The TRIM69-MST2 signaling axis regulates centrosome dynamics and chromosome segregation.

Stringent control of centrosome duplication and separation is important for preventing chromosome instability. Structural and numerical alterations in centrosomes are hallmarks of neoplastic cells and contribute to tumorigenesis. We show that a Centrosome Amplification 20 (CA20) gene signature is associated with high expression of the Tripartite Motif (TRIM) family member E3 ubiquitin ligase, TRIM69. TRIM69-ablation in cancer cells leads to centrosome scattering and chromosome segregation defects. We identify Serine/threonine-protein kinase 3 (MST2) as a new direct binding partner of TRIM69. TRIM69 redistributes MST2 to the perinuclear cytoskeleton, promotes its association with Polo-like kinase 1 (PLK1) and stimulates MST2 phosphorylation at S15 (a known PLK1 phosphorylation site that is critical for centrosome disjunction). TRIM69 also promotes microtubule bundling and centrosome segregation that requires PRC1 and DYNEIN. Taken together, we identify TRIM69 as a new proximal regulator of distinct signaling pathways that regulate centrosome dynamics and promote bipolar mitosis.

BIRC5 expression by race, age and clinical factors in breast cancer patients.

PURPOSE: Survivin/BIRC5 is a proliferation marker that is associated with poor prognosis in breast cancer and an attractive therapeutic target. However, BIRC5 has not been well studied among racially diverse populations where aggressive breast cancers are prevalent. EXPERIMENTAL DESIGN: We studied BIRC5 expression in association with clinical and demographic variables and as a predictor of recurrence in 2174 participants in the Carolina Breast Cancer Study (CBCS), a population-based study that oversampled Black (n = 1113) and younger (< 50 years; n = 1137) participants with breast cancer. For comparison, similar analyses were conducted in The Cancer Genome Atlas [TCGA N = 1094, Black (n = 183), younger (n = 295)]. BIRC5 was evaluated as a continuous and categorical variable (highest quartile vs. lower three quartiles). RESULTS: Univariate, continuous BIRC5 expression was higher in breast tumors from Black women relative to non-Black women in both estrogen receptor (ER)-positive and ER-negative tumors and in analyses stratified by stage (i.e., within Stage I, Stage II, and Stage III/IV tumors). Within CBCS and TCGA, BIRC5-high was associated with young age (< 50 years) and Black race, as well as hormone receptor-negative tumors, non-Luminal A PAM50 subtypes, advanced stage, and larger tumors (> 2 cm). Relative to BIRC5-low, BIRC5-high tumors were associated with poor 5-year recurrence-free survival (RFS) among ER-positive tumors, both in unadjusted models [HR (95% CI): 2.7 (1.6, 4.6)] and after adjustment for age and stage [Adjusted HR (95% CI): 1.87 (1.07, 3.25)]. However, this relationship was not observed among ER-negative tumors [Crude HR (95% CI): 0.7 (0.39, 1.2); Adjusted HR (95% CI): 0.67 (0.37, 1.2)]. CONCLUSION: Black and younger women with breast cancer have a higher burden of BIRC5-high tumors than older and non-Black women. Emerging anti-survivin treatment strategies may be an important future direction for equitable breast cancer outcomes.

Protein-based immune profiles of basal-like vs. luminal breast cancers.

Tumor-infiltrating lymphocytes play an important, but incompletely understood role in chemotherapy response and prognosis. In breast cancer, there appear to be distinct immune responses by subtype, but most studies have used limited numbers of protein markers or bulk sequencing of RNA to characterize immune response, in which spatial organization cannot be assessed. To identify immune phenotypes of Basal-like vs. Luminal breast cancer we used the GeoMx® (NanoString) platform to perform digital spatial profiling of immune-related proteins in tumor whole sections and tissue microarrays (TMA). Visualization of CD45, CD68, or pan-Cytokeratin by immunofluorescence was used to select regions of interest in formalin-fixed paraffin embedded tissue sections. Forty-four antibodies representing stromal markers and multiple immune cell types were applied to quantify the tumor microenvironment. In whole tumor slides, immune hot spots (CD45+) had increased expression of many immune markers, suggesting a diverse and robust immune response. In epithelium-enriched areas, immune signals were also detectable and varied by subtype, with regulatory T-cell (Treg) markers (CD4, CD25, and FOXP3) being higher in Basal-like vs. Luminal breast cancer. Extending these findings to TMAs with more patients (n = 75), we confirmed subtype-specific immune profiles, including enrichment of Treg markers in Basal-likes. This work demonstrated that immune responses can be detected in epithelium-rich tissue, and that TMAs are a viable approach for obtaining important immunoprofiling data. In addition, we found that immune marker expression is associated with breast cancer subtype, suggesting possible prognostic, or targetable differences.

Diffsig: Associating Risk Factors with Mutational Signatures.

BACKGROUND: Somatic mutational signatures elucidate molecular vulnerabilities to therapy, and therefore detecting signatures and classifying tumors with respect to signatures has clinical value. However, identifying the etiology of the mutational signatures remains a statistical challenge, with both small sample sizes and high variability in classification algorithms posing barriers. As a result, few signatures have been strongly linked to particular risk factors. METHODS: Here, we develop a statistical model, Diffsig, for estimating the association of one or more continuous or categorical risk factors with DNA mutational signatures. Diffsig takes into account the uncertainty associated with assigning signatures to samples as well as multiple risk factors' simultaneous effect on observed DNA mutations. RESULTS: We applied Diffsig to breast cancer data to assess relationships between five established breast-relevant mutational signatures and etiologic variables, confirming known mechanisms of cancer development. In simulation, our model was capable of accurately estimating expected associations in a variety of contexts. CONCLUSIONS: Diffsig allows researchers to quantify and perform inference on the associations of risk factors with mutational signatures. IMPACT: We expect Diffsig to provide more robust associations of risk factors with signatures to lead to better understanding of the tumor development process and improved models of tumorigenesis.

Diffsig: Associating Risk Factors With Mutational Signatures.

Somatic mutational signatures elucidate molecular vulnerabilities to therapy and therefore detecting signatures and classifying tumors with respect to signatures has clinical value. However, identifying the etiology of the mutational signatures remains a statistical challenge, with both small sample sizes and high variability in classification algorithms posing barriers. As a result, few signatures have been strongly linked to particular risk factors. Here we present Diffsig, a model and R package for estimating the association of risk factors with mutational signatures, suggesting etiologies for the pre-defined mutational signatures. Diffsig is a Bayesian Dirichlet-multinomial hierarchical model that allows testing of any type of risk factor while taking into account the uncertainty associated with samples with a low number of observations. In simulation, we found that our method can accurately estimate risk factor-mutational signal associations. We applied Diffsig to breast cancer data to assess relationships between five established breast-relevant mutational signatures and etiologic variables, confirming known mechanisms of cancer development. Diffsig is implemented as an R package available at: https://github.com/jennprk/diffsig.

Molecular and Clinical Characterization of Postpartum-Associated Breast Cancer in the Carolina Breast Cancer Study Phase I-III, 1993-2013.

BACKGROUND: Breast cancers in recently postpartum women may have worse outcomes, but studies examining tumor molecular features by pregnancy recency have shown conflicting results. METHODS: This analysis used Carolina Breast Cancer Study data to examine clinical and molecular tumor features among women less than 50 years of age who were recently (≤10 years prior) or remotely (>10 years prior) postpartum, or nulliparous. Prevalence odds ratios (POR) and 95% confidence intervals (CI) were estimated using multivariable models. RESULTS: Recently postpartum women (N = 618) were more frequently lymph node-positive [POR (95% CI): 1.66 (1.26-2.19)], estrogen receptor (ER)-negative [1.37 (1.02-1.83)], and IHC-based triple negative [1.57 (1.00-2.47)] compared with nulliparous (N = 360) women. Some differences were identified between recent versus remotely postpartum; smaller tumor size [0.67 (0.52-0.86)], p53 wildtype [0.53 (0.36-0.77)], and non-basal-like phenotype [0.53 (0.33-0.84)] were more common among recently postpartum. Recently postpartum (vs. nulliparous) had significant enrichment for adaptive immunity, T cells, B cells, CD8 T cells, activated CD8 T cells/natural killer (NK) cells, and T follicular helper (Tfh) cells and higher overall immune cell scores. These differences were attenuated in remotely (compared with recently) postpartum women. CONCLUSIONS: These results suggest a dominant effect of parity (vs. nulliparity) and a lesser effect of pregnancy recency on tumor molecular features, although tumor immune microenvironments were altered in association with pregnancy recency. IMPACT: Our study is unique in examining tumor immune microenvironment and RNA-based markers according to time since last childbirth. Future studies should examine the interplay between tumor features, postdiagnostic treatment, and outcomes among recently postpartum women. See related commentary by McDonald et al., p. 518.

Quantitative profiling of adaptation to cyclin E overproduction.

Cyclin E/CDK2 drives cell cycle progression from G1 to S phase. Despite the toxicity of cyclin E overproduction in mammalian cells, the cyclin E gene is overexpressed in some cancers. To further understand how cells can tolerate high cyclin E, we characterized non-transformed epithelial cells subjected to chronic cyclin E overproduction. Cells overproducing cyclin E, but not cyclins D or A, briefly experienced truncated G1 phases followed by a transient period of DNA replication origin underlicensing, replication stress, and impaired proliferation. Individual cells displayed substantial intercellular heterogeneity in cell cycle dynamics and CDK activity. Each phenotype improved rapidly despite high cyclin E-associated activity. Transcriptome analysis revealed adapted cells down-regulated a cohort of G1-regulated genes. Withdrawing cyclin E from adapted cells only partially reversed underlicensing indicating that adaptation is at least partly non-genetic. This study provides evidence that mammalian cyclin E/CDK inhibits origin licensing indirectly through premature S phase onset and provides mechanistic insight into the relationship between CDKs and licensing. It serves as an example of oncogene adaptation that may recapitulate molecular changes during tumorigenesis.

Obesity and Breast Cancer Metastasis across Genomic Subtypes.

BACKGROUND: Obese women have higher risk of aggressive breast tumors and distant metastasis. However, obesity has rarely been assessed in association with metastasis in diverse populations. METHODS: In the Carolina Breast Cancer Study Phase 3 (2008-2013), waist-to-hip ratio (WHR), body mass index (BMI), and molecular subtype [PAM50 risk-of-recurrence (ROR) score] were assessed. Obesity measures were evaluated in association with metastasis within five years of diagnosis, overall and stratified by race and ROR score. Absolute risk of metastasis and risk differences between strata were calculated using the Kaplan-Meier estimator, adjusted for age, grade, stage, race, and ER status. Relative frequency of metastatic site and multiplicity were estimated in association with obesity using generalized linear models. RESULTS: High-WHR was associated with higher risk of metastasis (5-year risk difference, RD, 4.3%; 95% confidence interval, 2.2-6.5). It was also associated with multiple metastases and metastases at all sites except brain. The 5-year risk of metastasis differed by race (11.2% and 6.9% in Black and non-Black, respectively) and ROR score (19.5% vs. 6.6% in high vs. low-to-intermediate ROR-PT). Non-Black women and those with low-to-intermediate ROR scores had similar risk in high- and low-WHR strata. However, among Black women and those with high ROR, risk of metastasis was elevated among high-WHR (RDBlack/non-Black = 4.6%, RDHigh/Low-Int = 3.1%). Patterns of metastasis were similar by BMI. CONCLUSIONS: WHR is associated with metastatic risk, particularly among Black women and those with high-risk tumors. IMPACT: Understanding how risk factors for metastasis interact may help in tailoring care plans and surveillance among patients with breast cancer.

DNA Damage Repair Classifier Defines Distinct Groups in Hepatocellular Carcinoma.

DNA repair pathways have been associated with variability in hepatocellular carcinoma (HCC) clinical outcomes, but the mechanism through which DNA repair varies as a function of liver regeneration and other HCC characteristics is poorly understood. We curated a panel of 199 genes representing 15 DNA repair pathways to identify DNA repair expression classes and evaluate their associations with liver features and clinicopathologic variables in The Cancer Genome Atlas (TCGA) HCC study. We identified two groups in HCC, defined by low or high expression across all DNA repair pathways. The low-repair group had lower grade and retained the expression of classical liver markers, whereas the high-repair group had more clinically aggressive features, increased p53 mutant-like gene expression, and high liver regenerative gene expression. These pronounced features overshadowed the variation in the low-repair subset, but when considered separately, the low-repair samples included three subgroups: L1, L2, and L3. L3 had high DNA repair expression with worse progression-free (HR 1.24, 95% CI 0.81-1.91) and overall (HR 1.63, 95% CI 0.98-2.71) survival. High-repair outcomes were also significantly worse compared with the L1 and L2 groups. HCCs vary in DNA repair expression, and a subset of tumors with high regeneration profoundly disrupts liver biology and poor prognosis.

RNA-Based Classification of Homologous Recombination Deficiency in Racially Diverse Patients with Breast Cancer.

BACKGROUND: Aberrant expression of DNA repair pathways such as homologous recombination (HR) can lead to DNA repair imbalance, genomic instability, and altered chemotherapy response. DNA repair imbalance may predict prognosis, but variation in DNA repair in diverse cohorts of breast cancer patients is understudied. METHODS: To identify RNA-based patterns of DNA repair expression, we performed unsupervised clustering on 51 DNA repair-related genes in the Cancer Genome Atlas Breast Cancer [TCGA BRCA (n = 1,094)] and Carolina Breast Cancer Study [CBCS (n = 1,461)]. Using published DNA-based HR deficiency (HRD) scores (high-HRD ≥ 42) from TCGA, we trained an RNA-based supervised classifier. Unsupervised and supervised HRD classifiers were evaluated in association with demographics, tumor characteristics, and clinical outcomes. RESULTS: : Unsupervised clustering on DNA repair genes identified four clusters of breast tumors, with one group having high expression of HR genes. Approximately 39.7% of CBCS and 29.3% of TCGA breast tumors had this unsupervised high-HRD (U-HRD) profile. A supervised HRD classifier (S-HRD) trained on TCGA had 84% sensitivity and 73% specificity to detect HRD-high samples. Both U-HRD and S-HRD tumors in CBCS had higher frequency of TP53 mutant-like status (45% and 41% enrichment) and basal-like subtype (63% and 58% enrichment). S-HRD high was more common among black patients. Among chemotherapy-treated participants, recurrence was associated with S-HRD high (HR: 2.38, 95% confidence interval = 1.50-3.78). CONCLUSIONS: HRD is associated with poor prognosis and enriched in the tumors of black women. IMPACT: RNA-level indicators of HRD are predictive of breast cancer outcomes in diverse populations.

The Landscape of Immune Microenvironments in Racially Diverse Breast Cancer Patients.

BACKGROUND: Immunotherapy is a rapidly evolving treatment option in breast cancer; However, the breast cancer immune microenvironment is understudied in Black and younger (<50 years) patients. METHODS: We used histologic and RNA-based immunoprofiling methods to characterize the breast cancer immune landscape in 1,952 tumors from the Carolina Breast Cancer Study (CBCS), a population-based study that oversampled Black (n = 1,030) and young women (n = 1,039). We evaluated immune response leveraging markers for 10 immune cell populations, compared profiles to those in The Cancer Genome Atlas (TCGA) Project [n = 1,095 tumors, Black (n = 183), and young women (n = 295)], and evaluated in association with clinical and demographic variables, including recurrence. RESULTS: Consensus clustering identified three immune clusters in CBCS (adaptive-enriched, innate-enriched, or immune-quiet) that varied in frequency by race, age, tumor grade and subtype; however, only two clusters were identified in TCGA, which were predominantly comprised of adaptive-enriched and innate-enriched tumors. In CBCS, the strongest adaptive immune response was observed for basal-like, HER2-positive (HER2+), triple-negative breast cancer (TNBC), and high-grade tumors. Younger patients had higher proportions of adaptive-enriched tumors, particularly among estrogen receptor (ER)-negative (ER-) cases. Black patients had higher frequencies of both adaptive-enriched and innate-enriched tumors. Immune clusters were associated with recurrence among ER- tumors, with adaptive-enriched showing the best and innate-enriched showing the poorest 5-year recurrence-free survival. CONCLUSIONS: These data suggest that immune microenvironments are intricately related to race, age, tumor subtype, and grade. IMPACT: Given higher mortality among Black and young women, more defined immune classification using cell-type-specific panels could help explain higher recurrence and ultimately lead to targetable interventions.

Adaptation and selection shape clonal evolution of tumors during residual disease and recurrence.

The survival and recurrence of residual tumor cells following therapy constitutes one of the biggest obstacles to obtaining cures in breast cancer, but it remains unclear how the clonal composition of tumors changes during relapse. We use cellular barcoding to monitor clonal dynamics during tumor recurrence in vivo. We find that clonal diversity decreases during tumor regression, residual disease, and recurrence. The recurrence of dormant residual cells follows several distinct routes. Approximately half of the recurrent tumors exhibit clonal dominance with a small number of subclones comprising the vast majority of the tumor; these clonal recurrences are frequently dependent upon Met gene amplification. A second group of recurrent tumors comprises thousands of subclones, has a clonal architecture similar to primary tumors, and is dependent upon the Jak/Stat pathway. Thus the regrowth of dormant tumors proceeds via multiple routes, producing recurrent tumors with distinct clonal composition, genetic alterations, and drug sensitivities.

CCL5 promotes breast cancer recurrence through macrophage recruitment in residual tumors.

Over half of breast-cancer-related deaths are due to recurrence 5 or more years after initial diagnosis and treatment. This latency suggests that a population of residual tumor cells can survive treatment and persist in a dormant state for many years. The role of the microenvironment in regulating the survival and proliferation of residual cells following therapy remains unexplored. Using a conditional mouse model for Her2-driven breast cancer, we identify interactions between residual tumor cells and their microenvironment as critical for promoting tumor recurrence. Her2 downregulation leads to an inflammatory program driven by TNFα/NFκB signaling, which promotes immune cell infiltration in regressing and residual tumors. The cytokine CCL5 is elevated following Her2 downregulation and remains high in residual tumors. CCL5 promotes tumor recurrence by recruiting CCR5-expressing macrophages, which may contribute to collagen deposition in residual tumors. Blocking this TNFα-CCL5-macrophage axis may be efficacious in preventing breast cancer recurrence.

Foxo-dependent Par-4 Upregulation Prevents Long-term Survival of Residual Cells Following PI3K-Akt Inhibition.

Tumor recurrence is a leading cause of death and is thought to arise from a population of residual cells that survive treatment. These residual cancer cells can persist, locally or at distant sites, for years or decades. Therefore, understanding the pathways that regulate residual cancer cell survival may suggest opportunities for targeting these cells to prevent recurrence. Previously, it was observed that the proapoptotic protein (PAWR/Par-4) negatively regulates residual cell survival and recurrence in mice and humans. However, the mechanistic underpinnings on how Par-4 expression is regulated are unclear. Here, it is demonstrated that Par-4 is transcriptionally upregulated following treatment with multiple drugs targeting the PI3K-Akt-mTOR signaling pathway, and identify the Forkhead family of transcription factors as mediators of this upregulation. Mechanistically, Foxo3a directly binds to the Par-4 promoter and activates its transcription following inhibition of the PI3K-Akt pathway. This Foxo-dependent Par-4 upregulation limits the long-term survival of residual cells following treatment with therapeutics that target the PI3K-Akt pathway. Taken together, these results indicate that residual breast cancer tumor cell survival and recurrence requires circumventing Foxo-driven Par-4 upregulation and suggest that approaches to enforce Par-4 expression may prevent residual cell survival and recurrence. Mol Cancer Res; 16(4); 599-609. ©2018 AACR.

Generation and comparison of CRISPR-Cas9 and Cre-mediated genetically engineered mouse models of sarcoma.

Genetically engineered mouse models that employ site-specific recombinase technology are important tools for cancer research but can be costly and time-consuming. The CRISPR-Cas9 system has been adapted to generate autochthonous tumours in mice, but how these tumours compare to tumours generated by conventional recombinase technology remains to be fully explored. Here we use CRISPR-Cas9 to generate multiple subtypes of primary sarcomas efficiently in wild type and genetically engineered mice. These data demonstrate that CRISPR-Cas9 can be used to generate multiple subtypes of soft tissue sarcomas in mice. Primary sarcomas generated with CRISPR-Cas9 and Cre recombinase technology had similar histology, growth kinetics, copy number variation and mutational load as assessed by whole exome sequencing. These results show that sarcomas generated with CRISPR-Cas9 technology are similar to sarcomas generated with conventional modelling techniques and suggest that CRISPR-Cas9 can be used to more rapidly generate genotypically and phenotypically similar cancers.

Akt1 and -2 inhibition diminishes terminal differentiation and enhances central memory CD8+ T-cell proliferation and survival.

The CD8 + T-cell response comprises terminally differentiated effector cells and antigen-experienced memory T cells. The latter encompass central (TCM) and effector (TEM) memory cells. TCM cells are superior in their protection against viral and bacterial challenges and mediation of antitumor immunity due to their higher proliferative ability upon antigen re-encounter. Defining a mechanism to enhance TCM cells and delay terminal differentiation of CD8 + T cells is crucial for cancer immune therapy, as it can promote a better tumor immune response. The differentiation of CD8 + memory T cells is thought to be coordinated by the phosphoinositide 3-kinase (PI3K)/Akt pathway. We, therefore, investigated the role of Akt isoforms in the differentiation and proliferation of memory CD8 + T cells. We found that Akt1 and Akt2, but not Akt3, drive the terminal differentiation of CD8 + T cells, and their inhibition enhances the therapeutically superior TCM phenotype. Furthermore, the inhibition of Akt1 and Akt2, but not Akt 3, delays CD8 + T-cell exhaustion and preserves naïve and TCM CD8 + T cells, thus enhancing their proliferative ability and survival and prolonging their cytokine and Granzyme B production ability. Here, we define a mechanism in which proliferative potential, function, and survival of CD8 + T cells are enhanced by maintaining a reservoir of TCM and naïve cells using only Akt1 and Akt2 inhibition. Therefore, our findings strongly suggest the utility of using Akt1 and Akt2 inhibitors to modulate CD8 + T cells, both for adoptive cell transfer and vaccine-based cancer immune therapies.

Distinct Transcript Isoforms of the Atypical Chemokine Receptor 1 (ACKR1)/Duffy Antigen Receptor for Chemokines (DARC) Gene Are Expressed in Lymphoblasts and Altered Isoform Levels Are Associated with Genetic Ancestry and the Duffy-Null Allele.

The Atypical ChemoKine Receptor 1 (ACKR1) gene, better known as Duffy Antigen Receptor for Chemokines (DARC or Duffy), is responsible for the Duffy Blood Group and plays a major role in regulating the circulating homeostatic levels of pro-inflammatory chemokines. Previous studies have shown that one common variant, the Duffy Null (Fy-) allele that is specific to African Ancestry groups, completely removes expression of the gene on erythrocytes; however, these individuals retain endothelial expression. Additional alleles are associated with a myriad of clinical outcomes related to immune responses and inflammation. In addition to allele variants, there are two distinct transcript isoforms of DARC which are expressed from separate promoters, and very little is known about the distinct transcriptional regulation or the distinct functionality of these protein isoforms. Our objective was to determine if the African specific Fy- allele alters the expression pattern of DARC isoforms and therefore could potentially result in a unique signature of the gene products, commonly referred to as antigens. Our work is the first to establish that there is expression of DARC on lymphoblasts. Our data indicates that people of African ancestry have distinct relative levels of DARC isoforms expressed in these cells. We conclude that the expression of both isoforms in combination with alternate alleles yields multiple Duffy antigens in ancestry groups, depending upon the haplotypes across the gene. Importantly, we hypothesize that DARC isoform expression patterns will translate into ancestry-specific inflammatory responses that are correlated with the axis of pro-inflammatory chemokine levels and distinct isoform-specific interactions with these chemokines. Ultimately, this work will increase knowledge of biological mechanisms underlying disparate clinical outcomes of inflammatory-related diseases among ethnic and geographic ancestry groups.