Young women’s breast cancer (YWBC) has poor prognosis and known interactions with parity. Women diagnosed within 5–10 years of childbirth, defined as postpartum breast cancer (PPBC), have poorer prognosis compared to age, stage, and biologic subtype-matched nulliparous patients. Genomic differences that explain this poor prognosis remain unknown. In this study, using RNA expression data from clinically matched estrogen receptor positive (ER+) cases (n = 16), we observe that ER+ YWBC can be differentiated based on a postpartum or nulliparous diagnosis. The gene expression signatures of PPBC are consistent with increased cell cycle, T-cell activation and reduced estrogen receptor and TP53 signaling. When applied to a large YWBC cohort, these signatures for ER+ PPBC associate with significantly reduced 15-year survival rates in high compared to low expressing cases. Cumulatively these results provide evidence that PPBC is a unique entity within YWBC with poor prognostic phenotypes.

During pregnancy, the rodent liver undergoes hepatocyte proliferation and increases in size, followed by weaning-induced involution via hepatocyte cell death and stromal remodeling, creating a prometastatic niche. These data suggest a mechanism for increased liver metastasis in breast cancer patients with recent childbirth. It is unknown whether the human liver changes in size and function during pregnancy and weaning. In this study, abdominal imaging was obtained in healthy women at early and late pregnancy and postwean. During pregnancy time points, glucose production and utilization and circulating bile acids were measured. Independently of weight gain, most women’s livers increased in size with pregnancy, then returned to baseline postwean. Putative roles for bile acids in liver growth and regression were observed. Together, the data support the hypothesis that the human liver is regulated by reproductive state with growth during pregnancy and volume loss postwean. These findings have implications for sex-specific liver diseases and for breast cancer outcomes.

In rodents, we identified a physiologic process within the normal liver that creates a pre-metastatic niche. This physiology is weaning-induced liver involution, characterized by hepatocyte cell death, immune influx, and extracellular matrix remodeling. Here, using weaning-induced liver involution as a model of a physiologically regulated pro-metastatic niche, we investigate how liver involution supports breast cancer metastasis. Liver metastases were induced in BALB/c immune competent hosts by portal vein injection of D2OR (low metastatic) or D2A1 (high metastatic) mouse mammary tumor cells. Tumor incidence and multiplicity increased in involution hosts with no evidence of a proliferation advantage. D2OR tumor cell extravasation, seeding, and early survival were not enhanced in the involuting group compared to the nulliparous group. Rather, the involution metastatic advantage was observed at 14 days post tumor cell injection. This metastatic advantage associated with induction of immune tolerance in the involution host liver, reproductive state dependent intra-tumoral immune composition, and CD8-dependent suppression of metastases in nulliparous hosts. Our findings suggest that the normal postpartum liver is in an immune suppressed state, which can provide a pro-metastatic advantage to circulating breast cancer cells. Potential relevance to women is suggested as a postpartum diagnosis of breast cancer is an independent predictor of liver metastasis.

BACKGROUND: During pregnancy, the rodent liver undergoes hepatocyte proliferation and increases in size, followed by weaning-induced involution via hepatocyte cell death and stromal remodeling, creating a pro-metastatic niche. These data suggest a mechanism for increased liver metastasis in postpartum breast cancer patients. OBJECTIVES: Investigate if the human liver changes in size and function during pregnancy and weaning. METHODS: Abdominal imaging was obtained in healthy women at early and late pregnancy, and post-wean. During pregnancy time points, endogenous glucose production was measured and fasting blood taken to measure bile acids. RESULTS: Independent of weight gain, most women's livers increased in size with pregnancy, returning to baseline post-wean. Putative roles for bile acids in liver growth were observed in pregnant women and rodents. CONCLUSIONS: The human liver is regulated by reproductive state with growth during pregnancy and volume loss post-wean. These findings may have broad implications for sexspecific liver diseases and cancer.

Background: Postpartum breast cancer (PPBC), variably defined as breast cancer diagnosed within 5-10 years after a patient’s last pregnancy, has overall increased risk of metastasis even after controlling for patient age, year of diagnosis, and tumor subtype and stage. Metastasis to the liver is specifically increased in PPBC compared to non-PPBC cases and likely contributes to the overall poor prognosis of PPBC. Our lab has previously shown that the rodent liver undergoes physiological remodeling after pregnancy, in a process reminiscent of weaning-induced mammary gland involution. Further, rodent models confirm that breast cancer cells delivered to the involuting liver grow more robustly than tumor cells delivered to nulliparous liver. Our rodent data suggest that weaning induced liver involution may be responsible for increased tumor cell seeding and ultimately higher rates of liver metastasis observed in women diagnosed with PPBC. Since weaning-induced liver involution is a recently recognized physiology, little is known of its molecular underpinnings. Here we employ RNA-seq of murine livers to better understand how reproductive status influences liver physiology with a focus on exploring the pro-metastatic niche formed after weaning. Study design: Livers were isolated from age-matched mice at the following reproductive time points: nulliparous (never pregnant), lactation, involution days 2, 4, 6, and 8, and fully regressed (4 weeks post-wean) and flash frozen for RNA isolation (n=3-6 mice per group). RNA-seq was utilized to identify gene expression differences between reproductive groups. We employed single-sample gene set enrichment analysis (ssGESA) to identify key pathways modulated by reproductive state. Results: We identify clear reproductive control of liver physiology. We find increases in proliferation, MTORC1, and anabolic metabolism pathways during lactation when compared to any other reproductive state. In the transition from lactation to involution, we find increases in apoptotic pathways, and a switch to catabolic metabolism. Correspondingly, during involution we see enrichment in pro-cancer pathways including increased TGFβ signaling, Epithelial to Mesenchymal Transition, and Inflammatory Responses Hallmark Pathways. Within the immune signatures changes of involution, we observe myeloid-derived cell signatures including enrichment in M2 associated genes, and increases in cytotoxic and helper T cell signatures that parallels a signature for disease-specific T cell exhaustion. Conclusion: This work highlights novel regulation of liver physiology by reproductive state, and finds pathways regulated during the natural physiological process of involution that have been previously identified as contributing to metastatic niches. This study has the potential to identify new targets for the prevention and treatment of breast cancer liver metastasis. Citation Format: Michelle K. Ozaki, Alexandra Q. Bartlett, Zheng Xia, Pepper Schedin. RNA-seq analysis of murine liver to identify breast cancer metastatic potential during liver involution [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 983.