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Therapeutic effect of tamoxifen (Tam) against ER+ breast cancer (BC) is known to be mediated by its binding to estrogen receptor-alpha (ERά/ESR1) and inhibiting estrogen signaling leading to altered gene expression. Besides this canonical mode of function, our pre-clinical studies had revealed a novel mechanism wherein ESR1 directly binds wild type p53 (wt TP53) resulting in repression of its tumor suppressor functions, and Tam blocks this inactivation of TP53. Although patients with Luminal Tumors expressing wt TP53 are known to be more responsive to tamoxifen therapy, the underlying mechanism in tumors has remained unknown. To test the hypothesis that abrogation of the ESR1-mediated functional inactivation of TP53 is one of the major mechanisms that underlie the early effects of Tam therapy, we conducted a window of opportunity clinical trial in newly diagnosed luminal breast cancer patients undergoing surgical therapy. Methods: 59 women with ER+ invasive BC were randomized to 20mg Tam daily for 28 days prior to surgery or standard of care (SOC). TP53 status was confirmed by massively parallel sequencing. ER+ wt TP53 tumors were included in the study. IHC was performed on FFPE tissue from tumors to compare expression of ESR1 and TP53 along with their selected downstream targets. ESR1–TP53 interaction in situ was determined by Proximity Ligation Assay (PLA).17β-estradiol and Tam metabolites were measured in the plasma, tumor, and surrounding normal tissue using LC-MS/MS. Global transcriptome analysis in tumors was conducted by RNA-seq. Proteome expression in resected tumors was analyzed by reverse phase protein array (RPPA) with 216 proteins. Findings: Importantly, IHC on tumor tissues showed that the levels of ESR1 and TP53 were not altered in response to Tam therapy, whereas ESR1–TP53 interaction was considerably disrupted by Tam (in situ PLA data). Differential gene expression (DGE) analysis using DESeq2 R package followed by GSEA pathway analysis showed that 307 genes were differentially expressed (p< 0.05) (log FC>1.5) in tumors from Tam-treated versus untreated patients in response to Tam therapy. Pathways representing TP53 signaling, stem cells, and low-grade luminal breast cancer were upregulated in the Tam treated group while those representing adipogenesis, invasive breast cancer, estradiol response, ras signaling, and E2F targets were downregulated. “Master Regulators” identified by iRegulon included several p53 targets. Integration of RNA-seq and RPPA data revealed that DEGs fall into three categories: (i) regulated by TP53, (ii) regulated by ESR1, and (iii) regulated by both TP53 and ESR1. Together, the data demonstrated that in addition to its conventional effects mediated by its binding to ESR1 and inhibiting estrogen signaling leading to altered gene expression, Tam disrupted the ESR1–TP53 interaction leading to functional reactivation of TP53 and reprogramming of gene expression. Conclusions: Our data 1) support ESR1–TP53 crosstalk in tumors as a novel mechanism underlying endocrine therapy response of luminal BC patients, and 2) highlight the importance of factoring TP53 into therapeutic strategies for ER+ BC patients, and 3) have implications in stratifying ER+ BC patients to those who will or will not be responsive to Tam therapy. Citation Format: Gokul M. Das, Swati A. Kulkarni, Chetan Oturkar, Spencer Rosario, Stephen B. Edge, Jianmin Wang, Wendy M. Swetzig, Alan D. Hutson, Benny Kaipparettu, Adrienne Groman, Araba Adjei, Andrew K. Goey, Carl D. Morrison, Shicha Kumar. PD10-05 Neoadjuvant tamoxifen therapy reactivates tumor suppressor protein p53 in luminal breast cancer patients: Results from a window-of-opportunity clinical trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD10-05.

PDF file - 41KB, The combination of Y15 and temozolomide decreases more significantly viability of U251 cells than each agent alone.

Supplementary tables 1s-4s. Supplementary Table 1s. Concordance of variant calls. Supplementary Table 2s. Breast cancer-related genes from the Cancer Gene Consensus database. Supplementary Table 3s. Concordance of variant calls in known breast cancer genes. Supplementary Table 4s. Discordant variant calls in breast cancer-related genes.

Purpose: Our previous work has shown low serum 25-hydroxyvitamin D concentrations in association with aggressive breast cancer subtypes. Vitamin D receptor (VDR) is central for vitamin D–mediated transcription regulation. Few studies have examined breast VDR expression with tumor characteristics or patient survival.Experimental Design: VDR expression in breast tumor tissue microarrays was determined by immunohistochemistry in 1,114 female patients as low, moderate, and strong expression based on an immunoreactive score, and examined with histopathologic tumor characteristics and survival outcomes including progression-free survival, breast cancer–specific survival, and overall survival.Results: A majority (58%) of breast tumors showed moderate or strong VDR expression. VDR expression was inversely related to aggressive tumor characteristics, including large tumor size, hormonal receptor (HR) negativity, and triple-negative subtype (P < 0.05). In addition, VDR expression was also inversely related to Ki-67 expression among patients older than 50 years. Nevertheless, VDR expression was not associated with any patient survival outcomes examined.Conclusions: In a large patient population, VDR expression is inversely associated with more aggressive breast cancer, but not with breast cancer survival outcomes. The present findings of VDR expression are consistent with our previous results of circulating vitamin D biomarkers, which provide two converging lines of evidence supporting the putative benefits of vitamin D against aggressive breast cancer. Because of the observational nature of our analyses, future studies are warranted to establish the causality of the reported associations. Clin Cancer Res; 23(1); 97–103. ©2016 AACR.

Background: Whole-exome sequencing (WES) has recently emerged as an appealing approach to systematically study coding variants. However, the requirement for a large amount of high-quality DNA poses a barrier that may limit its application in large cancer epidemiologic studies. We evaluated the performance of WES with low input amount and saliva DNA as an alternative source material.Methods: Five breast cancer patients were randomly selected from the Pathways Study. From each patient, four samples, including 3 μg, 1 μg, and 0.2 μg blood DNA and 1 μg saliva DNA, were aliquoted for library preparation using the Agilent SureSelect Kit and sequencing using Illumina HiSeq2500. Quality metrics of sequencing and variant calling, as well as concordance of variant calls from the whole exome and 21 known breast cancer genes, were assessed by input amount and DNA source.Results: There was little difference by input amount or DNA source on the quality of sequencing and variant calling. The concordance rate was about 98% for single-nucleotide variant calls and 83% to 86% for short insertion/deletion calls. For the 21 known breast cancer genes, WES based on low input amount and saliva DNA identified the same set variants in samples from a same patient.Conclusions: Low DNA input amount, as well as saliva DNA, can be used to generate WES data of satisfactory quality.Impact: Our findings support the expansion of WES applications in cancer epidemiologic studies where only low DNA amount or saliva samples are available. Cancer Epidemiol Biomarkers Prev; 24(8); 1207–13. ©2015 AACR.

Supplementary Figures - Figure S1 shows CC expression in AA and CA PCa cells. Figure S2 shows cell death and DEVDase activity in AA and CA PCa cells in response to docetaxel treatment. Figure S3 shows effect of CC knockdown on docetaxel-induced apoptotic cell death, caspase-3 and PARP cleavage in CA PCa cells; and mtDNA content in AA and CA PCa cells. Figure S4 shows levels of Nrf1, c-Myc, NF-κB, and PGC-1α in AA and CA PCa cells; and shows cell death, DEVDase activity, and CC expression in response to docetaxel treatment in the presence of c-Myc and NF-κB inhibitors in AA PCa cells. Figure S5 shows effect of Nrf1 knockdown on DEVDase activity in response to docetaxel treatment in the presence of c-Myc and NF-κB inhibitors or AKT activator in AA PCa cells. Figure S6 shows effect of genetic inhibition of c-Myc, NF-κB and PTEN on CC expression and DEVDase activity after docetaxel treatment in AA PCa cells. Figure S7 shows levels of p-Drp1S616 and p-Drp1S637 in primary prostate tumor (PT) and matching non-tumor (MN) tissue samples from AA and CA PCa patients. Figure S8 shows effect of Drp1 knockdown on docetaxel-induced DEVDase activity and apoptosis in CA PCa cells. Figure S9 shows knockdown of Drp1 and CC in AA PCa cells and its effect on DEVDase activity in response to docetaxel treatment in the presence of c-Myc and NF-κB inhibitors or AKT activator. Figure S10 shows expression levels of oxidative phosphorylation (OXPHOS) complexes III, IV and V in primary prostate tumor (PT) and matching non-tumor (MN) tissue samples from AA and CA PCa patients. Figure S11 shows levels of LDHA in primary prostate tumor (PT) and matching non-tumor (MN) tissue samples from AA and CA PCa patients.

Supplementary Figure 2s. Comparisons of quality metrics between concordant and discordant short insertion/deletions (indel).

Supplementary Figure 1s. Comparisons of quality metrics between concordant and discordant single nucleotide variants (SNVs).

Although African-American (AA) patients with prostate cancer tend to develop greater therapeutic resistance and faster prostate cancer recurrence compared with Caucasian-American (CA) men, the molecular mechanisms of this racial prostate cancer disparity remain undefined. In this study, we provide the first comprehensive evidence that cytochrome c deficiency in AA primary tumors and cancer cells abrogates apoptosome-mediated caspase activation and contributes to mitochondrial dysfunction, thereby promoting therapeutic resistance and prostate cancer aggressiveness in AA men. In AA prostate cancer cells, decreased nuclear accumulation of nuclear respiration factor 1 (Nrf1) and its subsequent loss of binding to the cytochrome c promoter mediated cytochrome c deficiency. The activation of cellular Myc (c-Myc) and NF-κB or inhibition of AKT prevented nuclear translocation of Nrf1. Genetic and pharmacologic inhibition of c-Myc and NF-κB or activation of AKT promoted Nrf1 binding to cytochrome c promoter, cytochrome c expression, caspase activation, and cell death. The lack of p-Drp1S616 in AA prostate cancer cells contributed to defective cytochrome c release and increased resistance to apoptosis, indicating that restoration of cytochrome c alone may be insufficient to induce effective apoptosis. Cytochrome c deficiency promoted the acquisition of glycolytic phenotypes and mitochondrial dysfunction, whereas cytochrome c restoration via inhibition of c-Myc and NF-κB or activation of AKT attenuated glycolysis in AA prostate cancer cells. Inhibition of c-Myc and NF-κB enhanced the efficacy of docetaxel in tumor xenografts. Therefore, restoring cytochrome c may overcome therapeutic resistance and prostate cancer aggressiveness in AA men. Overall, this study provides the first comprehensive experimental, mechanistic, and clinical evidence for apoptosome and mitochondrial dysfunction in prostate cancer racial disparity.Significance:Mechanistic insights on prostate cancer health disparity among American men provide novel approaches to restore mitochondrial function, which can address therapeutic resistance and aggressiveness in African-American men with prostate cancer.

Expression of the WWOX gene, encompassing the common chromosome fragile site FRA16D, is altered in a large fraction of cancers of various types, including prostate cancer. We have examined expression and biological functions of WWOX in prostate cancer. WWOX mRNA and protein expression were significantly reduced in prostate cancer-derived cells (LNCaP, DU145, and PC-3) compared with noncancer prostate cells (PWR-1E), and WWOX expression was reduced in 84% of prostate cancers, as assessed by immunohistochemical staining. Down-modulation of WWOX expression in the prostate cancer-derived cells is due to DNA hypermethylation in the WWOX regulatory region. Treatment with 5-aza-2′-deoxycytidine (AZA), a DNA methyltransferase inhibitor, and trichostatin A, a histone deacetylase inhibitor, led to increased WWOX mRNA and protein expression in prostate cancer-derived cells, most strikingly in DU145 cells. Transfection-mediated WWOX overexpression in DU145 cells suppressed colony growth (P = 0.0012), and WWOX overexpression by infection with Ad-WWOX virus induced apoptosis through a caspase-dependent mechanism and suppressed cell growth. Lastly, ectopic expression of WWOX by Ad-WWOX infection suppressed tumorigenicity of xenografts in nude mice, and intratumoral AZA treatment halted tumor growth. The data are consistent with a role for WWOX as a prostate cancer tumor suppressor and suggest that WWOX signal pathways should be further investigated in normal and cancerous prostate cells and tissues. (Cancer Res 2006; 66(13): 6477-81)

Supplementary Figure 1 from A Role for the WWOX Gene in Prostate Cancer

Supplementary data and figure legends from Combined Total Genome Loss of Heterozygosity Scan of Breast Cancer Stroma and Epithelium Reveals Multiplicity of Stromal Targets

Calcitriol, a regulator of calcium homeostasis with antitumor properties, is degraded by the product of the CYP24A1 gene, which is downregulated in human prostate cancer by unknown mechanisms. We found that CYP24A1 expression is inversely correlated with promoter DNA methylation in prostate cancer cell lines. Treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) activates CYP24A1 expression in prostate cancer cells. In vitro methylation of the CYP24A1 promoter represses its promoter activity. Furthermore, inhibition of histone deacetylases by trichostatin A (TSA) enhances the expression of CYP24A1 in prostate cancer cells. Quantitative chromatin immunoprecipitation-PCR (ChIP-qPCR) reveals that specific histone modifications are associated with the CYP24A1 promoter region. Treatment with TSA increases H3K9ac and H3K4me2 and simultaneously decreases H3K9me2 at the CYP24A1 promoter. ChIP-qPCR assay reveals that treatment with DAC and TSA increases the recruitment of vitamin D receptor to the CYP24A1 promoter. Reverse transcriptase-PCR analysis of paired human prostate samples revealed that CYP24A1 expression is downregulated in prostate malignant lesions compared with adjacent histologically benign lesions. Bisulfite pyrosequencing shows that CYP24A1 gene is hypermethylated in malignant lesions compared with matched benign lesions. Our findings indicate that repression of CYP24A1 gene expression in human prostate cancer cells is mediated in part by promoter DNA methylation and repressive histone modifications. Cancer Res; 70(14); 5953–62. ©2010 AACR.

Supplementary Table 4 from Tumor Suppressor Activity of CCAAT/Enhancer Binding Protein α Is Epigenetically Down-regulated in Head and Neck Squamous Cell Carcinoma

Supplementary Table 1 from Tumor Suppressor Activity of CCAAT/Enhancer Binding Protein α Is Epigenetically Down-regulated in Head and Neck Squamous Cell Carcinoma

Recent breast cancer studies have highlighted the importance of interactions between cancer epithelium and tumor stroma. Recently, the focus of solid tumor investigations has shifted from mutations in carcinomatous epithelium to disturbances of tissue organization in cancer. The genetic basis of this microenvironment, however, remains to be clarified. To begin to resolve this problem, a total genome loss of heterozygosity (LOH) scan was done on epithelial and stromal DNA from 134 sporadic invasive breast carcinomas. In addition to detecting more frequent LOH at three loci in stroma than in epithelium, we found strong evidence that LOH frequencies were significantly elevated in specific regions of each chromosome. We detected 57 markers, which were preferentially lost either in stroma (n = 38) or epithelium (n = 19), relative to the background LOH frequencies on their respective chromosomes. This multiplicity of stromal cell LOH, and hence loss of genetic material, provides a possible mechanism for interpatient variation in host-stromal response to invading adenocarcinoma cells. This is consistent with a model in which initial, random LOH occurs equally among epithelium and stroma, but subsequent clonal selection is driven by factors, which appear to be distinctly different between malignant epithelial and surrounding stromal cells. Genetic alterations in stroma did not mimic those in epithelium, but they could play a different and parallel role in carcinogenesis and tumor progression, probably by modifying some features specific to breast cancer.

Supplementary Figure Legend from A Role for the WWOX Gene in Prostate Cancer

Supplementary Table 2 from Tumor Suppressor Activity of CCAAT/Enhancer Binding Protein α Is Epigenetically Down-regulated in Head and Neck Squamous Cell Carcinoma

Supplementary Figure S1 from Combined Total Genome Loss of Heterozygosity Scan of Breast Cancer Stroma and Epithelium Reveals Multiplicity of Stromal Targets

Supplementary Figure 1 from Epigenetic Regulation of Vitamin D 24-Hydroxylase/CYP24A1 in Human Prostate Cancer

Tumor suppressor CCAAT enhancer binding protein α (C/EBPα) is a transcription factor involved in cell cycle control and cellular differentiation. In a recent study, microarray expression profiling on head and neck squamous cell carcinoma (HNSCC) samples identified significant C/EBPα down-regulation, correlating with poor prognosis. However, the mechanisms of C/EBPα down-regulation remained elusive. C/EBPα has been previously found to provide an antiproliferative role in lung cancer, and our laboratory showed that its down-regulation involves epigenetic mechanisms. This prompted us to investigate the involvement of epigenetics in down-regulating C/EBPα in HNSCC. Here, we show that C/EBPα is down-regulated in HNSCC by loss of heterozygosity and DNA methylation, but not by gene mutation. We found a consistently methylated upstream regulatory region (−1,399 bp to −1,253 bp in relation to the transcription start site) in 68% of the HNSCC tumor samples, and DNA demethylation using 5-aza-2′-deoxycytidine treatment was able to significantly restore C/EBPα mRNA expression in the HNSCC cell lines we tested. In addition, C/EBPα overexpression in a HNSCC cell line (SCC22B) revealed its ability to provide tumor suppressor activity in HNSCC in vitro and in vivo. In conclusion, we showed for the first time not only that C/EBPα has tumor suppressor activity in HNSCC, but also that it is down-regulated by DNA promoter methylation. [Cancer Res 2007;67(10):4657–64]

Supplementary Figure Legends 1-2 from Tumor Suppressor Activity of CCAAT/Enhancer Binding Protein α Is Epigenetically Down-regulated in Head and Neck Squamous Cell Carcinoma

Supplementary Figure 1 from Tumor Suppressor Activity of CCAAT/Enhancer Binding Protein α Is Epigenetically Down-regulated in Head and Neck Squamous Cell Carcinoma

To uncover underlying mechanisms associated with failure of indoleamine 2,3-dioxygenase 1 (IDO1) blockade in clinical trials, we conducted a pilot, window-of-opportunity clinical study in 17 patients with newly diagnosed advanced high-grade serous ovarian cancer before their standard tumor debulking surgery. Patients were treated with the IDO1 inhibitor epacadostat, and immunologic, transcriptomic, and metabolomic characterization of the tumor microenvironment was undertaken in baseline and posttreatment tumor biopsies. IDO1 inhibition resulted in efficient blockade of the kynurenine pathway of tryptophan degradation and was accompanied by a metabolic adaptation that shunted tryptophan catabolism toward the serotonin pathway. This resulted in elevated nicotinamide adenine dinucleotide (NAD + ), which reduced T cell proliferation and function. Because NAD + metabolites could be ligands for purinergic receptors, we investigated the impact of blocking purinergic receptors in the presence or absence of NAD + on T cell proliferation and function in our mouse model. We demonstrated that A2a and A2b purinergic receptor antagonists, SCH58261 or PSB1115, respectively, rescued NAD + -mediated suppression of T cell proliferation and function. Combining IDO1 inhibition and A2a/A2b receptor blockade improved survival and boosted the antitumor immune signature in mice with IDO1 overexpressing ovarian cancer. These findings elucidate the downstream adaptive metabolic consequences of IDO1 blockade in ovarian cancers that may undermine antitumor T cell responses in the tumor microenvironment.

Cougar (Puma concolor) sightings have increased markedly throughout much of Midwestern North America and breeding populations have re-established in areas where there has not been a viable population of cougars for much of the past century. Using satellite telemetry data, we estimated resource selection functions (RSF) to examine shifts in seasonal habitat selection of a recently re-established population of cougars relative to human activity in Cypress Hills Interprovincial Park (CHIP) in southwest Saskatchewan and southeast Alberta, Canada. We modeled human activity predictors, used in cougar RSFs, from data collected at a network of motion-triggered trail cameras on the road and trail system within CHIP. Using the same network of remote cameras, we quantified cougar use of trails to examine the potential for human–cougar encounters. Cougar habitat selection models that included human activity outperformed models including only environmental variables. Cougars avoided areas near roads and trails during seasonal peaks in human activity but selected those areas during seasons of low human activity. Within each season, we found cougars avoided areas of increased motorized and non-motorized human activity, particularly in spring (motorized and non-motorized) and summer (non-motorized). Although resource selection models did not include covariates for prey, selection results were consistent with the expected distribution of prey on the landscape. Cougar use of trails was prevalent throughout CHIP and use was concentrated during dusk and nighttime periods. Thus, the potential for human–cougar interactions are present throughout the year, although they are likely highest during periods of low human-use and in the evenings. Further, individual variation in habitat selection among cougars makes human–cougar interactions difficult to predict. Our results highlight the adaptable nature of cougars to varying levels of human disturbance, which will facilitate their continued eastward range expansion. © 2014 The Wildlife Society.

Gene amplification, a common mechanism for oncogene activation in cancers, has been used in the discovery of novel oncogenes. DNA amplification is frequently observed in head and neck squamous cell carcinomas (HNSCCs) where numerous amplification events and potential oncogenes have already been reported. Recently, we applied restriction landmark genome scanning (RLGS) to study gene amplifications in HNSCC and located novel amplified and uncharacterized regions in primary tumor samples. DNA amplification on 8q22.3, the location of 14-3-3[zeta] (YWHAZ, KCIP-1) is found in 30-40% HNSCC cases. Data obtained from fluorescent in-situ hybridization (FISH) and immunohistochemistry on HNSCC tissue microarrays confirmed frequent low-level 14-3-3[zeta] copy number gains and protein overexpression. 14-3-3[zeta] mRNA was frequently upregulated in patients' tumor tissues. Furthermore, 14-3-3[zeta] RNAi significantly suppressed the growth rate of HNSCC cell lines, and overexpression of 14-3-3[zeta] in HaCaT immortalized human skin keratinocytes promotes its growth, as well as morphological changes. Reduced 14-3-3[zeta] levels increased the G1/G0-phase proportion, decreased the S-phase proportion and the rate of DNA synthesis. Based on this evidence, we suggest that 14-3-3[zeta] is a candidate proto-oncogene and deserves further investigations into its role in HNSCC carcinogenesis.

Most cutaneous T-cell lymphomas are derived from mature postthymic T cells of the CD4 subtype. When other less common profiles are encountered, a diagnostic challenge is posed. Accurate categorization is critical because of the specificity of therapeutic regimens, including biologics. A 65-year-old woman was seen in 2001 because of a thigh mass manifesting an unusual phenotype eventually categorized as a mature postthymic CD8+ T-cell lymphoma with CD10 and weak CD20 expression. Molecular studies revealed T-cell receptor and heavy chain immunoglobulin rearrangement. Her cutaneous disease progressed despite several cycles of chemotherapy and radiation therapy. However, a therapeutic trial with denileukin diftitox resulted in a striking response. The importance of this case lies in the novel phenotype and dual T- and B-cell rearrangements. Rather than representing an aberrant phenotype, this tumor may represent the malignant counterpart of a benign population of weakly CD20+ T cells of the CD8 subset.

Thyroid cancer is common, occurring in 1% of the general population. The relative frequencies of two of the most common subtypes of thyroid carcinoma, follicular (FTC) and papillary (PTC), vary depending on the regional prevalence of iodine deficiency. Although PTC has been more extensively studied, the etiology of sporadic FTC is poorly understood. To further elucidate this, we conducted microarray expression comparison of FTC tumors and normal thyroid tissue. Three commonly down-regulated genes, caveolin-1, caveolin-2, and GDF10/BMP3b, were chosen for further study on the basis of their localization to two chromosomal regions, 7q31.1 and 10q11.1, that commonly show loss of heterozygosity in FTC. Two additional genes, glypican-3 and a novel chordin-like gene, were also analyzed in view of their involvement in bone morphogenetic protein signaling and possible interaction with GDF10. Each of these five genes was down-regulated inor=15 of 19 FTC tumors (79%) by semiquantitative reverse transcription-PCR. Caveolin-1 showed preferential down-regulation of its beta-isoform at both the mRNA and protein level, suggesting a distinct function for this isoform. Caveolin-1 is of particular functional interest because it has been shown to interact with PTEN, the tumor suppressor gene mutated in Cowden syndrome, an inherited multiple hamartoma syndrome that includes predisposition to FTC. Immunohistochemical analysis of 141 thyroid tumors of various histological types showed significantly fewer caveolin-1-positive tumors in FTCs, including insular type tumors, and Hurthle cell carcinomas in comparison with normal thyroid. PTC and anaplastic thyroid carcinomas did not show significant down-regulation, and thus, caveolin-1 may become a useful molecular marker to differentiate the various histologies of thyroid malignancies.