Patient-derived tumor organoids with p53 mutations, and not wild-type p53, are sensitive to synergistic combination PARP inhibitor treatment.

Poly (ADP-ribose) polymerase inhibitors (PARPi) are used for patients with BRCA1/2 mutations, but patients with other mutations may benefit from PARPi treatment. Another mutation that is present in more cancers than BRCA1/2 is mutation to the TP53 gene. In 2D breast cancer cell lines, mutant p53 (mtp53) proteins tightly associate with replicating DNA and Poly (ADP-ribose) polymerase (PARP) protein. Combination drug treatment with the alkylating agent temozolomide and the PARPi talazoparib kills mtp53 expressing 2D grown breast cancer cell lines. We evaluated the sensitivity to the combination of temozolomide plus PARPi talazoparib treatment to breast and lung cancer patient-derived tumor organoids (PDTOs). The combination of the two drugs was synergistic for a cytotoxic response in PDTOs with mtp53 but not for PDTOs with wtp53. The combination of talazoparib and temozolomide induced more DNA double-strand breaks in mtp53 expressing organoids than in wild-type p53 expressing organoids as shown by increased γ-H2AX protein expression. Moreover, breast cancer tissue microarrays (TMAs) showed a positive correlation between stable p53 and high PARP1 expression in sub-groups of breast cancers, which may indicate sub-classes of breast cancers sensitive to PARPi therapy. These results suggest that mtp53 could be a biomarker to predict response to the combination of PARPi talazoparib-temozolomide treatment.

Patient-derived tumor organoids with p53 mutations, and not wild-type p53, are sensitive to synergistic combination PARP inhibitor treatment.

Poly (ADP-ribose) polymerase inhibitors (PARPi) are used for patients with BRCA1/2 mutations, but patients with other mutations may benefit from PARPi treatment. Another mutation that is present in more cancers than BRCA1/2 is mutation to the TP53 gene. In 2D breast cancer cell lines, mutant p53 (mtp53) proteins tightly associate with replicating DNA and Poly (ADP-ribose) polymerase (PARP) protein. Combination drug treatment with the alkylating agent temozolomide and the PARPi talazoparib kills mtp53 expressing 2D grown breast cancer cell lines. We evaluated the sensitivity to the combination of temozolomide plus PARPi talazoparib treatment to breast and lung cancer patient-derived tumor organoids (PDTOs). The combination of the two drugs was synergistic for a cytotoxic response in PDTOs with mtp53 but not for PDTOs with wtp53. The combination of talazoparib and temozolomide induced more DNA double-strand breaks in mtp53 expressing organoids than in wild-type p53 expressing organoids as shown by increased γ-H2AX protein expression. Moreover, breast cancer tissue microarrays (TMAs) showed a positive correlation between stable p53 and high PARP1 expression in sub-groups of breast cancers, which may indicate sub-classes of breast cancers sensitive to PARPi therapy. These results suggest that mtp53 could be a biomarker to predict response to the combination of PARPi talazoparib-temozolomide treatment.

An enhancer variant associated with breast cancer susceptibility in Black women regulates TNFSF10 expression and antitumor immunity in triple-negative breast cancer.

Women of African ancestry have the highest mortality from triple-negative breast cancer (TNBC) of all racial groups. To understand the genomic basis of breast cancer in the populations, we previously conducted genome-wide association studies and identified single nucleotide polymorphisms (SNPs) associated with breast cancer in Black women. In this study, we investigated the functional significance of the top associated SNP rs13074711. We found the SNP served as an enhancer variant and regulated TNFSF10 (TRAIL) expression in TNBC cells, with a significant association between the SNP genotype and TNFSF10 expression in breast tumors. Mechanistically, rs13074711 modulated the binding activity of c-MYB at the motif and thereby controlled TNFSF10 expression. Interestingly, TNFSF10 expression in many cancers was consistently lower in African Americans compared with European Americans. Furthermore, TNFSF10 expression in TNBC was significantly correlated with the expression of antiviral immune genes and was regulated by type I interferons (IFNs). Accordingly, loss of TNFSF10 resulted in a profound decrease in apoptosis of TNBC cells in response to type I IFNs and poly(I:C), a synthetic analogue of double stranded virus. Lastly, in a syngeneic mouse model of breast cancer, TNFSF10-deficiency in breast tumors decreased tumor-infiltrated CD4+ and CD8+ T cell quantities. Collectively, our results suggested that TNFSF10 plays an important role in the regulation of antiviral immune responses in TNBC, and the expression is in part regulated by a genetic variant associated with breast cancer in Black women. Our results underscore the important contributions of genetic variants to immune defense mechanisms.

p50 mono-ubiquitination and interaction with BARD1 regulates cell cycle progression and maintains genome stability.

p50, the mature product of NFKB1, is constitutively produced from its precursor, p105. Here, we identify BARD1 as a p50-interacting factor. p50 directly associates with the BARD1 BRCT domains via a C-terminal phospho-serine motif. This interaction is induced by ATR and results in mono-ubiquitination of p50 by the BARD1/BRCA1 complex. During the cell cycle, p50 is mono-ubiquitinated in S phase and loss of this post-translational modification increases S phase progression and chromosomal breakage. Genome-wide studies reveal a substantial decrease in p50 chromatin enrichment in S phase and Cycln E is identified as a factor regulated by p50 during the G1 to S transition. Functionally, interaction with BARD1 promotes p50 protein stability and consistent with this, in human cancer specimens, low nuclear BARD1 protein strongly correlates with low nuclear p50. These data indicate that p50 mono-ubiquitination by BARD1/BRCA1 during the cell cycle regulates S phase progression to maintain genome integrity.

Gain-of-Function Mutant p53 R273H Interacts with Replicating DNA and PARP1 in Breast Cancer.

Over 80% of triple-negative breast cancers (TNBC) express mutant p53 (mtp53) and some contain oncogenic gain-of-function (GOF) p53. We previously reported that GOF mtp53 R273H upregulates the chromatin association of mini chromosome maintenance (MCM) proteins MCM2-7 and PARP and named this the mtp53-PARP-MCM axis. In this study, we dissected the function and association between mtp53 and PARP using a number of different cell lines, patient-derived xenografts (PDX), tissue microarrays (TMA), and The Cancer Genome Atlas (TCGA) database. Endogenous mtp53 R273H and exogenously expressed R273H and R248W bound to nascent 5-ethynyl-2´-deoxyuridine-labeled replicating DNA. Increased mtp53 R273H enhanced the association of mtp53 and PARP on replicating DNA. Blocking poly-ADP-ribose gylcohydrolase also enhanced this association. Moreover, mtp53 R273H expression enhanced overall MCM2 levels, promoted cell proliferation, and improved the synergistic cytotoxicity of treatment with the alkylating agent temozolomide in combination with the PARP inhibitor (PARPi) talazoparib. Staining of p53 and PARP1 in breast cancer TMAs and comparison with the TCGA database indicated a higher double-positive signal in basal-like breast cancer than in luminal A or luminal B subtypes. Higher PARP1 protein levels and PAR proteins were detected in mtp53 R273H than in wild-type p53-expressing PDX samples. These results indicate that mtp53 R273H and PARP1 interact with replicating DNA and should be considered as dual biomarkers for identifying breast cancers that may respond to combination PARPi treatments. SIGNIFICANCE: p53 gain-of-function mutant 273H and PARP1 interact with replication forks and could serve as potential biomarkers for breast cancer sensitivity to PARP inhibitors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/3/394/F1.large.jpg.

Recipient NK cell inactivation and intestinal barrier loss are required for MHC-matched graft-versus-host disease.

Previous studies have shown a correlation between pretransplant conditioning intensity, intestinal barrier loss, and graft-versus-host disease (GVHD) severity. However, because irradiation and other forms of pretransplant conditioning have pleiotropic effects, the precise role of intestinal barrier loss in GVHD pathogenesis remains unclear. We developed GVHD models that allowed us to isolate the specific contributions of distinct pretransplant variables. Intestinal damage was required for the induction of minor mismatch [major histocompatibility complex (MHC)-matched] GVHD, but was not necessary for major mismatch GVHD, demonstrating fundamental pathogenic distinctions between these forms of disease. Moreover, recipient natural killer (NK) cells prevented minor mismatch GVHD by limiting expansion and target organ infiltration of alloreactive T cells via a perforin-dependent mechanism, revealing an immunoregulatory function of MHC-matched recipient NK cells in GVHD. Minor mismatch GVHD required MyD88-mediated Toll-like receptor 4 (TLR4) signaling on donor cells, and intestinal damage could be bypassed by parenteral lipopolysaccharide (LPS) administration, indicating a critical role for the influx of bacterial components triggered by intestinal barrier loss. In all, the data demonstrate that pretransplant conditioning plays a dual role in promoting minor mismatch GVHD by both depleting recipient NK cells and inducing intestinal barrier loss.

Expression of polycomb targets predicts breast cancer prognosis.

Global changes in the epigenome are increasingly being appreciated as key events in cancer progression. The pathogenic role of enhancer of zeste homolog 2 (EZH2) has been connected to its histone 3 lysine 27 (H3K27) methyltransferase activity and gene repression; however, little is known about relationship of changes in expression of EZH2 target genes to cancer characteristics and patient prognosis. Here we show that through expression analysis of genomic regions with H3K27 trimethylation (H3K27me3) and EZH2 binding, breast cancer patients can be stratified into good and poor prognostic groups independent of known cancer gene signatures. The EZH2-bound regions were downregulated in tumors characterized by aggressive behavior, high expression of cell cycle genes, and low expression of developmental and cell adhesion genes. Depletion of EZH2 in breast cancer cells significantly increased expression of the top altered genes, decreased proliferation, and improved cell adhesion, indicating a critical role played by EZH2 in determining the cancer phenotype.

Regulation of Tcf7l1 DNA binding and protein stability as principal mechanisms of Wnt/ß-catenin signaling.

Wnt/ß-catenin signal transduction requires direct binding of ß-catenin to Tcf/Lef proteins, an event that is classically associated with stimulating transcription by recruiting coactivators. This molecular cascade plays critical roles throughout embryonic development and normal postnatal life by affecting stem cell characteristics and tumor formation. Here, we show that this pathway utilizes a fundamentally different mechanism to regulate Tcf7l1 (formerly named Tcf3) activity. ß-catenin inactivates Tcf7l1 without a switch to a coactivator complex by removing it from DNA, which leads to Tcf7l1 protein degradation. Mouse genetic experiments demonstrate that Tcf7l1 inactivation is the only required effect of the Tcf7l1-ß-catenin interaction. Given the expression of Tcf7l1 in pluripotent embryonic and adult stem cells, as well as in poorly differentiated breast cancer, these findings provide mechanistic insights into the regulation of pluripotency and the role of Wnt/ß-catenin in breast cancer.

Apc mutation enhances PyMT-induced mammary tumorigenesis.

The Adenomatous Polyposis Coli (APC) tumor suppressor gene is silenced by hypermethylation or mutated in up to 70% of human breast cancers. In mouse models, Apc mutation disrupts normal mammary development and predisposes to mammary tumor formation; however, the cooperation between APC and other mutations in breast tumorigenesis has not been studied. To test the hypothesis that loss of one copy of APC promotes oncogene-mediated mammary tumorigenesis, Apc(Min/+) mice were crossed with the mouse mammary tumor virus (MMTV)-Polyoma virus middle T antigen (PyMT) or MMTV-c-Neu transgenic mice. In the PyMT tumor model, the Apc(Min/+) mutation significantly decreased survival and tumor latency, promoted a squamous adenocarcinoma phenotype, and enhanced tumor cell proliferation. In tumor-derived cell lines, the proliferative advantage was a result of increased FAK, Src and JNK signaling. These effects were specific to the PyMT model, as no changes were observed in MMTV-c-Neu mice carrying the Apc(Min/+) mutation. Our data indicate that heterozygosity of Apc enhances tumor development in an oncogene-specific manner, providing evidence that APC-dependent pathways may be valuable therapeutic targets in breast cancer. Moreover, these preclinical model systems offer a platform for dissection of the molecular mechanisms by which APC mutation enhances breast carcinogenesis, such as altered FAK/Src/JNK signaling.

Wnt/beta-catenin pathway activation is enriched in basal-like breast cancers and predicts poor outcome.

Although Wnt/beta-catenin pathway activation has been implicated in mouse models of breast cancer, there is contradictory evidence regarding its importance in human breast cancer. In this study, invasive and in situ breast cancer tissue microarrays containing luminal A, luminal B, human epidermal growth factor receptor 2 (HER2)(+)/ER(-) and basal-like breast cancers were analyzed for beta-catenin subcellular localization. We demonstrate that nuclear and cytosolic accumulation of beta-catenin, a read-out of Wnt pathway activation, was enriched in basal-like breast cancers. In contrast, membrane-associated beta-catenin was observed in all breast cancer subtypes, and its expression decreased with tumor progression. Moreover, nuclear and cytosolic localization of beta-catenin was associated with other markers of the basal-like phenotype, including nuclear hormone receptor and HER2 negativity, cytokeratin 5/6 and vimentin expression, and stem cell enrichment. Importantly, this subcellular localization of beta-catenin was associated with a poor outcome and is more frequently observed in tumors from black patients. In addition, beta-catenin accumulation was more often observed in basal-like in situ carcinomas than other in situ subtypes, suggesting that activation of this pathway might be an early event in basal-like tumor development. Collectively, these data indicate that Wnt/beta-catenin activation is an important feature of basal-like breast cancers and is predictive of worse overall survival, suggesting that it may be an attractive pharmacological target for this aggressive breast cancer subtype.