Mitochondria lack nucleotide excision repair; however, mitochondrial DNA (mtDNA) is resistant to mutation accumulation following DNA damage. These observations suggest additional damage sensing or protection mechanisms. Transcription Factor A, Mitochondrial (TFAM) compacts mtDNA into nucleoids and binds differentially to certain forms of DNA damage. As such, TFAM has emerged as a candidate for protecting mtDNA or sensing damage. To examine the possibilities that TFAM might protect DNA from damage or act as a damage sensing protein for irreparable forms of mtDNA damage, we used live-cell imaging, cell-based assays, atomic force microscopy (AFM), and high-throughput protein-DNA binding assays to characterize the binding properties of TFAM to ultraviolet-C (UVC) irradiated DNA and the cellular consequences of UVC irradiation. Our cell data show increased TFAM mRNA after exposure and suggest an increase in mtDNA degradation and turnover without a loss in mitochondrial membrane potential that might trigger mitophagy. Our protein-DNA binding assays indicate a reduction in sequence specificity of TFAM following UVC irradiation and a redistribution of TFAM binding throughout the mitochondrial genome. Our AFM data show increased compaction of DNA by TFAM in the presence of damage. Despite the TFAM-mediated compaction of mtDNA, we do not observe any protective effect of increased compaction on DNA damage formation in cells or in vitro. Taken together, these studies indicate that UVC-induced DNA damage alters TFAM binding and promotes compaction by TFAM, suggesting that TFAM may act as a damage sensing protein, sequestering damaged genomes to prevent mutagenesis by facilitating removal or suppression of replication.

Mitochondria lack nucleotide excision repair; however, mitochondrial DNA (mtDNA) is resistant to mutation accumulation following DNA damage. These observations suggest additional damage sensing or protection mechanisms. Transcription Factor A, Mitochondrial (TFAM) compacts mtDNA into nucleoids and binds differentially to certain forms of DNA damage. As such, TFAM has emerged as a candidate for protecting mtDNA or sensing damage. To examine the possibilities that TFAM might protect DNA from damage or act as a damage sensing protein for irreparable forms of mtDNA damage, we used live-cell imaging, cell-based assays, atomic force microscopy (AFM), and high-throughput protein-DNA binding assays to characterize the binding properties of TFAM to ultraviolet-C (UVC) irradiated DNA and the cellular consequences of UVC irradiation. Our cell data show increased TFAM mRNA after exposure and suggest an increase in mtDNA degradation and turnover without a loss in mitochondrial membrane potential that might trigger mitophagy. Our protein-DNA binding assays indicate a reduction in sequence specificity of TFAM following UVC irradiation and a redistribution of TFAM binding throughout the mitochondrial genome. Our AFM data show increased compaction of DNA by TFAM in the presence of damage. Despite the TFAM-mediated compaction of mtDNA, we do not observe any protective effect of increased compaction on DNA damage formation in cells or in vitro . Taken together, these studies indicate that UVC-induced DNA damage alters TFAM binding and promotes compaction by TFAM, suggesting that TFAM may act as a damage sensing protein, sequestering damaged genomes to prevent mutagenesis by facilitating removal or suppression of replication.

Most prostate cancers express the androgen receptor (AR), and tumor growth and progression are facilitated by exceptionally low levels of systemic or intratumorally produced androgens. Thus, absolute inhibition of the androgen signaling axis remains the goal of current therapeutic approaches to treat prostate cancer (PCa). Paradoxically, high dose androgens also exhibit considerable efficacy as a treatment modality in patients with late-stage metastatic PCa. Here we show that low levels of androgens, functioning through an AR monomer, facilitate a non-genomic activation of the mTOR signaling pathway to drive proliferation. Conversely, high dose androgens facilitate the formation of AR dimers/oligomers to suppress c-MYC expression, inhibit proliferation and drive a transcriptional program associated with a differentiated phenotype. These findings highlight the inherent liabilities in current approaches used to inhibit AR action in PCa and are instructive as to strategies that can be used to develop new therapeutics for this disease and other androgenopathies.

The field of environmental epigenetics is uniquely suited to investigate biologic mechanisms that have the potential to link stressors to health disparities. However, it is common practice in basic epigenetic research to treat race as a covariable in large data analyses in a way that can perpetuate harmful biases without providing any biologic insight. In this article, we i) propose that epigenetic researchers open a dialogue about how and why race is employed in study designs and think critically about how this might perpetuate harmful biases; ii) call for interdisciplinary conversation and collaboration between epigeneticists and social scientists to promote the collection of more detailed social metrics, particularly institutional and structural metrics such as levels of discrimination that could improve our understanding of individual health outcomes; iii) encourage the development of standards and practices that promote full transparency about data collection methods, particularly with regard to race; and iv) encourage the field of epigenetics to continue to investigate how social structures contribute to biological health disparities, with a particular focus on the influence that structural racism may have in driving these health disparities.

ABSTRACTThe steroid hormone progesterone, acting through its nuclear progesterone receptor (PR), has complex physiologic activities with different levels of hormones manifesting distinct and sometimes opposing phenotypic responses in target tissues. However, most of what is currently known about the transcriptional activity of PR comes from studies performed using progestins at levels that are in the high physiologic range (≥10nM), relevant only in the luteal phase of the reproductive cycle and pregnancy in humans. These studies do not consider the non-linearity of responses to progestins that exist in physiology and are not informative as to the mechanisms by which low levels of progestins, as occurs during menopause, exert their biological activities. Thus, we undertook to define the mechanisms which enable cells to recognize and respond to different levels of progestins. Using a PR expressing cell model of luminal breast cancer (T47D cells) we demonstrated that low concentration progestins (0.1-0.3nM) drive proliferation while high dose progestins (≥10nM) inhibit proliferation. Using both unbiased and targeted approaches, we found that low dose progestins facilitate cell cycle entry by enhanced expression of CCND1 and SGK1, which are both required to initiate a signaling cascade that leads to increased phospho-Rb and E2F1 transcriptional activity. CCND1 cooperates with CDK4/6 to phosphorylate Rb, while SGK1 phosphorylates p21, thereby excluding it from the nucleus and inhibiting its anti-proliferative function. Expression ofCCND1andSGK1mRNAs are primary responses to low dose progestin treatment. However, these responses occur at very low levels of receptor occupancy and in the absence of receptor phosphorylation events that have been shown to be required for nuclear translocation and transcriptional activity. These findings challenge the assumption of linearity in response to progestin dose. Further, they suggest that concentrations of progestins found in post-menopausal women (0.1-0.3nM) have the potential to exert proliferative responses in PR expressing cancers.

Functional annotations (from DAVID) for genes with higher expression in cells with low spheroid-forming capacity relative to cells with high spheroid-forming capacity.

Figures S1, S2 and Tables S1,S2 and S7

Differentially expressed genes that exhibit increased expression in the low spheroid-forming capacity cell lines that discriminate between high and low potential to survive serum-free spheroid culture.

Functional annotations (from the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8) for genes with higher expression in cells with high spheroid-forming capacity relative to cells with low spheroid-forming capacity.

Differentially expressed genes that exhibit increased expression in the high spheroid-forming capacity cell lines that discriminate between high and low potential to survive serum-free spheroid culture.

AbstractSpheroids exhibit drug resistance and slow proliferation, suggesting involvement in cancer recurrence. The protein kinase C inhibitor UCN-01 (7-hydroxystaurosporine) has shown higher efficacy against slow proliferating and/or quiescent ovarian cancer cells. In this study, tumorigenic potential was assessed using anchorage-independent growth assays and spheroid-forming capacity, which was determined with ovarian cancer cell lines as well as primary ovarian cancers. Of 12 cell lines with increased anchorage-independent growth, 8 formed spheroids under serum-free culture conditions. Spheroids showed reduced proliferation (P < 0.0001) and Ki-67 immunostaining (8% vs. 87%) relative to monolayer cells. Spheroid formation was associated with increased expression of mitochondrial pathway genes (P ≤ 0.001) from Affymetrix HT U133A gene expression data. UCN-01, a kinase inhibitor/mitochondrial uncoupler that has been shown to lead to Puma-induced mitochondrial apoptosis as well as ATP synthase inhibitor oligomycin, demonstrated effectiveness against spheroids, whereas spheroids were refractory to cisplatin and paclitaxel. By live in vivo imaging, ovarian cancer xenograft tumors were reduced after primary treatment with carboplatin. Continued treatment with carboplatin was accompanied by an increase in tumor signal, whereas there was little or no increase in tumor signal observed with subsequent treatment with UCN-01 or oltipraz. Taken together, our findings suggest that genes involved in mitochondrial function in spheroids may be an important therapeutic target in preventing disease recurrence.

Abstract Purpose: Breast cancer diagnosed in young patients is often aggressive. Because primary breast tumors from young and older patients have similar mutational patterns, we hypothesized that the young host microenvironment promotes more aggressive metastatic disease. Experimental Design: Triple-negative or luminal B breast cancer cell lines were injected into young and older mice side-by-side to quantify lung, liver, and brain metastases. Young and older mouse brains, metastatic and naïve, were analyzed by flow cytometry. Immune populations were depleted using antibodies or a colony-stimulating factor-1 receptor (CSF-1R) inhibitor, and brain metastasis assays were conducted. Effects on myeloid populations, astrogliosis, and the neuroinflammatory response were determined. Results: Brain metastases were 2- to 4-fold higher in young as compared with older mouse hosts in four models of triple-negative or luminal B breast cancer; no age effect was observed on liver or lung metastases. Aged brains, naïve or metastatic, contained fewer resident CNS myeloid cells. Use of a CSF-1R inhibitor to deplete myeloid cells, including both microglia and infiltrating macrophages, preferentially reduced brain metastasis burden in young mice. Downstream effects of CSF-1R inhibition in young mice resembled that of an aged brain in terms of myeloid numbers, induction of astrogliosis, and Semaphorin 3A secretion within the neuroinflammatory response. Conclusions: Host microenvironmental factors contribute to the aggressiveness of triple-negative and luminal B breast cancer brain metastasis. CSF-1R inhibitors may hold promise for young brain metastasis patients.

Abstract Women diagnosed with breast cancer at a younger age (typically defined as < 40 years old) often have a poorer prognosis and an increased risk of brain metastasis compared to their older counterparts. Multivariate analyses accounting for differences in tumor characteristics have shown that age is an independent predictor of worse outcome. We therefore hypothesized that rather than intrinsic tumor properties, extrinsic microenvironmental factors contribute to age-related differences in aggressiveness. The effect of age was examined by injecting brain-selected breast cancer cells into young (2 – 6 months) and older (>12 months) mice. In four brain metastasis models examined, young mice developed 2- to 16-fold (p < 0.05) more brain metastases compared to older mice. The effect of age was not observed in mouse breast cancer models that metastasize to liver and lungs, suggesting that this is an organ-specific phenomenon. Flow cytometry-based immune-profiling of mouse brains showed that T-cells (CD4+, CD8+, and FOXP3+CD25+ regulatory T-cells), monocytes and neutrophils were elevated in brains with metastases, but the abundance of these populations did not vary dramatically with age. Furthermore, antibody-based depletion of T-cells, monocytes and neutrophils did not significantly alter brain metastasis development. Microglia, which are resident CNS myeloid cells, were 1.5-fold more abundant in young brains compared to older brains. Depletion of CNS myeloid cells using the colony stimulating factor-1-receptor inhibitor PLX3397 reduced brain metastatic tumor burden in young mice by 2.1-fold (p < 0.001). Importantly, loss of CNS myeloid cells/microglia, which are normally more activated in aged mice and thus may protect the older brain against metastasis, did not augment brain metastasis formation in older mice. These results suggest that the younger brain is more permissive for breast cancer metastasis and that targeting resident CNS myeloid cells may be an effective strategy to prevent brain metastasis development in younger patients.

Abstract Younger women (< 40 years old) diagnosed with breast cancer often have a poorer outcome and a higher risk of developing brain metastases compared to women diagnosed at an older age. Multi-variate analyses have shown that even after accounting for differences in primary tumor characteristics, young age is still an independent predictor of poorer outcome. We therefore hypothesize that rather than intrinsic tumor properties, age-related changes to microenvironmental factors can affect breast cancer metastasis. To test this hypothesis, human and mouse breast cancer cells were injected into young (< 6 month) and old (> 13 month) mice and metastatic tumor burden was quantified. Young mice injected with brain-seeking breast cancer cells (MDA-MB-231BR, 4T1-BR, and 99LN-BrM) developed significantly more brain metastases compared to their older counterparts. In contrast, age had no effect on lung metastatic tumor burden in five breast cancer models. The effect of age is organ-specific, and the young brain is more permissive for breast cancer metastasis. To gain mechanistic insight, the transcriptome of young and old mouse brains were analyzed by RNAseq, the metastatic microenvironment was analyzed by laser capture microdissection and mass spectrometry, immune populations have been identified by flow cytometry, and functional immune contributions analyzed by immunodepleting antibodies. Multiple brain immune subsets were altered with age. In vivo depletion experiments showed no significant contribution of CD4+ T-cells and GR1+ myeloid cells to baseline brain metastatic colonization. A subpopulation of microglia in aged metastatic brains had a high side-scatter profile, which is consistent with published reports that aged microglia are in a “pro-inflammatory” state. Depletion of microglia reduced baseline brain metastatic colonization by 50% and experiments are underway to determine their contribution to an age effect.

Objective Ovarian cancer cells often exist in vivo as multicellular spheroids. Spheroid formation in vitro has been used to enrich for cancer stem cell populations from primary tumors. Such spheroids exhibit drug resistance and slow proliferation, suggesting involvement in disease recurrence. Our objectives were to characterize cancer spheroid phenotypes, determine gene expression profiles associated with spheroid forming capacity and to evaluate the responsiveness of spheroids to commonly used and novel therapeutic agents. Methods Tumorigenic potential was assessed using anchorage independent growth assays in 24 cell lines. Spheroids from cell lines (N=12) and from primary cancers (N=8) were grown on non-adherent tissue culture plates in serum-free media. Cell proliferation was measured using MTT assays and Ki67 immunostaining. Affymetrix HT U133A gene expression data was used to identify differentially expressed genes based on spheroid forming capacity. Matched monolayers and spheroids (N=7 pairs) were tested for response to cisplatin, paclitaxel and 7-hydroxystaurosporine (UCN-01) while mitochondrial inhibition was performed using oligomycin. Xenograft tumors from intraperitoneal injection of CAOV2-GFP/LUC ovarian cancer cells into nude mice were treated with carboplatin to reduce tumor burden followed by secondary treatment with carboplatin, UCN-01, or Oltipraz. Tumor formation and response was monitored using live imaging. Results Of 12 cell lines with increased anchorage-independent growth, 8 also formed spheroids under serum-free spheroid culture conditions. Spheroids showed reduced proliferation (p<0.0001) and Ki67 immunostaining (8% versus 87%) relative to monolayer cells. Spheroid forming capacity was associated with increased mitochondrial pathway activity (p ≤ 0.001). The mitochondrial inhibitors, UCN-01 and Oligomycin, demonstrated effectiveness against spheroids, while spheroids were refractory to cisplatin and paclitaxel. By live in vivo imaging, ovarian cancer xenograft tumors were reduced after primary treatment with carboplatin. Continued treatment with carboplatin was accompanied by an increase in tumor signal while there was little or no increase in tumor signal observed with subsequent treatment with UCN-01 or Oltipraz. Conclusions Our findings suggest that the mitochondrial pathway in spheroids may be an important therapeutic target in preventing disease recurrence.

PURPOSE:The blood-tumor barrier (BTB) limits irinotecan distribution in tumors of the central nervous system. However, given that the BTB has increased passive permeability we hypothesize that liposomal irinotecan would improve local exposure of irinotecan and its active metabolite SN-38 in brain metastases relative to conventional irinotecan due to enhanced-permeation and retention (EPR) effect.METHODS:Female nude mice were intracardially or intracranially implanted with human brain seeking breast cancer cells (brain metastases of breast cancer model). Mice were administered vehicle, non-liposomal irinotecan (50 mg/kg), liposomal irinotecan (10 mg/kg and 50 mg/kg) intravenously starting on day 21. Drug accumulation, tumor burden, and survival were evaluated.RESULTS:Liposomal irinotecan showed prolonged plasma drug exposure with mean residence time (MRT) of 17.7 ± 3.8 h for SN-38, whereas MRT was 3.67 ± 1.2 for non-liposomal irinotecan. Further, liposomal irinotecan accumulated in metastatic lesions and demonstrated prolonged exposure of SN-38 compared to non-liposomal irinotecan. Liposomal irinotecan achieved AUC values of 6883 ± 4149 ng-h/g for SN-38, whereas non-liposomal irinotecan showed significantly lower AUC values of 982 ± 256 ng-h/g for SN-38. Median survival for liposomal irinotecan was 50 days, increased from 37 days (p<0.05) for vehicle.CONCLUSIONS:Liposomal irinotecan accumulates in brain metastases, acts as depot for sustained release of irinotecan and SN-38, which results in prolonged survival in preclinical model of breast cancer brain metastasis.

Brain metastases are devastating complications of cancer. The blood–brain barrier (BBB), which protects the normal brain, morphs into an inadequately characterized blood–tumor barrier (BTB) when brain metastases form, and is surrounded by a neuroinflammatory response. These structures contribute to poor therapeutic efficacy by limiting drug uptake. Here, we report that experimental breast cancer brain metastases of low- and high permeability to a dextran dye exhibit distinct microenvironmental gene expression patterns. Astrocytic sphingosine-1 phosphate receptor 3 (S1P3) is upregulated in the neuroinflammatory response of the highly permeable lesions, and is expressed in patients’ brain metastases. S1P3 inhibition functionally tightens the BTB in vitro and in vivo. S1P3 mediates its effects on BTB permeability through astrocytic secretion of IL-6 and CCL2, which relaxes endothelial cell adhesion. Tumor cell overexpression of S1P3 mimics this pathway, enhancing IL-6 and CCL-2 production and elevating BTB permeability. In conclusion, neuroinflammatory astrocytic S1P3 modulates BTB permeability.