SUMO post-translational modification of proteins or SUMOylation ensures normal cell function. Disruption of SUMO dynamics prompts various pathophysiological conditions, including cancer. The burden of deSUMOylating the large SUMO-proteome rests on 6 full-length mammalian SUMO-proteases or SENP. While multiple SENP isoforms exist, the function of these isoforms remains undefined. We now delineate the biological role of a novel SENP7 isoform SENP7S in mammary epithelial cells. SENP7S is the predominant SENP transcript in human mammary epithelia but is significantly reduced in precancerous ductal carcinoma in situ and all breast cancer subtypes. Like other SENP family members, SENP7S has SUMO isopeptidase activity but unlike full-length SENP7L, SENP7S is localized in the cytosol. In vivo, SUMOylated β-catenin and Axin1 are both SENP7S-substrates. With knockdown of SENP7S in mammary epithelial cells, Axin1-β-catenin interaction is lost and β-catenin escapes ubiquitylation-dependent proteasomal degradation. SUMOylated β-catenin accumulates at the chromatin and activates multiple oncogenes. Hence, non-tumorigenic MCF10-2A cells with reduced SENP7S exhibit greater cell proliferation and anchorage-dependent growth. SENP7S depletion directly potentiates tumorigenic properties of MCF10-2A cells with induction of anchorage-independent growth and self-renewal in 3D-spheroid conditions. Collectively, the results identify SENP7S as a novel mediator of β-catenin signaling and normal mammary epithelial cell physiology.

Background Hormone receptor positive (HR+) breast cancer (BCa) is the most frequently diagnosed subtype. Acquired and intrinsic resistance to conventional endocrine therapy (ET) commonly occurs and prompts incurable metastatic disease. Hence, ET-resistant (ET-R) HR+ BCa presents a therapeutic challenge. Previous studies show elevated androgen receptor (AR) that supports resistance to ET tamoxifen and correlates with HR+ BCa metastasis. Yet surprisingly, studies with AR-blocker enzalutamide (Enz) in ET-R HR+ BCa present conflicting results. We now report that a constitutively active, unique from canonical Enz-targeted, AR accumulates in endocrine resistant HR+ BCa cells. Methods AR protein profiles in acquired and intrinsic ET-R HR + -BCa were defined with cell-free modification tests, in-house in-vivo SUMOylation assays, and PLA imaging. Genomic activity of native AR and modified-AR mimetic was tested with reporter assays and limited transcriptome analysis. Spheroid growth and migration studies were used to evaluate inhibitory actions of Enz and combinatorial therapy. Results Sustained higher molecular weight SUMO-modified AR (SUMO-AR) persists in acquired and intrinsic ET-R BCa cell lines. Concurrently, SUMO isoforms and global SUMO-modified proteome also accumulates in the same cell lines. We identified AR as a novel substrate for the SUMO-E3 ligase HSPB1/Hsp27. Independent of ligand, SUMO-AR is resilient to ubiquitin-mediated proteasomal degradation, enriched in the nucleus, readily chromatin-bound, and transcriptionally active. Constitutive SUMO-AR initiates a gene-expression profile that favors epithelial-mesenchymal transition. Enz combined with a SUMO inhibitor attenuates migration and metastatic phenotype of ET-R HR+ BCa. Conclusion Targeting both unmodified and SUMO-modified AR prevents the metastatic progression of HR+ BCa with ET-R.

Epigenetic reprogramming allows cancer cells to bypass normal checkpoints and potentiate aberrant proliferation. Several chromatin regulators are subject to reversible SUMO-modification but little is known about how SUMOylation of chromatin-remodelers modulates the cancer epigenome. Recently, we demonstrated that SUMO-protease SENP7L is upregulated in aggressive BCa and maintains hypoSUMOylated heterochromatin protein 1-α (HP1α). Canonical models define HP1α as a “reader” of repressive H3K9m3 marks that supports constitutive heterochromatin. It is unclear how SUMOylation affects HP1α function in BCa cells. This report shows HP1α SUMO-dynamics are closely regulated in a complex with SENP7L and SUMO-E3 Polycomb-2 (PC2/CBX4). This complex accumulates at H3K9m3 sites, hypoSUMOylates HP1α and PC2, and reduces PC2's SUMO-E3 activity. HyperSUMO conditions cause complex dissociation, SUMOylation of PC2 and HP1α, and recruitment of SUMOylated HP1α to multiple DNA-repair genes including Rad51C. SUMOylated HP1α's enrichment at euchromatin requires chromatin-bound non-coding RNA (ncRNA), reduces Rad51C protein, and increases DNA-breaks in BCa cells. Hence, HP1α SUMOylation and consistently low SENP7L increase efficacy of DNA-damaging chemotherapeutic agents. BCa patients on chemotherapy that express low SENP7L exhibit greater survival rates than patients with high SENP7L. Collectively, these studies suggest that SUMOylated HP1α is a critical epigenetic-regulator of DNA-repair in BCa that could define chemotherapy responsiveness.

Preclinical model systems are essential research tools that help us understand the biology of invasive lobular carcinoma of the breast (ILC). The number of well-established ILC models are increasing but remain limited. Lower incidence of ILC, under-representation of ILC patients in clinical trials, and intrinsic ILC tumor characteristics all contribute to this challenge. Hence, there is significant need to continually develop better model systems to recapitulate the essential characteristics of ILC biology, genetics, and histology, and empower preclinical therapeutic studies to be translated back into the clinic. In this Perspective, we highlight recent advances in in vivo experimental models, which recapitulate key features of ILC biology and disease progression and potentially reshape the future of ILC translational research. We assert that all existing in vitro and in vivo ILC preclinical models have their strengths and weaknesses, and that it is necessary to bridge key deficiencies in each model context as we move forward with ILC research. Thus, unlocking the mysteries of ILC will be best achieved by choosing the right combination of preclinical model systems.

In HR+ (hormone receptor positive) breast cancer, endocrine therapies that directly antagonize estrogen receptor or reduce synthesis of its ligand can be highly effective, but endocrine-resistance occurs frequently. Metabolic dysregulation is a mechanistic driver of endocrine-resistant HR+ breast cancer, but most existing methods cannot be multiplexed with assays for biomarker expression. Spectral phasor imaging of multiplex immunofluorescence (mIF) staining for expression of hormone receptors and other biomarkers combined with phasor lifetime analysis of NADH to quantify metabolic state will provide integrated spatial maps of metabolism and biomarker expression in endocrine-resistant HR breast cancer. We used the HCI-013 (013) and HCI-013EI (013EI) patient-derived xenograft (PDX) system, derived from a HR+, invasive lobular metastatic breast cancer which harbors the Y537S ESR1 mutation. Tumors were stained with a multiplex immunofluorescence (mIF) panel for pan-cytokeratin, ER, PR, HER2, and Ki67, detected by spectrally separate OPAL dyes. Hyperspectral phasor fluorescence spectra were obtained using successive single photon excitation at 488 nm, 561 nm and 640 nm and using multiphoton excitation at 1024 nm. Single photon excitation excites specific dyes, and multiphoton excites the entire panel of dyes simultaneously. The emission was collected using a Zeiss 880 laser scanning microscope equipped with a 32 channel spectral detector. Phasor coordinates of each component were resolved into fractional intensities of the five genes. In the same tissues and sections, phasor fluorescence lifetime imaging (FLIM) of NADH was used to quantify metabolic heterogeneity. NADH is present in two forms: free and protein-bound NADH. They have different fluorescence lifetimes and the ratio of these two species are correlated with NAD+/NADH ratio. Free NADH is associated with increased glycolysis. We observed greater metabolic heterogeneity in 013EI PDX samples, with similar levels of free NADH (glycolysis) at the tumor border, but significantly increased bound NADH (oxidative phosphorylation) in the tumor core. Spectral phasor data show differential expression of PR and PanCK (for 640 nm excitation), and HER2 and ERalpha (for 561 nm excitation) in the edge and center of cancer in 013 and 013EI variants. Our results show how a combination of spectral phasor and phasor-FLIM can be used to define metabolic changes in endocrine-resistant, HR+ PDX models and correlate them with biomarker expression. Ongoing work is quantitatively mapping these measurements at single-pixel resolution, and applying this imaging workflow to RNAscope in situ hybridization assays for gene expression. Combining multiphoton spectral (RNAscope) and FLIM (NADH) will allow pixelwise correlation of the gene expression and metabolic changes. Citation Format: Belen Torrado, Alexander Vallmitjana, Ayodeji Olukoya, Shaymaa Bahnassy, Hillary Stires, Aaron Rozeboom, Rebecca B. Riggins, Suman Ranjit. Integrated lifetime and spectral phasor imaging of biomarker expression and metabolism in hormone receptor positive breast cancer models [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 2461.