA damage response in cells of Ataxia Telangiectasia patients may be linked with frequent chromothripsis. Furthermore, we show that ATM loss is associated with increased chromothripsis prevalence in additional tumor entities.

The onset of p53 loss of heterozygosity is differentially induced in various stem cell types and may involve the loss of either allele.

p53 loss of heterozygosity (p53LOH) is frequently observed in Li-Fraumeni syndrome (LFS) patients who carry a mutant (Mut) p53 germ-line mutation. Here, we focused on elucidating the link between p53LOH and tumor development in stem cells (SCs). Although adult mesenchymal stem cells (MSCs) robustly underwent p53LOH, p53LOH in induced embryonic pluripotent stem cells (iPSCs) was significantly attenuated. Only SCs that underwent p53LOH induced malignant tumors in mice. These results may explain why LFS patients develop normally, yet acquire tumors in adulthood. Surprisingly, an analysis of single-cell sub-clones of iPSCs, MSCs and ex vivo bone marrow (BM) progenitors revealed that p53LOH is a bi-directional process, which may result in either the loss of wild-type (WT) or Mut p53 allele. Interestingly, most BM progenitors underwent Mutp53LOH. Our results suggest that the bi-directional p53LOH process may function as a cell-fate checkpoint. The loss of Mutp53 may be regarded as a DNA repair event leading to genome stability. Indeed, gene expression analysis of the p53LOH process revealed upregulation of a specific chromatin remodeler and a burst of DNA repair genes. However, in the case of loss of WTp53, cells are endowed with uncontrolled growth that promotes cancer.

RNAi screening in glioma stem-like cells identifies PFKFB4 as a key molecule important for cancer cell survival.

The concept of cancer stem-like cells (CSCs) has gained considerable attention in various solid tumors including glioblastoma, the most common primary brain tumor. This sub-population of tumor cells has been intensively investigated and their role in therapy resistance as well as tumor recurrence has been demonstrated. In that respect, development of therapeutic strategies that target CSCs (and possibly also the tumor bulk) appears a promising approach in patients suffering from primary brain tumors. In the present study, we utilized RNA interference (RNAi) to screen the complete human kinome and phosphatome (682 and 180 targets, respectively) in order to identify genes and pathways relevant for the survival of brain CSCs and thereby potential therapeutical targets for glioblastoma. We report of 46 putative candidates including known survival-related kinases and phosphatases. Interestingly, a number of genes identified are involved in metabolism, especially glycolysis, such as PDK1 and PKM2 and, most prominently PFKFB4. In vitro studies confirmed an essential role of PFKFB4 in the maintenance of brain CSCs. Furthermore, high PFKFB4 expression was associated with shorter survival of primary glioblastoma patients. Our findings support the importance of the glycolytic pathway in the maintenance of malignant glioma cells and brain CSCs and imply tumor metabolism as a promising therapeutic target in glioblastoma.

MicroRNA miR-885-5p targets CDK2 and MCM5, activates p53 and inhibits proliferation and survival.

Several microRNA (miRNA) loci are found within genomic regions frequently deleted in primary neuroblastoma, including miR-885-5p at 3p25.3. In this study, we demonstrate that miR-885-5p is downregulated on loss of 3p25.3 region in neuroblastoma. Experimentally enforced miR-885-5p expression in neuroblastoma cell lines inhibits proliferation triggering cell cycle arrest, senescence and/or apoptosis. miR-885-5p leads to the accumulation of p53 protein and activates the p53 pathway, resulting in upregulation of p53 targets. Enforced miR-885-5p expression consistently leads to downregulation of cyclin-dependent kinase (CDK2) and mini-chromosome maintenance protein (MCM5). Both genes are targeted by miR-885-5p via predicted binding sites within the 3'-untranslated regions (UTRs) of CDK2 and MCM5. Transcript profiling after miR-885-5p introduction in neuroblastoma cells reveals alterations in expression of multiple genes, including several p53 target genes and a number of factors involved in p53 pathway activity. Taken together, these data provide evidence that miR-885-5p has a tumor suppressive role in neuroblastoma interfering with cell cycle progression and cell survival.

Basement membrane component laminin-5 is a target of the tumor suppressor Smad4.

The tumor suppressor Smad4 is involved in carcinogenesis mainly of the pancreas and colon. Functional inactivation of Smad4 is a genetically late event that occurs upon transition from premalignant stages to invasive and metastatic growth. Smad4 encodes an intracellular messenger common to all signalling cascades induced by members of the transforming growth factor-beta (TGF-beta) superfamily of cytokines. Despite extensive knowledge about the mechanisms of TGF-beta/Smad signal transduction, little is known about Smad4 targets involved in the transition to malignancy. The hallmark of invasive growth is a breakdown of the basement membrane (BM), a specialized sheet of extracellular matrix produced through cooperation of epithelial and stromal cells. Laminin-5, a heterotrimeric epithelial-derived BM component, is commonly lost in carcinomas but not in premalignant tumors. Herein, we report that in human colon and pancreatic tumor cells, Smad4 functions as a positive transcriptional regulator of all three genes encoding laminin-5. Coordinate re-expression of the three laminin-5 chains induced by reconstitution of Smad4 leads to secretion and deposition of the heterotrimeric molecule in BM-like structures. These data define the expression control of an essential BM component as a novel function for the tumor suppressor Smad4.

NoBP, a nuclear fibroblast growth factor 3 binding protein, is cell cycle regulated and promotes cell growth.

Secreted and nuclear forms of fibroblast growth factor 3 (FGF3) have opposing effects on cells. The secreted form stimulates cell growth and transformation, while the nuclear form inhibits DNA synthesis and cell proliferation. By using the yeast two-hybrid system we have identified a nucleolar FGF3 binding protein (NoBP) which coimmunoprecipitated and colocalized with FGF3 in transfected COS-1 cells. Characterization of the NoBP binding domain of FGF3 exactly matched the sequence requirements of FGF3 for its translocation into the nucleoli, suggesting that NoBP might be the nucleolar binding partner of FGF3 essential for its nucleolus localization. Carboxyl-terminal domains of NoBP contain linear nuclear and nucleolar targeting motifs which are capable of directing a heterologous protein beta-galactosidase to the nucleus and the nucleoli. While NoBP expression was detected in all analyzed proliferating established cell lines, NoBP transcription was rapidly downregulated in the promyelocytic leukemia cell line HL60 when induced to differentiate. Analysis on the expression pattern of NoBP mRNA throughout the cell cycle in HeLa cells synchronized by lovastatin demonstrated a substantial upregulation during the late G(1)/early S phase. NoBP overexpression conferred a proliferating effect onto NIH 3T3 cells and can counteract the inhibitory effect of nuclear FGF3, suggesting a role of NoBP in controlling proliferation in cells. We propose that NoBP is the functional target of nuclear FGF3 action.