Aggressive Metastatic Breast Cancer Research Articles

Chronic interferon-stimulated gene transcription promotes oncogene-induced breast cancer.

The MRE11 complex (comprising MRE11, RAD50, and NBS1) is integral to the maintenance of genome stability. We previously showed that a hypomorphic Mre11 mutant mouse strain (Mre11 ATLD1/ATLD1 ) was highly susceptible to oncogene-induced breast cancer. Here we used a mammary organoid system to examine which MRE11-dependent responses are tumor-suppressive. We found that Mre11 ATLD1/ATLD1 organoids exhibited an elevated interferon-stimulated gene (ISG) signature and sustained changes in chromatin accessibility. This Mre11 ATLD1/ATLD1 phenotype depended on DNA binding of a nuclear innate immune sensor, IFI205. Ablation of Ifi205 in Mre11 ATLD1/ATLD1 organoids restored baseline and oncogene-induced chromatin accessibility patterns to those observed in WT. Implantation of Mre11 ATLD1/ATLD1 organoids and activation of the oncogene led to aggressive metastatic breast cancer. This outcome was reversed in implanted Ifi205 -/- Mre11 ATLD1/ATLD1 organoids. These data reveal a connection between innate immune signaling and tumor development in the mammary epithelium. Given the abundance of aberrant DNA structures that arise in the context of genome instability syndromes, the data further suggest that cancer predisposition in those contexts may be partially attributable to chronic innate immune transcriptional programs.

Disintegrin-like Protein Strategy to Inhibit Aggressive Triple-Negative Breast Cancer.

Venoms are a rich source of bioactive compounds, and among them is leberagin-C (Leb-C), a disintegrin-like protein derived from the venom of Macrovipera lebetina transmediterrannea snakes. Leb-C has shown promising inhibitory effects on platelet aggregation. Previous studies have demonstrated that this SECD protein specifically targets α5β1, αvβ3, and αvβ6 integrins through a mimic mechanism of RGD disintegrins. In our current study, we focused on exploring the potential effects of Leb-C on metastatic breast cancer. Our findings revealed that Leb-C disrupted the adhesion, migration, and invasion capabilities of MDA-MB-231 breast cancer cells and its highly metastatic D3H2LN sub-population. Additionally, we observed significant suppression of adhesion, migration, and invasion of human umbilical vein endothelial cells (HUVECs). Furthermore, Leb-C demonstrated a strong inhibitory effect on fibroblast-growth-factor-2-induced proliferation of HUVEC. We conducted in vivo experiments using nude mice and found that treatment with 2 µM of Leb-C resulted in a remarkable 73% reduction in D3H2LN xenograft tumor size. Additionally, quantification of intratumor microvessels revealed a 50% reduction in tumor angiogenesis in xenograft after 21 days of twice-weekly treatment with 2 µM of Leb-C. Collectively, these findings suggest the potential utility of this disintegrin-like protein for inhibiting aggressive and resistant metastatic breast cancer.

A Case of an Aggressive Triple Negative Metastatic Metaplastic Breast Cancer

Introduction: We present a case of a 69 years old Bulgarian patient with a Triple Negative (TN) MBC. One year after the Breast-Conserving Surgery (BCS), adjuvant chemo- and radiotherapy she was diagnosed with metastases in the sigmoid colon, mesosigmoid, loco-regional lymph nodes and in the brain. Case report: The patient was initially diagnosed with locally advanced TNBC of the left breast in March 2021. She had undergone 6 courses of neoadjuvant chemotherapy and BCS with axillary lymph node dissection on 15.07.2021. The patient had received 4 courses of CarboTAx and whole breast radiation therapy postoperatively until December 2021. Eight months after that she was diagnosed with large bowel lesion in the sigmoid and a second small one in the mesosigmoid on the follow up PET/CT. She was admitted and had undergone laparoscopic left hemicolectomy. Due to signs of central system disorder (headache, impaired vision and vertigo) she was sent to additional examination with MRI (17.10.2022). Several heterogenous formations were found in the supratentorial space. Discussion: TNBC presents an aggressive subgroup, associated with worse prognosis and in about 30-40% of the cases early metastases and recurrence are observed. MBCs have poor response rates to cytotoxic chemotherapy compared to other types of BC. Conclusion: The presented case is here to show the unpredictable biological behaviour of this pathology regardless of the adequate treatment plan and careful follow up and to remind the physicians to be permanently aware of the numerous manifestations of the disease.

Targeted Two-Step Delivery of Oncotheranostic Nano-PLGA for HER2-Positive Tumor Imaging and Therapy In Vivo: Improved Effectiveness Compared to One-Step Strategy

Therapy for aggressive metastatic breast cancer remains a great challenge for modern biomedicine. Biocompatible polymer nanoparticles have been successfully used in clinic and are seen as a potential solution. Specifically, researchers are exploring the development of chemotherapeutic nanoagents targeting the membrane-associated receptors of cancer cells, such as HER2. However, there are no targeting nanomedications that have been approved for human cancer therapy. Novel strategies are being developed to alter the architecture of agents and optimize their systemic administration. Here, we describe a combination of these approaches, namely, the design of a targeted polymer nanocarrier and a method for its systemic delivery to the tumor site. Namely, PLGA nanocapsules loaded with a diagnostic dye, Nile Blue, and a chemotherapeutic compound, doxorubicin, are used for two-step targeted delivery using the concept of tumor pre-targeting through the barnase/barstar protein "bacterial superglue". The first pre-targeting component consists of an anti-HER2 scaffold protein, DARPin9_29 fused with barstar, Bs-DARPin9_29, and the second component comprises chemotherapeutic PLGA nanocapsules conjugated to barnase, PLGA-Bn. The efficacy of this system was evaluated in vivo. To this aim, we developed an immunocompetent BALB/c mouse tumor model with a stable expression of human HER2 oncomarkers to test the potential of two-step delivery of oncotheranostic nano-PLGA. In vitro and ex vivo studies confirmed HER2 receptor stable expression in the tumor, making it a feasible tool for HER2-targeted drug evaluation. We demonstrated that two-step delivery was more effective than one-step delivery for both imaging and tumor therapy: two-step delivery had higher imaging capabilities than one-step and a tumor growth inhibition of 94.9% in comparison to 68.4% for the one-step strategy. The barnase*barstar protein pair has been proven to possess excellent biocompatibility, as evidenced by the successful completion of biosafety tests assessing immunogenicity and hemotoxicity. This renders the protein pair a highly versatile tool for pre-targeting tumors with various molecular profiles, thereby enabling the development of personalized medicine.

Extracellular Matrix Glycation and Crosslinking in Mammary Tumor Progression.

Breast cancer progression is accompanied by profound extracellular matrix (ECM) remodeling. A greater abundance of aligned fibrillar collagen is characteristic of invasive and aggressive breast cancers and has been associated with elevated activity of collagen crosslinking enzymes, such as lysyl oxidase (LOX) and lysyl hydroxylases (LH) and the formation of more mature collagen matrix crosslinks. Aligned collagen fibers can facilitate metastatic dissemination of tumor cells, and LOX inhibitors have been used to inhibit tumor progression and metastasis in experimental models. Thus, a better understanding of how matrix crosslinking alters tumor cell phenotypes, and behaviors would improve our ability to effectively treat aggressive metastatic breast cancer. Herein described is an experimental approach to glycate and crosslink a collagen-I/basement membrane extract ECM to study the impact of ECM crosslinking on mammary tumor progression in vivo. Moreover, glycation of collagen by sugars to form advanced glycation end products naturally occurs during aging, extending the potential relevance of this approach to research on mechanisms of aging involved in disease progression.

Targeting primary and metastatic tumor growth in an aggressive breast cancer by engineered tryptophan auxotrophic Salmonella Typhimurium

The global cancer burden is growing and accounted for 10 million deaths in 2020. The resurgence of chemo- and radiation resistance have contributed to the treatment failures in many cancer types. Therefore, alternative strategies are desired for the effective cancer therapy. Bacteria-mediated cancer therapy presents an attarctive alternative option for the treatment and diagnosis of cancers. Herein, we describe an engineered Salmonella Typhimurium (ST) auxotrophic for tryptophan as a cancer therapeutic. The tryptophan auxotrophy was sufficient to render ST avirulent and highly safe to mice. The auxotroph recovered from the infected tumors had improved ability to target and colonize the tumors. We show that tryptophan auxotrophy reduced the fitness of ST in healthy tissues, but not in the tumors. We evaluated the auxotroph in highly aggressive metastatic 4T1 breast cancer model to inhibit primary tumor growth and lung metastases. The therapy greatly suppressed the primary growth with tumor-free survival of 40% mice. Importantly, therapy abolished the metastatic dissemination of tumor to lungs. Further, therapy markedly diminished the macrophage population in the tumors that may have contributed to the therapeutic benefit recorded. Collectively, results highlight the therapeutic efficacy of the tryptophan auxotrophic ST in an aggressive metastatic cancer model.

Gene Dosage Analysis on the Single-Cell Transcriptomes Linking Cotranslational Protein Targeting to Metastatic Triple-Negative Breast Cancer.

Many recent efforts have been put into the association between expression heterogeneity and different cell types and states using single-cell RNA transcriptome analysis. There is only limited understanding of gene dosage effects for the genetic heterogeneity at the single-cell level. By focusing on concordant copy number variation (CNV) and expression, we presented a computational framework to explore dosage effect for aggressive metastatic triple-negative breast cancer (TNBC) at the single-cell level. In practice, we collected CNV and single-cell expression data from the same patients with independent technologies. By focusing on 47,198 consistent copy number gains (CNG) and gene up-regulation from 1145 single cells, ribosome proteins with important roles in protein targeting were enriched. Independent validation in another metastatic TNBC dataset further prioritized signal recognition particle-dependent protein targeting as the top functional module. More interesting, the increased ribosome gene copies in TNBC may associate with their enhanced stemness and metastatic potential. Indeed, the prioritization of a well-upregulated functional module confirmed by high copy numbers at the single-cell level and contributing to patient survival may indicate the possibility of targeted therapy based on ribosome proteins for TNBC.

Cyto-KIK: A phase II trial of cytoreductive surgery in kidney cancer plus immunotherapy (nivolumab) and targeted kinase inhibition (cabozantinib).

TPS4598 Background: Despite recent therapeutic advancements in metastatic renal cell carcinoma (mRCC), only 5-10% of patients will achieve a complete response (CR) to therapy. Cytoreductive nephrectomy removes a large portion of the tumor which may be a source of immunosuppression driven by tumor cell-intrinsic factors in the tumor microenvironment. A pre-clinical orthotopic mouse model of aggressive metastatic triple negative breast cancer showed that neoadjuvant anti-PD-1 checkpoint inhibition generated enhanced and sustained antitumor immune responses with improved survival compared to adjuvant therapy (Liu J et al. Cancer Discov. 2016:1382). Clinical validation of improved outcomes with neoadjuvant compared to adjuvant immune checkpoint inhibitors has been demonstrated in trials for patients with non-small cell lung cancer, advanced melanoma, and recurrent glioblastoma (Forde, P.M., et al. N Engl J Med. 2018:1976; Amaria, R.N., et al Nat Med. 2018:1649; Cloughesy T.F., et al. Nat Med 2019:477). Recent data from a phase III trial in subjects with untreated mRCC, demonstrated the superiority of combination cabozantinib and nivolumab over sunitinib and established a new standard of care for mRCC (Choueiri T.K., et al. Annals of Onc, 2020;31 (suppl; abstr 6960). We hypothesize that if tumor specific immune responses to immunotherapy are greatest prior to nephrectomy, then treatment with nivolumab (nivo) and cabozantinib (cabo) prior to cytoreductive nephrectomy will lead to maximal peripheral and intra-tumoral specific immune responses and higher rates of CR during the course of treatment. Methods: This is an open label phase II, multicenter clinical trial of combination nivo and cabo prior to cytoreductive nephrectomy in patients with mRCC (NCT04322955). 48 treatment- naïve subjects with radiological or histological diagnosis of mRCC will be enrolled with the primary endpoint of CR rate according to RECIST version 1.1. Subjects will receive cabo (40mg) daily and nivo (480mg) every 4 weeks for 12 weeks prior to nephrectomy and a 3+3 design will be used to evaluate the safety of the interval (21 or 14 days) between the discontinuation of cabo and nephrectomy. Post-operatively, subjects will resume treatment with cabo and nivo until evidence of disease progression. Secondary endpoints include median size reduction of the primary tumor, response rate, PFS, OS, and surgical outcomes using the Clavien-Dindo classification system. Tissue based assays will quantify treatment related changes in the renal tumor microenvironment through polychromatic immunofluorescence, single cell RNA sequencing of the biopsy and nephrectomy specimen, and multiplex assessment of circulating serum cytokines. Dynamic contrast-enhanced MRI will be performed in a subset of subjects to assess radiologic correlates of response. The study is currently open to enrollment. Clinical trial information: NCT04322955.

Phase II trial of cytoreductive surgery in kidney cancer plus immunotherapy (nivolumab) and targeted kinase inhibition (cabozantinib) (Cyto-KIK).

TPS371 Background: Despite recent therapeutic advancements in metastatic renal cell carcinoma (mRCC), only 5-10% of patients will achieve a complete response (CR) to therapy. Cytoreductive nephrectomy removes a large portion of the tumor which may be a source of immunosuppression driven by tumor cell-intrinsic factors in the tumor microenvironment. A pre-clinical orthotopic mouse model of aggressive metastatic triple negative breast cancer showed that neoadjuvant anti-PD-1 checkpoint inhibition generated enhanced and sustained antitumor immune responses with improved survival compared to adjuvant therapy (Liu J et al. Cancer Discov. 2016:1382). Clinical validation of improved outcomes with neoadjuvant compared to adjuvant immune checkpoint inhibitors has been demonstrated in trials for patients with non-small cell lung cancer, advanced melanoma, and recurrent glioblastoma (Forde, P.M., et al. N Engl J Med. 2018:1976; Amaria, R.N., et al Nat Med. 2018:1649; Cloughesy T.F., et al. Nat Med 2019:477). Recent data from a phase III trial in subjects with untreated mRCC, demonstrated the superiority of combination cabozantinib and nivolumab over sunitinib and established a new standard of care for mRCC (Choueiri T.K., et al. Annals of Onc, 2020;31 (suppl; abstr 6960). We hypothesize that if tumor specific immune responses to immunotherapy are greatest prior to nephrectomy, then treatment with nivolumab (nivo) and cabozantinib (cabo) prior to cytoreductive nephrectomy will lead to maximal peripheral and intra-tumoral specific immune responses and higher rates of CR during the course of treatment. Methods: This is an open label phase II, multicenter clinical trial of combination nivo and cabo prior to cytoreductive nephrectomy in patients with mRCC (NCT04322955). 48 treatment- naïve subjects with radiological or histological diagnosis of mRCC will be enrolled with the primary endpoint of CR rate according to RECIST version 1.1. Subjects will receive cabo (40mg) daily and nivo (480mg) every 4 weeks for 12 weeks prior to nephrectomy and a 3+3 design will be used to evaluate the safety of the interval (21 or 14 days) between the discontinuation of cabo and nephrectomy. Post-operatively, subjects will resume treatment with cabo and nivo until evidence of disease progression. Secondary endpoints include median size reduction of the primary tumor, response rate, PFS, OS, and surgical outcomes using the Clavien-Dindo classification system. Tissue based assays will quantify treatment related changes in the renal tumor microenvironment through polychromatic immunofluorescence, single cell RNA sequencing of the biopsy and nephrectomy specimen, and multiplex assessment of circulating serum cytokines. Dynamic contrast-enhanced MRI will be performed in a subset of subjects to assess radiologic correlates of response. The study is currently open to enrollment. Clinical trial information: AAAS6927 .

Linking insurance claims across time to characterize treatment, monitoring, and end-of-life care in metastatic breast cancer.

7063 Background: Treatment and monitoring options for metastatic breast cancer (MBC) are increasing, but little is known about patterns or predictors of their use. Insurance claims are potentially informative, but tracking patient care timelines using episodic claims data has been cumbersome. Methods: We used an advanced cohort engine, implementing a temporal query language, to link IBM Marketscan claims over time for > 125 million US individuals from 2007-2014. To select the most common MBC subtype (ER+, HER2-), our criteria were: ≥2 MBC ICD codes, ≥1 year of follow-up, and ≥1 CPT or medication code for endocrine therapy and 0 for HER2-targeted therapy. We defined aggressive MBC as ≥1 ICD code for visceral or central nervous system metastasis < 1 year after the first MBC code. Geographic area was defined by 9 Census-Bureau designated regions. End-of-record was used as a surrogate for death in a Cox regression. We used multivariate logistic regression to determine correlation of factors, including disease aggressiveness and geography, with treatment, monitoring, and end-of-life events. Results: 7,335 women met criteria for ER+, HER2- MBC, with median age 59. Nearly half (46%) had aggressive disease, which correlated with shorter survival (hazard ratio (HR) 1.5 [1.4-1.6], P < 0.001). Treatment: first-line was endocrine therapy for 64% versus chemotherapy for 36%. Monitoring: 79% were imaged mostly by CT versus 21% by PET-CT, with median between-scan interval of 81 days; 63% received CA 15-3 serum tumor markers. End-of-life: 10% had a hospice code, of whom 19% had an ICU stay and 34% chemotherapy in the prior 3 months. Correlates of care: Disease aggressiveness correlated with first-line chemotherapy (odds ratio (OR) 2.0 [1.8-2.2], P < 0.001), PET/CT (OR 1.6 [1.4-1.8], P < 0.001), more frequent scans (OR 2.3 [2.1-2.6], P < 0.001), and chemotherapy < 3 months pre-hospice (OR 1.2 [1.1-1.4], P < 0.001), but not with CA 15-3 monitoring or ICU stay. Disease aggressiveness did not vary by region (χ2 P = 0.8), but region was significantly associated with treatment, monitoring, and end-of-life care (P < 0.001). Conclusions: Approximately two-thirds of ER+, HER2- MBC patients receive first-line endocrine therapy and are monitored with CA 15-3 serum tumor markers; 10% had evidence of hospice use, a likely underestimate due to differential follow-up. MBC care patterns vary by geography while disease aggressiveness does not, suggesting that care is not optimally tailored to individuals. These insights from claims data can inform quality improvement for MBC care.

Abstract 408: Expression of CCR5 associated with HER2 in circulating tumor cells (CTCs) is a novel biomarker for patients with metastatic breast cancer (MBC)

Abstract Introduction: The Chemokine C-C motif ligand 5 (CCL5) and its receptor 5 (CCR5) play a significant role in solid tumors, particularly triple negative breast cancer (TNBC) and HER2+ subtypes with prognostic implications. CCL5-CCR5 axis was reported to govern cancer stem cells expansion and play key role in MBC progression. Our group recently reported that overexpression of HER2 was associated with CTC-clusters which caused poor prognosis of patients with MBC. Herein, we first reported the expression of CCR5 in CTCs of MBC patients, and described the correlation between CCR5 and HER2 expression in CTCs. Methods: Whole blood samples (7.5ml/each) were collected from stage III/IV MBC patients before systemic therapy. CTCs enumeration was performed in FDA approved CELLTRACKS ANALYZERII® System (Menarini) by using CTC Kit contains antibodies targeting the Epithelial Cell Adhesion Molecule (EpCAM) antigen for capturing CTCs, and immunofluorescent staining reagents including anti-CK-PE (specific for epithelial cells), DAPI (for nucleus), anti-CD45-APC (specific for leukocytes), and anti-HER-2/neu-FLU. The CTCs were classified as CK+, EpCAM+, DAPI+ and CD45-. After confirming CTCs were positive by CellSearch system, CCR5 expression were evaluated by using CXC kit for multiple staining includes anti-CCR5-PE (R&D Systems), Anti-CK-FITC, DAPI and Anti-CD45-APC. The correlation between HER2 expression and CCR5 expression was analyzed by Kruskal-Wallis test was used for statistics. Results: CTCs were found positive (≥5) in all seven MBC patients with a range of numbers between 124 and 442, and HER expression was identified in 6 out 7 cases (between 57 and 149 total cells). The ratios of HER2+ CTCs/total CTCs were varied between 28.5% and 88.1%. Meanwhile, CCR5 expression were found in 3 out 7 patients (Group 1), with CCR5+ CTCs/ total CTCs ratios were 4.45%, 43.1% and 59.1% respectively. There were 4 patients without CCR5 expression in CTCs (Group 2). The average HER2+ CTCs was 127.6 with HER2+ CTC/Total CTC as average of 59.4% in Group 1, which were significantly higher in compared with the corresponding numbers as 80.3 and 29.97% respectively in Group 2, which indicated that upregulation of CCR5 was positively associated with high level of HER2 expression in CTCs. Conclusions: Our data provides the first evidence of strong expression of CCR5 in CTCs of MBC as potential new marker. The significant correlation between overexpression of HER2 in CTCs and high level of CCR5 indicated that CCR5 may contributes to more aggressive MBC subtypes with HER2 expression, which promotes carcinogenesis and metastasis at least partly by maintaining and increasing cancer stem cells leading to increased invasion. We conclude that the further understanding of the molecular interactions of CCR5 and HER2 in CTCs will be important to elucidate the mechanism of metastasis of MBC and predict prognosis. Citation Format: Qiang Zhang, Lorezo Gerratana, Ami N. Shah, Andrew A. Davis, Lisa Flaum, Youbin Zhang, Richard G. Pestell, Firas Wehbe, Amir Behdad, Leonidas Platanias, William Gradishar, Massimo Cristofanilli. Expression of CCR5 associated with HER2 in circulating tumor cells (CTCs) is a novel biomarker for patients with metastatic breast cancer (MBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 408.

Abstract P3-01-10: Associations between plasma Interleukin 2 (IL-2) and HER2 expression in circulating tumor cell (CTC) and MYC alterations in circulation tumor DNA (ctDNA) open a new insight on immune microenvironment for patients with metastatic breast cancer (MBC)

Abstract Introduction: Overexpression of HER2 has been reported to be associated with metastasis and poor prognosis of patients with MBC. We reported in AACR 2018 that HER2 overexpression is associated with CTC-cluster. Preclinical data suggested that MYC and HER2 cooperate to drive stem cell phenotype and poor prognosis in MBC (Nair R). Furthermore, IL-2 upregulates the transcription of MYC (Grigorieva I) and gets involved into its alterations. We reasoned that further understanding of interactions of HER2 in CTC and MYC will be important to elucidate the mechanism of metastasis of MBC. Herein, we report a significant correlation between the plasma IL-2 level and HER2 expression in CTCs, and the IL-2 related MYC ctDNA alterations in MBC. Methods: This study enrolled 43patients with stage III/IV BCa at the Northwestern Memorial Hospital (2016-2017) that had longitudinally detection of CTCs and ctDNA. Whole blood samples (7.5ml/each) were collected for CTCs enumeration by using CELLTRACKS ANALYZERII® System (Menarini) contains antibodies of anti-EpCAM for capturing CTCs, anti-CK-PE for epithelial cells, DAPI for nucleus, anti-CD45-APC for leukocytes and anti-HER-2/neu-FLU. The CTCs were classified based on phenotype as CK+, EpCAM+, DAPI+ and CD45-.Plasma ctDNA was analyzed using the Guardant360 TM NGS-based assay (Guardant Health), a 73 genes panel. ELISA for IL-2 was performed by using patients' plasma. Database of IL-2, HER2, CTCs and ctDNA was linked with clinical database and analyzed by Kruskal-Wallis test. Results: CTCs ≥ 5 were found in 20 patients (46%). There were 15 patients that had HER2 negative CTCs (Group 1), and 5 patients had HER2 positive (Group 2) CTCs. The level of IL-2 was much higher in Group 1 (88.17pg) compared to Group 2 (66.81pg), indicating that patients with HER2 positive CTCs have significant lower IL-2 than patients with negative CTCs (P=0.02). Meanwhile, ctDNA MYC alterations were detected in 10 patients (including 1 L114R mutation, 7 CNV and 2 SNV) who have the average IL-2 level as 94.00pg. There were 11 patients without any alterations of MYC had average IL-2 level of 70.17pg, which indicated that patients with alterations in the ctDNA MYC have significant higher level of IL-2 in compared with patients without MYC alterations (P=0.02). Conclusions: Findings of the correlation between overexpression of HER2 in CTCs and low IL-2 level indicated that low immunity may contribute to more aggressive MBC. And the higher level of IL-2 appear associated predominantly with MYC genomic alterations indicated that overexpression of MYC may also stimulate the immune response by upregulating IL-2 via a reverse feedback pathway. We postulated that increasing IL-2 suppresses the HER2 expression in CTC and breaks cooperation between HER2 and MYC. Although the interactions between them still unknown, our results suggest that IL-2 related immune microenvironment acts as a key player to suppress HER2- and MYC-mediated progress in MBC, including the formation of CTC-cluster. Monitoring and administration of IL-2 may benefit pretreated MBC patients and predict disease metastasis. Citation Format: Zhang Q, Gerratana L, Zhang Y, Flaum L, Shah A, Davis A, Behdad A, Gradishar W, Platanias L, Cristofanilli M. Associations between plasma Interleukin 2 (IL-2) and HER2 expression in circulating tumor cell (CTC) and MYC alterations in circulation tumor DNA (ctDNA) open a new insight on immune microenvironment for patients with metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-10.

Abstract P5-05-01: Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive aggressive metastatic breast cancer

Abstract Background: Metabolic rewiring is one of the central hallmarks of cancer progression and survival to support anabolic and energetic demands. Tumor cells constantly alter their metabolic state in response to oncogenic stimuli, nutrient availability, and interaction with immune cells however the precise regulation that precedes the metabolic alteration is poorly understood. Here we report a direct interaction of glycolytic enzyme PFKFB4 with transcriptional coregulator SRC-3. PFKFB4 functions as a critical regulator of Warburg effect and our study reveals that upon glucose stimulation PFKFB4 activates SRC-3 driving an invasive-metastatic breast cancer. Methods: Molecular experiments were performed to understand the transcriptional activation of SRC-3 by PFKFB4 enzyme. Chromatin immunoprecipitation and gene expression studies were performed to investigate the functions of PFKFB4/SRC-3 crosstalk on transcriptional regulation. Metabolomics and isotope tracing studies were performed to identify the metabolic adaptations regulated by PFKFB4/SRC-3 in breast tumors. PFKFB4-knockout was established using CRISPR-Cas9 system and functional studies were carried out to define its role in tumor cell proliferation, invasion-migration, and breast to lung metastasis. Human breast tumor samples were evaluated to identify the clinical importance of PFKFB4/SRC-3 crosstalk in patients. Results:Molecular studies revealed that PFKFB4 enzyme phosphorylates SRC-3 at serine 857 (S857) enhancing its transcriptional activity, whereas either suppression of PFKFB4 or ectopic expression of a phosphorylation-deficient SRC-3 mutant S857A (SRC-3S857A) significantly abolished SRC-3-mediated transcriptional output (p<0.000001). Functionally, PFKFB4-driven SRC-3 activation drives glucose flux towards the pentose phosphate pathway enabling purine synthesis by transcriptionally upregulating the expression of enzyme transketolase (TKT). Deletion of PFKFB4 by CRISPR-Cas9 system resulted in significantly reduced proliferation (p<0.05) and migration-invasion (p<0.001) compared to wildtype breast tumor cells. Ablation of SRC-3 or PFKFB4 suppressed in vivo breast tumor growth and prevents metastasis to the lung from an orthotopic setting (p<0.0001). PFKFB4 and phosphorylated SRC-3 levels are significantly increased in breast tumors (p=0.02), whereas, in patients with the basal subtype, PFKFB4 and SRC-3 drive a common protein signature that correlates with the poor survival of TNBC patients (p=0.03). Conclusion:Our data suggest that the Warburg pathway enzyme PFKFB4 acts as a molecular fulcrum that couples sugar metabolism to transcriptional activation by stimulating SRC-3 to promote aggressive metastatic tumors. It also provides first evidence how Warburg pathway drives aggressive breast tumorigenesis by directly activating powerful oncogene SRC-3. Our work suggests that targeting the PFKFB4–SRC-3 axis may be therapeutically valuable in breast tumors that are notably dependent on glucose metabolism. (This work is funded by grants from Susan G. Komen and NCI to S.D.) Citation Format: Dasgupta S, Anand V, John H, Sawant Dessai A, Katsuta E, Takabe K, O'Malley B. Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive aggressive metastatic breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-05-01.

Phase II, Multicenter, Single-arm Trial of Eribulin as First-line Therapy for Patients With Aggressive Taxane-pretreated HER2-Negative Metastatic Breast Cancer: The MERIBEL Study

BackgroundEribulin has efficacy in patients with progression after ≥ 1 chemotherapeutic regimen for metastatic breast cancer (MBC). A short disease-free interval (DFI) and previous use of taxanes in the neoadjuvant or adjuvant setting have been associated with worse outcomes for patients receiving first-line chemotherapy for HER2-negative MBC. The aim of the present trial was to evaluate the efficacy and safety of eribulin as first-line therapy for patients with HER2-negative MBC with these poor prognostic factors. Patients and MethodsEribulin monotherapy was administered until disease progression or unacceptable toxicity. The principal selection criteria were HER2 negativity without previous chemotherapy for MBC, the previous use of taxanes for early-stage breast cancer, and a DFI of < 36 months (subsequently amended to 48 months). The primary endpoint was the investigator-assessed time to progression. The secondary endpoints included overall survival, progression-free survival, objective response rate, clinical benefit rate, duration of response, and toxicity profile. A total of 53 patients were enrolled and received ≥ 1 dose of eribulin. ResultsThe median patient age was 47 years (range, 23-82.8 years). The median DFI was 15.7 months (range, 0.1-46.4 months). The median investigator-assessed time to progression was 4.1 months (range, 0.2-27.8 months; 95% confidence interval, 3.2-6.2 months). The objective response and clinical benefit rate was 20.8% and 26.4%, respectively. All-grade and grade 3/4 adverse events developed in 96.2% and 69.8% of patients, respectively. The most common treatment-related adverse events were neutropenia, leukopenia, alopecia, nausea, and anemia. ConclusionEribulin is effective and safe as first-line therapy for aggressive taxane-pretreated HER2-negative MBC.

Predictive tools for bevacizumab therapy in patients with aggressive HER 2-negative metastatic breast cancer: 2-years results from an observational study.

12043Background: The determination of monitoring Circulating Tumor Cells (CTC) in patients (pts) with metastatic breast cancer (MBC) is a well-established prognostic marker of efficacy/tolerance. W...

Combinational siRNA delivery using hyaluronic acid modified amphiphilic polyplexes against cell cycle and phosphatase proteins to inhibit growth and migration of triple-negative breast cancer cells.

The manuscript investigated the efficacy of a tailored polymeric siRNA delivery system formulation as well as combinational siRNA therapy in metastatic breast cancer cells to inhibit malignant cell growth and migration. The siRNA delivery was undertaken by non-viral means with PEI/HA. We identified six phosphatases that could be critical targets to inhibit migration of highly aggressive metastatic breast cancer cells. We further report on specifically targeting cell cycle and phosphatase proteins to decrease both malignant cell growth and migration simultaneously. Clinical gene therapy against metastatic breast cancer with effective and safe delivery systems is urgently needed to realize the potential of molecular medicine in this deadly disease and our studies in this manuscript is intended to facilitate this endeavor.

Abstract P3-05-01: Increased glycolysis directly activates transcriptional coactivator SRC-3 to promote aggressive metastatic breast cancer

Abstract Reprogramming of metabolic pathways is one of the central hallmarks of cancer cell growth and survival to support anabolic and energetic demands. Rapidly proliferating tumor cells utilize increased amounts of glucose for energy generation and macromolecule biosynthesis, yet it is still poorly understood how increased glucose-flux accentuates aggressive metastatic tumor. Understanding the molecular and genetic regulators of these metabolic-programs is critical to discover new drug targets for cancer therapy. Here, we report that the oncogenic transcriptional coregulator steroid receptor coactivator-3 (SRC-3) is a critical sensor of increased tumor glycolysis and re-programs transcriptional responses to support breast cancer growth and metastasis. From an unbiased kinome-wide RNAi library screen, we identified the glycolytic enzyme PFKFB4 as a direct stimulator of SRC-3 activity. PFKFB4 is a bifunctional metabolic enzyme that contains both 'kinase' and 'phosphatase' domain, yet the functional role of these regulatory domains in tumor metabolism is not clear. Glucose stimulation promotes PFKFB4-dependent phosphorylation of SRC-3 (pSRC-3) which then enhances transcriptional coactivation by this coregulator. Mechanistic studies revealed that SRC-3 and PFKFB4 mutually cooperate to maintain high levels of purine biosynthesis by transcriptionally upregulating rate limiting enzymes transketolase (TKT), adenosine monophosphate deaminase-1 (AMPD1) and xanthine dehydrogenase (XDH). Knockdown of PFKFB4 or SRC-3 significantly decreased the expression of these enzymes resulting in reduced proliferation of breast tumor cells, and this functional deficiency could be rescued by exogenous addition of purines. Chromatin immunoprecipitation (ChIP) studies identified increased occupancy of pSRC-3 on target gene promoters, which overlapped with activating transcription factor 4 (ATF4) binding sites in the proximal promoter region. Immunoprecipitation of ATF4 identified increased interaction with pSRC-3 in glycolytic breast tumor cells, whereas knockdown of PFKFB4 or expression of a phospho-deficient SRC-3 mutant significantly reduced the SRC-3/ATF4 association. These findings suggest that upon enhanced glycolysis, PFKFB4 induced activation of SRC-3 by phosphorylation promotes expression of purine biosynthetic genes primarily by coactivating ATF4. Importantly, ablation of either SRC-3 or PFKFB4 suppresses in vivo breast tumor growth and metastasis, and these tumorigenic-effects cannot be simulated by a phospho-deficient SRC-3 mutant suggesting the PFKFB4-SRC-3 axis may be therapeutically exploited for treating breast cancer patients. [S.D. is supported by Susan G. Komen Award PDF14300468]. Citation Format: Dasgupta S, Zhu B, Nikolai BC, Putluri N, Foulds CE, Tsai SY, Tsai M-J, O'Malley BW. Increased glycolysis directly activates transcriptional coactivator SRC-3 to promote aggressive metastatic breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-05-01.

Irreversible inhibition of Δ16HER2 is necessary to suppress Δ16HER2-positive breast carcinomas resistant to Lapatinib

HER2 tyrosine kinase receptor is a validated target in breast cancer therapy. However, increasing evidence points to a major role of Δ16HER2 splice variant commonly coexpressed with HER2 and identified as a clinically important HER2 molecular alteration promoting aggressive metastatic breast cancer. Consistently, mice transgenic for the human Δ16HER2 isoform (Δ16HER2 mice) develop invasive mammary carcinomas with early onset and 100% penetrance. The present study provides preclinical evidence that Δ16HER2 expression confers de novo resistance to standard anti-HER2-therapies such as Lapatinib and acquired resistance to the selective Src inhibitor Saracatinib in breast cancer. Of note, Dacomitinib, an irreversible small molecule pan-HER inhibitor, was able to completely suppress Δ16HER2-driven breast carcinogenesis. Thus, only Dacomitinib may offer benefit in this molecularly defined patient subset by irreversibly inhibiting Δ16HER2 activation.

Capecitabine in elderly patients with metastatic breast cancer.

Capecitabine is the reference treatment for anthracycline- and/or taxane-pretreated metastatic breast cancer (MBC). This study examined its efficacy, tolerability and impact on the quality of life of elderly patients with MBC. Between January 2002 and December 2009, 75 consecutive elderly patients with MBC received first-line chemotherapy with capecitabine 1000 mg/m2 twice daily for 14 days every 3 weeks. Endpoints were efficacy, tolerability and clinical-benefit response measured every 3 cycles. Median age was 76 years (range 65-88); median ECOG performance status was 1 (range 0-2); 51 patients (68%) had received adjuvant chemotherapy and all patients had received hormonal therapy. Median exposure was 6 cycles. After 3 cycles, 11 patients (14.7%) had a partial response, one patient experienced a complete response, and 49 patients (65.3%) had stable disease, amounting to a disease control rate of 81.3%. Stable disease was maintained in 45 patients (60%) after 6 cycles, in 21 patients (28%) after 9 cycles, and in 13 patients (17.3%) after 12 cycles. A clinical-benefit response was experienced by 42 patients (56%), indicating a positive impact on quality of life. Treatment was well tolerated, the most common grade 3 events being diarrhea (12%) hand-foot syndrome (8%), and mucositis (8%). Adverse events were managed with dose adjustments and supportive therapy when required. Our results indicate that capecitabine is active and well tolerated in elderly patients with MBC. This dosing regimen warrants further study in the first-line setting for patients with less aggressive MBC who are not candidates for combination therapy.

Features of aggressive breast cancer

BackgroundAggressive breast cancer is a term commonly used in literature to describe breast cancer with a poor prognosis. Identifying and understanding the factors associated with aggressiveness could be helpful to the management of patients with breast cancer. Breast cancer is a heterogeneous disease, both clinically and biologically, which may be responsible for the wide range of survival durations for patients with metastatic disease. AimThe goal of this study was to identify the factors most often described in association with aggressive metastatic breast cancer (MBC). MethodsA systematic review was performed by querying PubMed from January 1, 2012 to June 1, 2014 for “metastatic breast cancer” (“aggressive” or “poor prognosis” or “high risk”). The level of evidence to support each potential prognostic factor of aggressive MBC was also reviewed. ResultsThe identified factors were grouped into 3 principle categories: clinical, biological, and patient related. Because patient-related factors may not be indicative of inherent cancer aggressiveness, this review focused only on clinical and biological factors. The factors with the highest levels of evidence to support associations with survival in metastatic breast cancer were visceral metastases, number of metastatic sites, disease-free interval, presence of CTCs, triple-negative disease, and tumour grade. ConclusionIdentification of these factors and understanding their contribution to the aggressiveness of MBC and disease progression may lead to more personalized treatment in this patient population.