Tamoxifen is one of the most widely used anticancer drugs in the world. It is a safe drug with generally well-tolerated side effects and has been prescribed for the treatment of early-stage and advanced-stage or metastatic estrogen receptor α (ERα/ESR1)-positive breast cancer. Tamoxifen therapy also provides a 38% reduction of the risk of developing breast cancer in women at high risk. With the advent of newer medications targeting ERα-positive breast cancer, tamoxifen is now mainly used as adjuvant therapy for lower-risk premenopausal breast cancer and cancer prevention. It is widely accepted that tamoxifen as a selective estrogen receptor modulator exerts its therapeutic effect by competitively binding to ERα, leading to the recruitment of corepressors and inhibition of transcription of genes involved in the proliferation of breast cancer epithelium. As such, expression of ERα in breast tumors has been considered necessary for tumors to be responsive to tamoxifen therapy. However, ERα-independent effects of tamoxifen in various in vitro and in vivo contexts have been reported over the years. Importantly, the recent discovery that ERα and estrogen receptor β (ERβ/ESR2) can bind tumor suppressor protein p53 with functional consequences has provided new insights into the mechanisms underlying response to tamoxifen therapy and resistance. Furthermore, these findings have paved the way for broadening the use of tamoxifen by potentially repurposing it to treat triple negative (negative for ERα, human epidermal growth factor receptor 2, and progesterone receptor) breast cancer. Herein, we summarize these developments and discuss their mechanistic underpinnings and clinical implications.

Triple negative breast cancer (TNBC) is an aggressive invasive cancer with fewer treatment options and worse prognosis. The current care of TNBC consists of chemotherapy regimens such as anthracyclines, and taxanes with an evolving role for immunotherapy, antibody-drug conjugates, and drugs targeting DNA damage and repair. However, these regimens are not very effective in majority of patients and have serious adverse effects. Therefore, there is an unmet need for rationally designed therapies. Although the importance of p53 and estrogen receptors in breast cancer is known for years, much less is known about functional integration between p53, estrogen receptors, and p73 signaling in TNBC. We have discovered a novel estrogen receptor beta (ERβ)-p53-p73 axis that can be exploited to develop new therapeutic strategies against TNBC. Binding and inactivating tumor suppressor p73 is a major gain-of-oncogenic function of mutant p53. We used MDA-MB468 and isogenic TNBC cell line (MDA-MB231-LM) models differing in ERβ and p53 expression status, cell line-derived xenografts (CDXs), and patient tumor-derived xenografts (PDXs) to analyze functional interactions between these proteins and their impact on cell proliferation, cell death, migration, invasion, and therapeutic response. Various experimental approaches including knocking down and overexpressing genes, Co-IP, chromatin immunoprecipitation (ChIP), ChIP-Seq, RNA-seq, quantitative real time PCR, reverse phase protein array (RPPA), proximity ligation assay (PLA), immunofluorescence, immunohistochemistry, flow cytometry, and PK/PD and synergy analysis were used for comprehensive mechanistic studies and for determining translational opportunities. Conventionally, Tam has not been used for TNBC therapy. We show that tamoxifen (Tam) synergizes with doxorubicin (Doxo) to increase apoptosis and decrease cell proliferation of TNBC cells in vitro, and to inhibit growth of TNBC CDX and PDX models in vivo. These therapeutic responses are dependent on binding of ERβ to mutant p53. Mechanistically, Tam enhances ERβ-mediated sequestration of p73-bound mutant p53, thereby disrupting p73-mutant p53 complex resulting in reactivation of p73, leading to apoptosis and inhibition of proliferation in synergy with Doxo that induces DNA damage. Importantly, RNA-seq analysis showed Tam and Doxo combination targeted unique set of genes regulating pathways including metastasis and DNA damage repair (DDR). An IRB-approved phase II clinical trial is set to start to test this therapy in patients. As large percentage of TNBCs express both mutant p53 and ERβ, successful translation of our novel findings on repurposing Tam will have huge clinical impact with an effective and safe therapy without financial toxicity to the patients. Gokul M. Das, Chetan C. Oturkar, Junhoung Park, Christina Adams, Iqbal Aijaz, Utpal K. Upadhyay, Melissa Dolan, Michalis Mastri, Alexander Caradori, Masanori Oshi, Yoshihisa Tokumaru, Yalii Zhang, Jianmin Wang, Brent Boleslav, Donald E. Mager, Tagari Samanta, Sujkin Yang, Seung Y. Han, Richa Mishra, John Ebos, Kazuaki Takabe, Kristopher Attwood, Benny A. Kaipparettu. Synergistic effect of tamoxifen and doxorubicin on a novel estrogen receptorβ-p53-p73 axis: A new therapeutic strategy against triple negative breast cancer cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB005.

Tissue-resident macrophages (TRMs) are abundant immune cells within pre-metastatic sites, yet their functional contributions to metastasis remain incompletely understood. Here, we show that alveolar macrophages (AMs), the main TRMs of the lung, are susceptible to downregulation of the immune stimulatory transcription factor IRF8, impairing anti-metastatic activity in models of metastatic breast cancer. G-CSF is a key tumor-associated factor (TAF) that acts upon AMs to reduce IRF8 levels and facilitate metastasis. Translational relevance of IRF8 downregulation was observed among macrophage precursors in breast cancer and a CD68hiIRF8loG-CSFhi gene signature suggests poorer prognosis in triple-negative breast cancer (TNBC), a G-CSF-expressing subtype. Our data highlight the underappreciated, pro-metastatic roles of AMs in response to G-CSF and identify the contribution of IRF8-deficient AMs to metastatic burden. AMs are an attractive target of local neoadjuvant G-CSF blockade to recover anti-metastatic activity.

The canonical mechanism behind tamoxifen's therapeutic effect on estrogen receptor α/ESR1+ breast cancers is inhibition of ESR1-dependent estrogen signaling. Although ESR1+ tumors expressing wild-type p53 were reported to be more responsive to tamoxifen (Tam) therapy, p53 has not been factored into choice of this therapy and the mechanism underlying the role of p53 in Tam response remains unclear. In a window-of-opportunity trial on patients with newly diagnosed stage I-III ESR1+/HER2/wild-type p53 breast cancer who were randomized to arms with or without Tam prior to surgery, we reveal that the ESR1-p53 interaction in tumors was inhibited by Tam. This resulted in functional reactivation of p53 leading to transcriptional reprogramming that favors tumor-suppressive signaling, as well as downregulation of oncogenic pathways. These findings illustrating the convergence of ESR1 and p53 signaling during Tam therapy enrich mechanistic understanding of the impact of p53 on the response to Tam therapy.

Natural killer (NK) cells are cytotoxic lymphocytes that accumulate within the tumor microenvironment and are generally considered to be antitumorigenic. Using single-cell RNA sequencing and functional analysis of multiple triple-negative breast cancer (TNBC) and basal tumor samples, we observed a unique subcluster of Socs3 high CD11b − CD27 − immature NK cells that were present only in TNBC samples. These tumor-infiltrating NK cells expressed a reduced cytotoxic granzyme signature and, in mice, were responsible for activating cancer stem cells through Wnt signaling. NK cell–mediated activation of these cancer stem cells subsequently enhanced tumor progression in mice, whereas depletion of NK cells or Wnt ligand secretion from NK cells by LGK-974 decreased tumor progression. In addition, NK cell depletion or inhibition of their function improved anti–programmed cell death ligand 1 (PD-L1) antibody or chemotherapy response in mice with TNBC. Furthermore, tumor samples from patients with TNBC and non-TNBC revealed that increased numbers of CD56 bright NK cells were present in TNBC tumors and were correlated to poor overall survival in patients with TNBC. Together, our findings identify a population of protumorigenic NK cells that may be exploited for both diagnostic and therapeutic strategies to improve outcomes for patients with TNBC.

INTRODUCTION:Chronic hepatitis C infection can result in insulin resistance (IR). We have previously shown that it occurs through the interaction of pathways for glucose homeostasis, insulin signaling, and autophagy. But it is not known how soon the pathways are activated and how IR is related to the signals generated by catabolic and anabolic conditions occurring in infected cells. We have extended our studies to a cell culture system mimicking acute infection and to downstream pathways involving energy-sensor AMPK and nutrient-sensor mTOR that are active in catabolic and anabolic processes within the infected cells.METHODS:Huh7 liver cells in culture were infected with hepatitis C virus (HCV). We performed proteomics analysis of key proteins in infected cells by Western blotting and IP experiments, with or without IFNα exposure as a component of conventional therapeutic strategy.RESULTS:We present evidence that (a) IRS-1 Ser312, Beclin-1, protein conjugate Atg12-Atg5 or GS Ser641 are up-regulated early in infection presumably by activating the same pathways as utilized for persistent infection; (b) Bcl-XL, an inhibitor of both autophagy and apoptosis, is present in a core complex with IRS-1 Ser312 and Beclin-1 during progression of IR; (c) AMPK level remains about the same in infected cells where it is activated by phosphorylation at Thr172 concomitant with increased autophagy, a hallmark of catabolic conditions; (d) an mTOR level that promotes anabolism is increased rather than decreased under an expanded autophagy; (e) hypophosphorylation of translational repressor 4E-BP1 downstream of mTOR is suggestive of reduced protein synthesis; and (f) β-catenin, is up-regulated but not phosphorylated suggesting indirectly our previous contention that its kinase, GSK-3β, is mostly in an inactive state.CONCLUSION:We report that in the development of IR following chronic infection, anabolic and catabolic pathways are activated early, and the metabolic interaction occurs possibly in a core complex with IRS-1 Ser312, Beclin-1, and autophagy inhibitor Bcl-XL. Induction of autophagy is usually controlled by a two-edged mechanism acting in opposition under anabolic and catabolic conditions by AMPK/mTOR/4E-BP1 pathway with GSK-3β-mediated feedback loops. However, we have observed an up-regulation of mTOR along with an up-regulation of AMPK caused by HCV infection is a deviation from the normal scenario described above which might be of therapeutic interest.

The absence of effective therapeutic targets and aggressive nature of triple-negative breast cancer (TNBC) renders this disease subset difficult to treat. Although estrogen receptor beta (ERβ) is expressed in TNBC, studies on its functional role have yielded inconsistent results. However, recently, our preclinical studies, along with other observations, have shown the potential therapeutic utility of ERβ in the context of mutant p53 expression. The current case study examines the efficacy of the selective estrogen receptor modulator tamoxifen in p53-mutant TNBC with brain metastases. Significant increase in ERβ protein expression and anti-proliferative interaction between mutant p53 and ERβ were observed after cessation of tamoxifen therapy, with significant regression of brain metastases. This case study provides supporting evidence for the use of tamoxifen in p53-mutant, ERβ+TNBC, especially in the setting of brain metastasis.

Supplementary Methods, Figure Legend from Estrogen Receptor α Inhibits p53-Mediated Transcriptional Repression: Implications for the Regulation of Apoptosis

Supplementary Figure 1 from Estrogen Receptor α Inhibits p53-Mediated Transcriptional Repression: Implications for the Regulation of Apoptosis

Abstract Breast cancer (BC) is one of the most commonly diagnosed cancers worldwide and the most common in women in the United States. The majority of the BC are estrogen receptor alpha positive (ER+) and are likely to respond to endocrine therapy. Tamoxifen and fulvestrant are the most widely used hormonal treatments for ER+ BC. However, nearly half of patients receiving endocrine therapy suffer from the risk of recurrence due to either intrinsic or acquired resistance. Thus, it is vital to understand the mechanisms of resistance to endocrine therapy in ER+ BC. Metabolic reprogramming is one of the significant hallmarks of cancer and it is now known that many tumors utilize mitochondrial oxidative phosphorylation (OXPHOS) for their energy needs. Our lab previously reported that metastatic triple-negative breast cancer (TNBC), which does not express ER, has a high energy dependency on mitochondrial fatty acid beta-oxidation (FAO). In this project, we developed a gene signature by integrating the transcriptomic, metabolomic, and lipidomic data from FAO rate-limiting gene (CPT1) modulated TNBC cells. Our signature represented the FAO-regulated gene set relevant to metabolic or lipidomic changes. We discovered a significant reliance on this TNBC signature in endocrine-resistant ER+ BC. Clinical data suggests that our gene signature predicts the survival of endocrine-treated ER+ BC patients. Though increased fatty acid consumption is reported in tamoxifen-resistant cells, the role of FAO in endocrine treatment resistance is largely unclear. We then analyzed the metabolic reprogramming in ER+ BC cell lines after tamoxifen or fulvestrant therapy. Molecular, genetic, and metabolic analyses suggested that endocrine therapy-induce AMPK-FAO-OXPHOS signal activation in ER+ BC cells. Furthermore, the knockdown of CPT1, the rate-limiting enzyme of FAO, or treatment with FAO inhibitors significantly enhanced the response to endocrine therapies. We have also reported that FAO induces the autophosphorylation of the c-Src proto-oncogene in TNBC tumors. Thus, we analyzed this phenomenon in the endocrine-resistant ER+ BC cells. As expected, endocrine therapy-induced FAO activated the Src pathway in ER+ BC also. Moreover, in vitro, and in vivo studies confirmed that endocrine-resistant ER+ BC cells have increased sensitivity to FAO, OXPHOS, and Src inhibitors. Finally, analysis of clinical data suggests that low expression of FAO rate-limiting genes in the ER+ primary tumors have better recurrence-free and distant metastasis-free survival after endocrine therapy. Overall, our findings suggest that the gene signature generated from FAO-modulated TNBC cells predicts response to endocrine therapy in ER+ BC. Moreover, metabolic reprogramming in endocrine-resistant ER+ tumors induce FAO, OXPHOS, and Src pathways, providing potential targets to overcome endocrine-resistance ER+ BC patients. Citation Format: Songyeon Ahn, Junhyoung Park, Sandra L. Grimm, Badrajee WB Piyarathna, Nagireddy Putluri, Gokul Das, Cristian Coarfa, Benny A. Kaipparettu. Metabolomic rewiring in endocrine therapy resistant estrogen receptor positive breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4852.

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.

Triple-negative breast cancer (TNBC) is an aggressive subtype without any effective targeted therapies and rapidly become resistant to generic chemotherapy. Therefore, there is an urgent need to develop new therapeutic strategies. Estrogen receptor beta (ER beta) levels are high in about 60-70% of TNBCs. Recent reports including the Cancer Genome Atlas (TCGA) show that about 80% of TNBC express mutant p53 and it is the most predominant driver in these cancers. We have previously reported (JNCI, 2019, 111:1202-1215) that ER-beta binds p53 and exerts proliferative versus anti-proliferative/tumor suppressive functions depending on the wild type and mutant p53 status in TNBC, respectively. In the current work we used multiple approaches such as immunoprecipitation, in situ proximity ligation assay (PLA), and gene expression analysis by quantitative real-time PCR (qRT-PCR) to show that tamoxifen (Tam) increases ER beta-p53 interaction resulting in decrease of mutant p53 binding to p73 leading to cell cycle arrest, increased apoptosis, decreased proliferation, and increased expression of anti-proliferation genes. Importantly, ER beta antagonist PHTPP decreases the ER beta-p53 interaction in TNBC cells, whereas ER beta agonist DPN did not have any effect. Importantly, Tam synergized with doxorubicin (Dox) to decrease the IC50 of Dox more than 3-fold. This synergism was absent in an isogenic cell line where TP53 gene was knocked out. The fact that mutant p53 expression was necessary for Tam to synergize with Dox, along with our observation that upregulation of anti-proliferation gene expression was dependent on both ER beta and p73, strongly suggests that ER beta-mutant p53-p73 axis is the target of the novel effect of Tam. RNA-seq and reverse phase protein array (RPPA) analysis of isogenic TNBC cells differing in p53 mutational status without and with ER beta depletion revealed important cellular pathways impacted by the synergistic effect of Tam plus Dox combination treatment. To test the effect of Tam plus Dox combination therapy in vivo, we used isogenic MDA-MB-231 cell line-derived xenograft (CDX) and TNBC patient-derived xenograft (PDX) tumors. Consistent with our observations in the TNBC cell models, combination therapy inhibited progression of both CDX and PDX tumors more effectively compared to monotherapies. Furthermore, the antitumor effect was dependent on expression of mutant p53 in tumors. Our study has revealed a novel ER beta-mutant p53-p73 axis that could be targeted by Tam in combination with chemotherapy, raising the possibility of repurposing Tam to treat molecularly stratified TNBC that expresses both ERβ and mutant p53. Besides the potential for relatively faster entry of a safe and less expensive therapy to the clinic, our discovery can be exploited to reduce toxic adverse effects by reducing the dose of Dox in treatment regimens. Citation Format: Gokul M. Das, Chetan C. Oturkar, Christina Adams, Jung H. Park, Melissa Dolan, Michalis Mastri, Manasori Oshi, Yoshihisa Tokumaru, Utpal K. Mukhopadhyay, Kalyani Abha, Kwang H. Jung, Sukjin Yang, Suna Kim, John Ebos, Kazuaki Takabe, Benny A. Kaipparettu. Combination of tamoxifen and doxorubicin targets estrogen receptor beta-mutant p53-p73 axis: A novel therapeutic strategy for triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3986.

Metabolic reprogramming is associated with myeloid-derived suppressor cell (MDSC) immunosuppressive function. Here, we outline the process for acquiring MDSCs from human and murine sources for subsequent analysis of fatty acid oxidation, oxidative phosphorylation, and glycolysis using the Seahorse XFe 96 Analyzer. Murine MDSCs can be isolated directly from tumor-bearing mice or derived through IL-6 and GM-CSF culture of bone marrow cells from non-tumor-bearing mice. To generate human MDSCs, peripheral blood mononuclear cells (PBMCs) can be cultured with IL-6 and GM-CSF. For complete details on the use and execution of this protocol, please refer to Mohammadpour et al. (2021).

High grade serous ovarian cancer (HGSOC) is the most common and lethal subtype of epithelial ovarian cancer. Prevalence (~96%) of mutant p53 is a hallmark of HGSOC. Estrogen receptor-beta (ERβ) has been reported to be another important player in HGSOC, although the pro-versus anti-tumorigenic role of its different isoforms remains unsettled. However, whether there is functional interaction between ERβ and mutant p53 in HGSOC is unknown. ERβ1 and ERβ2 mRNA and protein analysis in HGSOC cell lines demonstrated that ERβ2 is the predominant isoform in HGSOC. Specificity of ERβ2 antibody was ascertained using cells depleted of ERβ2 and ERβ1 separately with isoform-specific siRNAs. ERβ2-mutant p53 interaction in cell lines was confirmed by co-immunoprecipitation and in situ proximity ligation assay (PLA). Expression levels of ERβ2, ERα, p53, and FOXM1 proteins and ERβ2-mutant p53 interaction in patient tumors were determined by immunohistochemistry (IHC) and PLA, respectively. ERβ2 levels correlate positively with FOXM1 levels and negatively with progression-free survival (PFS) and overall survival (OS). Quantitative chromatin immunoprecipitation (qChIP) and mRNA expression analysis revealed that ERβ2 and mutant p53 co-dependently regulated FOXM1 gene transcription. The combination of ERβ2-specific siRNA and PRIMA-1MET that converts mutant p53 to wild type conformation increased apoptosis. Our work provides the first evidence for a novel ERβ2-mutant p53-FOXM1 axis that can be exploited for new therapeutic strategies against HGSOC.

Luminal breast cancer (LBC) driven by dysregulated estrogen receptor-alpha (ERα) signaling accounts for 70% of the breast cancer cases diagnosed. Although endocrine therapy (ET) is effective against LBC, about one-third of these patients fail to respond to therapy owing to acquired or inherent resistance mechanisms. Aberrant signaling via ERα, oncogenes, growth factor receptors, and mutations in tumor suppressors such as p53 impinge on downstream regulators such as AMPK and mTOR. While both AMPK and mTOR have been reported to play important roles in determining sensitivity of LBC to ET, how the ERα-p53 crosstalk impinges on regulation of AMPK and mTOR, thereby influencing therapeutic efficacy remains unknown. Here, we have addressed this important issue using isogenic breast cancer cell lines, siRNA-mediated RNA knockdown, and different modes of drug treatments. Interaction of p53 with ERα and AMPK was determined by in situ proximity ligation assay (PLA), and endogenous gene transcripts were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Further, the effect of concurrent and sequential administration of Fulvestrant–Everolimus combination on colony formation was determined. The studies showed that in cells expressing wild type p53, as well as in cells devoid of p53, ERα represses AMPK, whereas in cells harboring mutant p53, repression of AMPK is sustained even in the absence of ERα. AMPK is a major negative regulator of mTOR, and to our knowledge, this is the first study on the contribution of AMPK-dependent regulation of mTOR by ERα. Furthermore, the studies revealed that independent of the p53 mutation status, combination of Fulvestrant and Everolimus may be a viable first line therapeutic strategy for potentially delaying resistance of ERα+/HER2− LBC to ET.

c-Src (Src) is a proto-oncogene involved in signaling that culminates in the control of multiple biological functions. Src is also one of the most frequently upregulated pathways in triple negative breast cancer (TNBC). Dysregulation of Src has been detected in TNBC and is strongly associated with tumor metastasis and poor prognosis. However, even after promising preclinical studies, Src inhibitors did not show major clinical advantage in unselected TNBC populations. We have previously published that metastatic TNBC has high energy-dependency to mitochondrial fatty acid beta-oxidation (FAO) and FAO activates Src by inducing autophosphorylation at Y419. However, our recent analysis suggests that as observed with the Src inhibitors, TNBC tumors treated with FAO inhibitors also develop drug-resistance and exhibit continuous tumor growth. Evaluation of their drug resistance mechanism revealed that while short-term inhibition of FAO or Src induces autophagic and apoptotic cell deaths, long-term inhibition results in autophagy-mediated drug resistance and survival. Further analyses suggest that FAO/Src inhibitors promote interferon regulatory factor 1 (IRF1) expression and activate mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway via the induction of cellular reactive oxygen species (ROS) in TNBC. Activated MEK/ERK then suppresses IRF1 expression and induces survival pathways for drug resistance and tumor survival. Validation of in vitro findings using in vivo TNBC models confirmed that combination of FAO/Src inhibitors with MEK/ERK inhibitor or ROS scavenger provide significant benefit to overcome the therapeutic resistance of TNBC. These findings open-up new therapeutic opportunities to manage TNBC patients with currently non-targetable metastatic tumors. Citation Format: Kwanghwa Jung, Junhyoung Park, Tirupataiah Sirupangi, Dongya Jia, Nishant Gandhi, Shivanand Pudakalakatti, Jessica Elswood, Weston Porter, Nagireddy Putluri, Xiang H.-F Zhang, Xi Chen, Pratip K. Bhattacharya, Chad J. Creighton, Michael T. Lewis, Jeffrey M. Rosen, Lee-Jun C. Wong, Gokul M. Das, C. Kent Osborne, Mothaffar F Rimawi, Benny Abraham Kaipparettu. Autophagy-mediated survival mechanism to c-Src inhibitor therapy in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-06-12.

Abstract That endocrine therapy or HER2 targeted therapies are not effective against TNBC makes it the most difficult breast cancer subtype to treat. Although these tumors initially respond to chemotherapy, there is high risk of relapse. Therefore, there is urgent need to develop new therapeutic strategies against these aggressive cancers. Reports including the Cancer Genome Atlas (TCGA) show that about 80% of TNBC express mutant p53 and it is a major driver of these cancers. Furthermore, 60-80% of TNBC express estrogen receptor-beta (ERβ). We have shown that tumor suppressor protein p53 status (wild type versus mutant) is a determinant of pro-versus anti-tumorigenic roles of ERβin breast cancer (JNCI, 2019, 111:1202-1215). These observations, along with our novel discovery that Tamoxifen (Tam) increases interaction between ERβ and mutant p53 leading to decreased binding of mutant p53 to tumor suppressor p73 resulting in reactivation of p73 in TNBC cells, led us to hypothesize that Tam can be repurposed to treat TNBC stratified based on combined expression of mutant p53 and ERβ. Experimental procedures to test this hypothesis include use of cell culture, TNBC cell line-derived xenograft (CDX), and patient tumor-derived organoid (PDO) models, proximity ligation assay (PLA), co-immunoprecipitation (Co-IP), quantitative chromatin immunoprecipitation (qChIP), quantitative real time PCR (qPCR), RNAi-mediated knockdown, gene knockout with CRISPR, RNA-seq, and bioinformatics data analysis. Our data show that Tam when combined with the widely used chemotherapeutic agent doxorubicin (Doxo) reduces several fold the IC50 of Doxo in TNBC cells. Importantly, Tam was unable to decrease the IC50 of Doxo in isogenic TNBC cells where mutant p53 was knocked out demonstrating that the effect of Tam mediated by ERβ and p73 is directed against the oncogenic gain-of-functions of mutant p53. Consistent with this, the combination treatment elicited robust antitumor effect on CDX tumors in vivo. The drug combination also increases apoptosis as a consequence of restored p73 activity. Conversely, PHTPP (an ERβ-specific antagonist) disrupts the ERβ-mutant p53 interaction enabling mutant p53 to bind and inactivate p73. Our finding that Tam in combination with Doxo activates p73 leading to robust apoptotic response and inhibition of tumor growth suggest that Tam, a drug that is well tolerated with relatively less side effects, can be repurposed in combination with Doxo to treat TNBCs expressing mutant p53 and ERβ and this approach can reach the clinic relatively fast as both the drugs are FDA-approved and have been in the clinic for several years to treat other forms of cancer. Furthermore, based on our data that Tam decreases the IC50 of Doxo considerably, there is potential for using Doxo at a dose much lower than what is currently used in the management of TNBC, thereby reducing major side effects of Doxo. Citation Format: Chetan C. Oturkar, Christina Adams, Utpal K. Mukhopadhyay, Alexander Caradori, Manasori Oshi, Yoshihisa Tokumaru, Chad J. Creighton, Jun H. Park, Nishant Gandhi, Kazuaki Takabe, Kristopher M. Attwood, Benny A. Kaipparettu, Gokul M. Das. Drug repurposing to treat triple negative breast cancer (TNBC) based on a novel ER beta-p53-p73 signaling axis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5213.

Cervical cancer is well known to cause metastasis in liver. Not usually they present in a pattern of acute hepatitis with the liver enzymes trending above the range of thousands. Here we present an interesting case of a patient who presented as acute hepatitis in the setting of metastatic cervical cancer to the liver. She presented with elevated liver enzymes above thousands. Her imaging studies revealed new metastatic lesions in the liver. She was subsequently started on chemotherapy but unfortunately, two months later developed septic shock and expired. To our knowledge this is the only case describing a malignancy with metastasis to the liver presenting as an acute hepatitis picture. All the other cases that have been described in the literature presented with acute liver failure.