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 profiling of in vivo ductal carcinoma in situ progression models identified B cell lymphoma-9 as a molecular driver of breast cancer invasion.

INTRODUCTION: There are an estimated 60,000 new cases of ductal carcinoma in situ (DCIS) each year. A lack of understanding in DCIS pathobiology has led to overtreatment of more than half of patients. We profiled the temporal molecular changes during DCIS transition to invasive ductal carcinoma (IDC) using in vivo DCIS progression models. These studies identified B cell lymphoma-9 (BCL9) as a potential molecular driver of early invasion. BCL9 is a newly found co-activator of Wnt-stimulated ß-catenin-mediated transcription. BCL9 has been shown to promote progression of multiple myeloma and colon carcinoma. However BCL9 role in breast cancer had not been previously recognized. METHODS: Microarray and RNA sequencing were utilized to characterize the sequential changes in mRNA expression during DCIS invasive transition. BCL9-shRNA knockdown was performed to assess the role of BCL9 in in vivo invasion, epithelial-mesenchymal transition (EMT) and canonical Wnt-signaling. Immunofluorescence of 28 patient samples was used to assess a correlation between the expression of BCL9 and biomarkers of high risk DCIS. The cancer genome atlas data were analyzed to assess the status of BCL9 gene alterations in breast cancers. RESULTS: Analysis of BCL9, by RNA and protein showed BCL9 up-regulation to be associated with DCIS transition to IDC. Analysis of patient DCIS revealed a significant correlation between high nuclear BCL9 and pathologic characteristics associated with DCIS recurrence: Estrogen receptor (ER) and progesterone receptor (PR) negative, high nuclear grade, and high human epidermal growth factor receptor2 (HER2). In vivo silencing of BCL9 resulted in the inhibition of DCIS invasion and reversal of EMT. Analysis of the TCGA data showed BCL9 to be altered in 26 % of breast cancers. This is a significant alteration when compared to HER2 (ERBB2) gene (19 %) and estrogen receptor (ESR1) gene (8 %). A significantly higher proportion of basal like invasive breast cancers compared to luminal breast cancers showed BCL9 amplification. CONCLUSION: BCL9 is a molecular driver of DCIS invasive progression and may predispose to the development of basal like invasive breast cancers. As such, BCL9 has the potential to serve as a biomarker of high risk DCIS and as a therapeutic target for prevention of IDC.

A novel role of microRNA146b in promoting mammary alveolar progenitor cell maintenance.

In this report, we have shown that miR146b promotes the maintenance of pregnancy-derived mammary luminal alveolar progenitors. MiR146b expression was significantly higher in the mammary glands of pregnant and lactating mice than in virgin mice. Furthermore, miR146b levels were significantly higher in mouse mammary glands exposed to the sex hormones, estrogen and progesterone, compared with those of untreated control animals. Pregnancy-derived primary mouse mammary epithelial cells in which miR146b was knocked down showed a significant reduction in the number of hollow acinar organoid structures formed on three-dimensional Matrigel and in ß-casein expression. This demonstrates that miR146b promotes the maintenance of pregnancy-derived mammary luminal alveolar progenitors. It has been shown that mouse mammary luminal progenitors give rise to hollow organoid structures, whereas solid organoid structures are derived from stem cells. Among several miR146b targets, miR146b knockdown resulted in preferential STAT3ß overexpression. In the primary mouse mammary epithelial cells, overexpression of STAT3ß isoform caused mammary epithelial cell death and a significant reduction in ß-casein mRNA expression. Therefore, we conclude that during pregnancy miR146b is involved in luminal alveolar progenitor cell maintenance, at least partially, by regulating STAT3ß.

Emerging functions of microRNA-146a/b in development and breast cancer: microRNA-146a/b in development and breast cancer.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression through translational repression or mRNA degradation. These molecules play critical roles in regulating normal developmental processes, but when deregulated, are causally linked to the pathogenesis of numerous diseases, including cancer. MicroRNA-146a and -146b are encoded by two different genes, but differ by only two bases and appear to function redundantly in many systems. Initial studies branded miR-146a/b as important mediators of inflammatory signaling, documenting the ability of these miRNAs to influence differentiation, proliferation, apoptosis and effector immune mechanisms within the hematopoietic system. Numerous contemporary studies now implicate miR-146a/b as pleiotropic regulators of tumorigenesis, as a polymorphism in miR-146a and altered expression of both miR-146a/b have been linked with cancer risk, tumor histogenesis and invasive and metastatic capacity in diverse cancers. Despite the numerous reports concerning miR-146a/b in human cancers, the mechanistic contributions of these miRNAs in both normal and neoplastic mammary gland development and biology remains poorly characterized.

Metastasis from tongue squamous cell carcinoma to the kidney.

Metastasis to the kidney from other primary sites is extremely rare. Previous studies reported the lung as the most common primary site. Distant metastasis from the tongue to the kidney is exceedingly rare. Herein, we describe a case of metastatic squamous cell carcinoma to the kidney in a 71-year-old male with a detailed discussion of differentiating it from potential mimickers. The patient underwent a total glossectomy and bilateral cervical lymph node dissection. A diagnosis of well-differentiated squamous cell carcinoma of the tongue was rendered and the tumor was staged pT3 pN3b. Within two years of initial presentation, the patient developed widely metastatic disease, including pulmonary nodules, renal masses, left adrenal mass, and pancreatic mass. Accurate diagnosis of a secondary involvement of the kidney by a metastatic tumor requires the appropriate correlation of clinical and imaging findings as well as morphologic and immunohistochemical clues.

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.

NEMO, a Transcriptional Target of Estrogen and Progesterone, Is Linked to Tumor Suppressor PML in Breast Cancer.

The beneficial versus detrimental roles of estrogen plus progesterone (E+P) in breast cancer remains controversial. Here we report a beneficial mechanism of E+P treatment in breast cancer cells driven by transcriptional upregulation of the NFκB modulator NEMO, which in turn promotes expression of the tumor suppressor protein promyelocytic leukemia (PML). E+P treatment of patient-derived epithelial cells derived from ductal carcinoma in situ (DCIS) increased secretion of the proinflammatory cytokine IL6. Mechanistic investigations indicated that IL6 upregulation occurred as a result of transcriptional upregulation of NEMO, the gene that harbored estrogen receptor (ER) binding sites within its promoter. Accordingly, E+P treatment of breast cancer cells increased ER binding to the NEMO promoter, thereby increasing NEMO expression, NFκB activation, and IL6 secretion. In two mouse xenograft models of DCIS, we found that RNAi-mediated silencing of NEMO increased tumor invasion and progression. This seemingly paradoxical result was linked to NEMO-mediated regulation of NFκB and IL6 secretion, increased phosphorylation of STAT3 on Ser727, and increased expression of PML, a STAT3 transcriptional target. In identifying NEMO as a pivotal transcriptional target of E+P signaling in breast cancer cells, our work offers a mechanistic explanation for the paradoxical antitumorigenic roles of E+P in breast cancer by showing how it upregulates the tumor suppressor protein PML. Cancer Res; 77(14); 3802-13. ©2017 AACR.

Metastasis from tongue squamous cell carcinoma to the kidney

Metastasis to the kidney from other primary sites is extremely rare. Previous studies reported the lung as the most common primary site. Distant metastasis from the tongue to the kidney is exceedingly rare. Herein, we describe a case of metastatic squamous cell carcinoma to the kidney in a 71-year-old male with a detailed discussion of differentiating it from potential mimickers. The patient underwent a total glossectomy and bilateral cervical lymph node dissection. A diagnosis of well-differentiated squamous cell carcinoma of the tongue was rendered and the tumor was staged pT3 pN3b. Within two years of initial presentation, the patient developed widely metastatic disease, including pulmonary nodules, renal masses, left adrenal mass, and pancreatic mass. Accurate diagnosis of a secondary involvement of the kidney by a metastatic tumor requires the appropriate correlation of clinical and imaging findings as well as morphologic and immunohistochemical clues.