Mouse intraductal modeling of primary ductal carcinoma in situ.

Mouse intraductal modeling enables efficient in vivo propagation of pre-invasive breast cancer lesions and provides a suitable micro-environment for creating patient-derived tumor xenograft models of estrogen-receptor-positive breast cancer. Here, we present a protocol for mouse intraductal modeling of primary ductal carcinoma in situ (DCIS). We describe steps for processing primary DCIS tissues and performing intraductal injections. We then detail procedures for processing intraductal lesions for 3D whole-mount imaging or serial transplantation using magnetic bead sorting. For complete details on the use and execution of this protocol, please refer to Hutten et al. (2023).1.

Transcriptome analysis reveals differences in cell cycle, growth and migration related genes that distinguish fibroblasts derived from pre-invasive and invasive breast cancer.

Background/Introduction: As the most common form of pre-invasive breast cancer, ductal carcinoma in situ (DCIS) affects over 50,000 women in the US annually. Despite standardized treatment involving lumpectomy and radiation therapy, up to 25% of patients with DCIS experience disease recurrence often with invasive ductal carcinoma (IDC), indicating that a subset of patients may be under-treated. As most DCIS cases will not progress to invasion, many patients may experience over-treatment. By understanding the underlying processes associated with DCIS to IDC progression, we can identify new biomarkers to determine which DCIS cases may become invasive and improve treatment for patients. Accumulation of fibroblasts in IDC is associated with disease progression and reduced survival. While fibroblasts have been detected in DCIS, little is understood about their role in DCIS progression. Goals: We sought to determine 1) whether DCIS fibroblasts were similar or distinct from normal and IDC fibroblasts at the transcriptome level, and 2) the contributions of DCIS fibroblasts to breast cancer progression. Methods: Fibroblasts underwent transcriptome profiling and pathway analysis. Significant DCIS fibroblast-associated genes were further analyzed in existing breast cancer mRNA databases and through tissue array immunostaining. Using the sub-renal capsule graft model, fibroblasts from normal breast, DCIS and IDC tissues were co-transplanted with DCIS.com breast cancer cells. Results: Through transcriptome profiling, we found that DCIS fibroblasts were characterized by unique alterations in cell cycle and motility related genes such as PKMYT1, TGF-α, SFRP1 and SFRP2, which predicted increased cell growth and invasion by Ingenuity Pathway Analysis. Immunostaining analysis revealed corresponding increases in expression of stromal derived PKMYT1, TGF-α and corresponding decreases in expression of SFRP1 and SFRP2 in DCIS and IDC tissues. Grafting studies in mice revealed that DCIS fibroblasts enhanced breast cancer growth and invasion associated with arginase-1+ cell recruitment. Conclusion: DCIS fibroblasts are phenotypically distinct from normal breast and IDC fibroblasts, and play an important role in breast cancer growth, invasion, and recruitment of myeloid cells. These studies provide novel insight into the role of DCIS fibroblasts in breast cancer progression and identify some key biomarkers associated with DCIS progression to IDC, with important clinical implications.

A living biobank of patient-derived ductal carcinoma in situ mouse-intraductal xenografts identifies risk factors for invasive progression.

Ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive breast cancer (IBC). Due to a lack of biomarkers able to distinguish high- from low-risk cases, DCIS is treated similar to early IBC even though the minority of untreated cases eventually become invasive. Here, we characterized 115 patient-derived mouse-intraductal (MIND) DCIS models reflecting the full spectrum of DCIS observed in patients. Utilizing the possibility to follow the natural progression of DCIS combined with omics and imaging data, we reveal multiple prognostic factors for high-risk DCIS including high grade, HER2 amplification, expansive 3D growth, and high burden of copy number aberrations. In addition, sequential transplantation of xenografts showed minimal phenotypic and genotypic changes over time, indicating that invasive behavior is an intrinsic phenotype of DCIS and supporting a multiclonal evolution model. Moreover, this study provides a collection of 19 distributable DCIS-MIND models spanning all molecular subtypes.

Human Ductal Carcinoma In Situ: Advances and Future Perspectives.

Due to widespread adoption of screening mammography, there has been a significant increase in new diagnoses of ductal carcinoma in situ (DCIS). However, DCIS outcomes remain unclear. A large fraction of human DCIS (>50%) may not need the multimodality treatment options currently offered to all DCIS patients. More importantly, while we may be overtreating many, we cannot identify those most at risk of invasion or metastasis following a DCIS diagnosis. This review summarizes the studies that have furthered our understanding of DCIS pathology and mechanisms of invasive progression by using advanced technologies including spatial genomics, transcriptomics, and multiplex proteomics. This review also highlights a need for rethinking DCIS with a more focused view on epithelial states and programs and their cross talk with the microenvironment.

Short-term PI3K Inhibition Prevents Breast Cancer in Preclinical Models.

Antiestrogen medication is the only chemoprevention currently available for women at a high risk of developing breast cancer; however, antiestrogen therapy requires years to achieve efficacy and has adverse side effects. Therefore, it is important to develop an efficacious chemoprevention strategy that requires only a short course of treatment. PIK3CA is commonly activated in breast atypical hyperplasia, the known precancerous precursor of breast cancer. Targeting PI3K signaling in these precancerous lesions may offer a new strategy for chemoprevention. Here, we first established a mouse model that mimics the progression from precancerous lesions to breast cancer. Next, we demonstrated that a short-course prophylactic treatment with the clinically approved PI3K inhibitor alpelisib slowed early lesion expansion and prevented cancer formation in this model. Furthermore, we showed that alpelisib suppressed ex vivo expansion of patient-derived atypical hyperplasia. Together, these data indicate that the progression of precancerous breast lesions heavily depends on the PI3K signaling, and that prophylactic targeting of PI3K activity can prevent breast cancer. PREVENTION RELEVANCE: PI3K protein is abnormally high in breast precancerous lesions. This preclinical study demonstrates that the FDA-approved anti-PI3K inhibitor alpelisib can prevent breast cancer and thus warrant future clinical trials in high-risk women.

Towards the development of DCIS risk prediction models.

Ductal carcinoma in situ (DCIS) has a variable natural history, with a poorly understood biology. In this issue of Cancer Cell, Strand et. al. use two well-annotated DCIS repositories to identify an 812-gene classifier that associates with high risk of DCIS recurrence and define novel DCIS subtypes that informs future studies.

Elevated NRAS expression during DCIS is a potential driver for progression to basal-like properties and local invasiveness.

BACKGROUND: Ductal carcinoma in situ (DCIS) is the most common type of in situ premalignant breast cancers. What drives DCIS to invasive breast cancer is unclear. Basal-like invasive breast cancers are aggressive. We have previously shown that NRAS is highly expressed selectively in basal-like subtypes of invasive breast cancers and can promote their growth and progression. In this study, we investigated whether NRAS expression at the DCIS stage can control transition from luminal DCIS to basal-like invasive breast cancers. METHODS: Wilcoxon rank-sum test was performed to assess expression of NRAS in DCIS compared to invasive breast tumors in patients. NRAS mRNA levels were also determined by fluorescence in situ hybridization in patient tumor microarrays (TMAs) with concurrent normal, DCIS, and invasive breast cancer, and association of NRAS mRNA levels with DCIS and invasive breast cancer was assessed by paired Wilcoxon signed-rank test. Pearson's correlation was calculated between NRAS mRNA levels and basal biomarkers in the TMAs, as well as in patient datasets. RNA-seq data were generated in cell lines, and unsupervised hierarchical clustering was performed after combining with RNA-seq data from a previously published patient cohort. RESULTS: Invasive breast cancers showed higher NRAS mRNA levels compared to DCIS samples. These NRAShigh lesions were also enriched with basal-like features, such as basal gene expression signatures, lower ER, and higher p53 protein and Ki67 levels. We have shown previously that NRAS drives aggressive features in DCIS-like and basal-like SUM102PT cells. Here, we found that NRAS-silencing induced a shift to a luminal gene expression pattern. Conversely, NRAS overexpression in the luminal DCIS SUM225 cells induced a basal-like gene expression pattern, as well as an epithelial-to-mesenchymal transition signature. Furthermore, these cells formed disorganized mammospheres containing cell masses with an apparent reduction in adhesion. CONCLUSIONS: These data suggest that elevated NRAS levels in DCIS are not only a marker but can also control the emergence of basal-like features leading to more aggressive tumor activity, thus supporting the therapeutic hypothesis that targeting NRAS and/or downstream pathways may block disease progression for a subset of DCIS patients with high NRAS.

NR2F1 Is a Barrier to Dissemination of Early-Stage Breast Cancer Cells.

Cancer cells can disseminate during very early and sometimes asymptomatic stages of tumor progression. Though biological barriers to tumorigenesis have been identified and characterized, the mechanisms that limit early dissemination remain largely unknown. We report here that the orphan nuclear receptor nuclear receptor subfamily 2, group F, member 1 (NR2F1)/COUP-TF1 serves as a barrier to early dissemination. NR2F1 expression was decreased in patient ductal carcinoma in situ (DCIS) samples. High-resolution intravital imaging of HER2+ early-stage cancer cells revealed that loss of function of NR2F1 increased in vivo dissemination and was accompanied by decreased E-cadherin expression, activation of wingless-type MMTV integration site family, member 1 (WNT)-dependent ß-catenin signaling, disorganized laminin 5 deposition, and increased expression of epithelial-mesenchymal transition (EMT) genes such as twist basic helix-loop-helix transcription factor 1 (TWIST1), zinc finger E-box binding homeobox 1 (ZEB1), and paired related homeobox 1 (PRRX1). Furthermore, downregulation of NR2F1 promoted a hybrid luminal/basal phenotype. NR2F1 expression was positively regulated by p38α signaling and repressed by HER2 and WNT4 pathways. Finally, early cancer cells with NR2F1LOW/PRRX1HIGH staining were observed in DCIS samples. Together, these findings reveal the existence of an inhibitory mechanism of dissemination regulated by NR2F1 in early-stage breast cancer cells. SIGNIFICANCE: During early stages of breast cancer progression, HER2-mediated suppression of NR2F1 promotes dissemination by inducing EMT and a hybrid luminal/basal-like program.

Mouse-INtraDuctal (MIND): an in vivo model for studying the underlying mechanisms of DCIS malignancy.

Due to widespread adoption of screening mammography, there has been a significant increase in new diagnoses of ductal carcinoma in situ (DCIS). However, DCIS prognosis remains unclear. To address this gap, we developed an in vivo model, Mouse-INtraDuctal (MIND), in which patient-derived DCIS epithelial cells are injected intraductally and allowed to progress naturally in mice. Similar to human DCIS, the cancer cells formed in situ lesions inside the mouse mammary ducts and mimicked all histologic subtypes including micropapillary, papillary, cribriform, solid, and comedo. Among 37 patient samples injected into 202 xenografts, at median duration of 9 months, 20 samples (54%) injected into 95 xenografts showed in vivo invasive progression, while 17 (46%) samples injected into 107 xenografts remained non-invasive. Among the 20 samples that showed invasive progression, nine samples injected into 54 xenografts exhibited a mixed pattern in which some xenografts showed invasive progression while others remained non-invasive. Among the clinically relevant biomarkers, only elevated progesterone receptor expression in patient DCIS and the extent of in vivo growth in xenografts predicted an invasive outcome. The Tempus XT assay was used on 16 patient DCIS formalin-fixed, paraffin-embedded sections including eight DCISs that showed invasive progression, five DCISs that remained non-invasive, and three DCISs that showed a mixed pattern in the xenografts. Analysis of the frequency of cancer-related pathogenic mutations among the groups showed no significant differences (KW: p > 0.05). There were also no differences in the frequency of high, moderate, or low severity mutations (KW; p > 0.05). These results suggest that genetic changes in the DCIS are not the primary driver for the development of invasive disease. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Expression of CCL2/CCR2 signaling proteins in breast carcinoma cells is associated with invasive progression.

Ductal carcinoma in situ (DCIS) is the most common type of pre-invasive breast cancer diagnosed in women. Because the majority of DCIS cases are unlikely to progress to invasive breast cancer, many women are over-treated for DCIS. By understanding the molecular basis of early stage breast cancer progression, we may identify better prognostic factors and design treatments tailored specifically to the predicted outcome of DCIS. Chemokines are small soluble molecules with complex roles in inflammation and cancer progression. Previously, we demonstrated that CCL2/CCR2 chemokine signaling in breast cancer cell lines regulated growth and invasion through p42/44MAPK and SMAD3 dependent mechanisms. Here, we sought to determine the clinical and functional relevance of CCL2/CCR2 signaling proteins to DCIS progression. Through immunostaining analysis of DCIS and IDC tissues, we show that expression of CCL2, CCR2, phospho-SMAD3 and phospho-p42/44MAPK correlate with IDC. Using PDX models and an immortalized hDCIS.01 breast epithelial cell line, we show that breast epithelial cells with high CCR2 and high CCL2 levels form invasive breast lesions that express phospho-SMAD3 and phospho-p42/44MAPK. These studies demonstrate that increased CCL2/CCR2 signaling in breast tissues is associated with DCIS progression, and could be a signature to predict the likelihood of DCIS progression to IDC.

BCL9/STAT3 regulation of transcriptional enhancer networks promote DCIS progression.

The molecular processes by which some human ductal carcinoma in situ (DCIS) lesions advance to the more aggressive form, while others remain indolent, are largely unknown. Experiments utilizing a patient-derived (PDX) DCIS Mouse INtraDuctal (MIND) animal model combined with ChIP-exo and RNA sequencing revealed that the formation of protein complexes between B Cell Lymphoma-9 (BCL9), phosphoserine 727 STAT3 (PS-727-STAT3) and non-STAT3 transcription factors on chromatin enhancers lead to subsequent transcription of key drivers of DCIS malignancy. Downregulation of two such targets, integrin ß3 and its associated metalloproteinase, MMP16, resulted in a significant inhibition of DCIS invasive progression. Finally, in vivo targeting of BCL9, using rosemary extract, resulted in significant inhibition of DCIS malignancy in both cell line and PDX DCIS MIND animal models. As such, our studies provide compelling evidence for future testing of rosemary extract as a chemopreventive agent in breast cancer.

Gas6 expression is reduced in advanced breast cancers.

Growth arrest-specific gene 6 (Gas6) is a cytokine that binds to receptor tyrosine kinases Tyro3, Axl, and Mer. Numerous studies have suggested that macrophage-derived Gas6 interacts with Axl to promote cancer progression, and Axl has been associated with poor clinical outcome. However, the expression and relevance of Gas6 in human breast cancer patients has not been studied. Analysis of tissue microarrays showed that Gas6 was highly expressed in ductal carcinoma in situ (DCIS) but markedly decreased in invasive breast cancer. Gas6 and Axl were weakly correlated, suggesting that their functions may not exclusively rely on each other. Analyses of publicly available databases showed significantly improved overall and relapse-free survival in patients with high Gas6 mRNA, particularly in luminal A breast cancers. These findings indicate that tumor-derived Gas6 is not overexpressed in invasive breast cancer, and may not be a negative prognostic factor in human breast cancer.

Postpartum breast cancer progression is driven by semaphorin 7a-mediated invasion and survival.

Young women diagnosed with breast cancer (BC) have poor prognosis due to increased rates of metastasis. In addition, women diagnosed within 10 years of most recent childbirth are approximately three times more likely to develop metastasis than age- and stage-matched nulliparous women. We define these cases as postpartum BC (PPBC) and propose that the unique biology of the postpartum mammary gland drives tumor progression. Our published results revealed roles for SEMA7A in breast tumor cell growth, motility, invasion, and tumor-associated lymphangiogenesis, all of which are also increased in preclinical models of PPBC. However, whether SEMA7A drives progression in PPBC remains largely unexplored. Our results presented herein show that silencing of SEMA7A decreases tumor growth in a model of PPBC, while overexpression is sufficient to increase growth in nulliparous hosts. Further, we show that SEMA7A promotes multiple known drivers of PPBC progression including tumor-associated COX-2 expression and fibroblast-mediated collagen deposition in the tumor microenvironment. In addition, we show for the first time that SEMA7A-expressing cells deposit fibronectin to promote tumor cell survival. Finally, we show that co-expression of SEMA7A/COX-2/FN predicts for poor prognosis in breast cancer patient cohorts. These studies suggest SEMA7A as a key mediator of BC progression, and that targeting SEMA7A may open avenues for novel therapeutic strategies.

Long noncoding RNA BHLHE40-AS1 promotes early breast cancer progression through modulating IL-6/STAT3 signaling.

Ductal carcinoma in situ (DCIS) is a nonobligate precursor to invasive breast cancer. Only a small percentage of DCIS cases are predicted to progress; however, there is no method to determine which DCIS lesions will remain innocuous from those that will become invasive disease. Therefore, DCIS is treated aggressively creating a current state of overdiagnosis and overtreatment. There is a critical need to identify functional determinants of progression of DCIS to invasive ductal carcinoma (IDC). Interrogating biopsies from five patients with contiguous DCIS and IDC lesions, we have shown that expression of the long noncoding RNA BHLHE40-AS1 increases with disease progression. BHLHE40-AS1 expression supports DCIS cell proliferation, motility, and invasive potential. Mechanistically, BHLHE40-AS1 modulates interleukin (IL)-6/signal transducer and activator of transcription 3 (STAT3) activity and a proinflammatory cytokine signature, in part through interaction with interleukin enhancer-binding factor 3. These data suggest that BHLHE40-AS1 supports early breast cancer progression by engaging STAT3 signaling, creating an immune-permissive microenvironment.

MNK1/NODAL Signaling Promotes Invasive Progression of Breast Ductal Carcinoma In Situ.

The mechanisms by which breast cancers progress from relatively indolent ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) are not well understood. However, this process is critical to the acquisition of metastatic potential. MAPK-interacting serine/threonine-protein kinase 1 (MNK1) signaling can promote cell invasion. NODAL, a morphogen essential for embryogenic patterning, is often reexpressed in breast cancer. Here we describe a MNK1/NODAL signaling axis that promotes DCIS progression to IDC. We generated MNK1 knockout (KO) or constitutively active MNK1 (caMNK1)-expressing human MCF-10A-derived DCIS cell lines, which were orthotopically injected into the mammary glands of mice. Loss of MNK1 repressed NODAL expression, inhibited DCIS to IDC conversion, and decreased tumor relapse and metastasis. Conversely, caMNK1 induced NODAL expression and promoted IDC. The MNK1/NODAL axis promoted cancer stem cell properties and invasion in vitro. The MNK1/2 inhibitor SEL201 blocked DCIS progression to invasive disease in vivo. In clinical samples, IDC and DCIS with microinvasion expressed higher levels of phospho-MNK1 and NODAL versus low-grade (invasion-free) DCIS. Cumulatively, our data support further development of MNK1 inhibitors as therapeutics for preventing invasive disease. SIGNIFICANCE: These findings provide new mechanistic insight into progression of ductal carcinoma and support clinical application of MNK1 inhibitors to delay progression of indolent ductal carcinoma in situ to invasive ductal carcinoma.

ATM-dependent activation of SIM2s regulates homologous recombination and epithelial-mesenchymal transition.

There is increasing evidence that genomic instability is a prerequisite for cancer progression. Here we show that SIM2s, a member of the bHLH/PAS family of transcription factors, regulates DNA damage repair through enhancement of homologous recombination (HR), and prevents epithelial-mesenchymal transitions (EMT) in an Ataxia-telangiectasia mutated (ATM)-dependent manner. Mechanistically, we found that SIM2s interacts with ATM and is stabilized through ATM-dependent phosphorylation in response to IR. Once stabilized, SIM2s interacts with BRCA1 and supports RAD51 recruitment to the site of DNA damage. Loss of SIM2s through the introduction of shSIM2 or the mutation of SIM2s at one of the predicted ATM phosphorylation sites (S115) reduces HR efficiency through disruption of RAD51 recruitment, resulting in genomic instability and induction of EMT. The EMT induced by the mutation of S115 is characterized by a decrease in E-cadherin and an induction of the basal marker, K14, resulting in increased invasion and metastasis. Together, these results identify a novel player in the DNA damage repair pathway and provides a link in ductal carcinoma in situ progression to invasive ductal carcinoma through loss of SIM2s, increased genomic instability, EMT, and metastasis.

Modeling Human Ductal Carcinoma In Situ in the Mouse.

Breast cancer development is a multi-step process in which genetic and molecular heterogeneity occurs at multiple stages. Ductal carcinoma arises from pre-invasive lesions such as atypical ductal hyperplasia (ADH) and ductal carcinoma in situ (DCIS), which progress to invasive and metastatic cancer. The feasibility of obtaining tissue samples from all stages of progression from the same patient is low, and thus molecular studies dissecting the mechanisms that mediate the transition from pre-invasive DCIS to invasive carcinoma have been hampered. In the past 25 years, numerous mouse models have been developed that partly recapitulate the histological and biological properties of early stage lesions. In this review, we discuss in vivo model systems of breast cancer progression from syngeneic mouse models to human xenografts, with particular focus on how accurately these models mimic human disease.

MIND model for triple-negative breast cancer in syngeneic mice for quick and sequential progression analysis of lung metastasis.

Mouse models of breast cancer with specific molecular subtypes (e.g., ER or HER2 positive) in an immunocompetent or an immunocompromised environment significantly contribute to our understanding of cancer biology, despite some limitations, and they give insight into targeted therapies. However, an ideal triple-negative breast cancer (TNBC) mouse model is lacking. What has been missing in the TNBC mouse model is a sequential progression of the disease in an essential native microenvironment. This notion inspired us to develop a TNBC-model in syngeneic mice using a mammary intraductal (MIND) method. To achieve this goal, Mvt-1and 4T1 TNBC mouse cell lines were injected into the mammary ducts via nipples of FVB/N mice and BALB/c wild-type immunocompetent mice, respectively. We established that the TNBC-MIND model in syngeneic mice could epitomize all breast cancer progression stages and metastasis into the lungs via lymphatic or hematogenous dissemination within four weeks. Collectively, the syngeneic mouse-TNBC-MIND model may serve as a unique platform for further investigation of the underlying mechanisms of TNBC growth and therapies.