Sonali Jindal

Cyclooxygenase-2-dependent lymphangiogenesis promotes nodal metastasis of postpartum breast cancer.

Breast involution following pregnancy has been implicated in the high rates of metastasis observed in postpartum breast cancers; however, it is not clear how this remodeling process promotes metastasis. Here, we demonstrate that human postpartum breast cancers have increased peritumor lymphatic vessel density that correlates with increased frequency of lymph node metastases. Moreover, lymphatic vessel density was increased in normal postpartum breast tissue compared with tissue from nulliparous women. In rodents, mammary lymphangiogenesis was upregulated during weaning-induced mammary gland involution. Furthermore, breast cancer cells exposed to the involuting mammary microenvironment acquired prolymphangiogenic properties that contributed to peritumor lymphatic expansion, tumor size, invasion, and distant metastases. Finally, in rodent models of postpartum breast cancer, cyclooxygenase-2 (COX-2) inhibition during the involution window decreased normal mammary gland lymphangiogenesis, mammary tumor-associated lymphangiogenesis, tumor cell invasion into lymphatics, and metastasis. Our data indicate that physiologic COX-2-dependent lymphangiogenesis occurs in the postpartum mammary gland and suggest that tumors within this mammary microenvironment acquire enhanced prolymphangiogenic activity. Further, our results suggest that the prolymphangiogenic microenvironment of the postpartum mammary gland has potential as a target to inhibit metastasis and suggest that further study of the therapeutic efficacy of COX-2 inhibitors in postpartum breast cancer is warranted.

Obesity and Overfeeding Affecting Both Tumor and Systemic Metabolism Activates the Progesterone Receptor to Contribute to Postmenopausal Breast Cancer

Obese postmenopausal women have increased risk of breast cancers with poorer clinical outcomes than their lean counterparts. However, the mechanisms underlying these associations are poorly understood. Rodent model studies have recently identified a period of vulnerability for mammary cancer promotion, which emerges during weight gain after the loss of ovarian function (surgical ovariectomy; OVX). Thus, a period of transient weight gain may provide a life cycle-specific opportunity to prevent or treat postmenopausal breast cancer. We hypothesized that a combination of impaired metabolic regulation in obese animals prior to OVX plus an OVX-induced positive energy imbalance might cooperate to drive tumor growth and progression. To determine if lean and obese rodents differ in their metabolic response to OVX-induced weight gain, and whether this difference affects later mammary tumor metabolism, we performed a nutrient tracer study during the menopausal window of vulnerability. Lean animals preferentially deposited excess nutrients to mammary and peripheral tissues rather than to the adjacent tumors. Conversely, obese animals deposited excess nutrients into the tumors themselves. Notably, tumors from obese animals also displayed increased expression of the progesterone receptor (PR). Elevated PR expression positively correlated with tumor expression of glycolytic and lipogenic enzymes, glucose uptake, and proliferation markers. Treatment with the antidiabetic drug metformin during ovariectomy-induced weight gain caused tumor regression and downregulation of PR expression in tumors. Clinically, expression array analysis of breast tumors from postmenopausal women revealed that PR expression correlated with a similar pattern of metabolic upregulation, supporting the notion that PR+ tumors have enhanced metabolic capacity after menopause. Our findings have potential explanative power in understanding why obese, postmenopausal women display an increased risk of breast cancer.

Wound healing‐like immune program facilitates postpartum mammary gland involution and tumor progression

Women diagnosed with breast cancer within 5 years postpartum have poor survival rates. The process of postpartum mammary gland involution, whereby the lactating gland remodels to its prepregnant state, promotes breast cancer progression in xenograft models. Macrophage influx occurs during mammary gland involution, implicating immune modulation in the promotion of postpartum breast cancer. Herein, we characterize the postpartum murine mammary gland and find an orchestrated influx of immune cells similar to that which occurs during wound healing. Further, the normal involuting gland may be in an immunosuppressed state as discerned by the transient presence of Foxp3+ regulatory T cells and IL‐10+ macrophages with T cell suppressive function. To determine the influence of the postpartum immune microenvironment on mammary tumor promotion, we developed an immune‐competent model. In this model, mammary tumors in the involution group are sixfold larger than nulliparous group tumors, have decreased CD4+ and CD8+ T cell infiltrates and contain a greater number of macrophages with the ability to inhibit T cell activation. Targeting involution with a neutralizing antibody against the immunosuppressive cytokine IL‐10 reduces tumor growth in involution group mice but not in nulliparous mice, implicating the involution microenvironment as the primary target of αIL‐10 treatment. Relevance to women is implicated, as we find postlactational human breast tissue has transient high IL‐10+ and Foxp3+ immune cell infiltrate. These data show an immune modulated microenvironment within the normal involuting mammary gland suggestive of immunosuppression, that when targeted reduces tumor promotion, revealing possible immune‐based strategies for postpartum breast cancer.

Physiological COX-2 Expression in Breast Epithelium Associates with COX-2 Levels in Ductal Carcinoma in Situ and Invasive Breast Cancer in Young Women

Cyclooxygenase-2 (COX-2) overexpression is implicated in increased risk and poorer outcomes in breast cancer in young women. We investigated COX-2 regulation in normal premenopausal breast tissue and its relationship to malignancy in young women. Quantitative COX-2 immunohistochemistry was performed on adjacent normal and breast cancer tissues from 96 premenopausal women with known clinical reproductive histories, and on rat mammary glands with distinct ovarian hormone exposures. COX-2 expression in the normal breast epithelium varied more than 40-fold between women and was associated with COX-2 expression levels in ductal carcinoma in situ and invasive cancer. Normal breast COX-2 expression was independent of known breast cancer prognostic indicators, including tumor stage and clinical subtype, indicating that factors regulating physiological COX-2 expression may be the primary drivers of COX-2 expression in breast cancer. Ovarian hormones, particularly at pregnancy levels, were identified as modulators of COX-2 in normal mammary epithelium. However, serial breast biopsy analysis in nonpregnant premenopausal women suggested relatively stable baseline levels of COX-2 expression, which persisted independent of menstrual cycling. These data provide impetus to investigate how baseline COX-2 expression is regulated in premenopausal breast tissue because COX-2 levels in normal breast epithelium may prove to be an indicator of breast cancer risk in young women, and predict the chemopreventive and therapeutic efficacy of COX-2 inhibitors in this population.

Mechanism and preclinical prevention of increased breast cancer risk caused by pregnancy

While a first pregnancy before age 22 lowers breast cancer risk, a pregnancy after age 35 significantly increases life-long breast cancer risk. Pregnancy causes several changes to the normal breast that raise barriers to transformation, but how pregnancy can also increase cancer risk remains unclear. We show in mice that pregnancy has different effects on the few early lesions that have already developed in the otherwise normal breast—it causes apoptosis evasion and accelerated progression to cancer. The apoptosis evasion is due to the normally tightly controlled STAT5 signaling going astray—these precancerous cells activate STAT5 in response to pregnancy/lactation hormones and maintain STAT5 activation even during involution, thus preventing the apoptosis normally initiated by oncoprotein and involution. Short-term anti-STAT5 treatment of lactation-completed mice bearing early lesions eliminates the increased risk after a pregnancy. This chemoprevention strategy has important implications for preventing increased human breast cancer risk caused by pregnancy. DOI: http://dx.doi.org/10.7554/eLife.00996.001

Myoepithelial cell differentiation markers in ductal carcinoma in situ progression.

We describe a preclinical model that investigates progression of early-stage ductal carcinoma in situ (DCIS) and report that compromised myoepithelial cell differentiation occurs before transition to invasive disease. Human breast cancer MCF10DCIS.com cells were delivered into the mouse mammary teat by intraductal injection in the absence of surgical manipulations and accompanying wound-healing confounders. DCIS-like lesions developed throughout the mammary ducts with full representation of human DCIS histologic patterns. Tumor cells were incorporated into the normal mammary epithelium, developed ductal intraepithelial neoplasia and DCIS, and progressed to invasive carcinoma, suggesting the model provides a rigorous approach to study early stages of breast cancer progression. Mammary glands were evaluated for myoepithelium integrity with immunohistochemical assays. Progressive loss of the myoepithelial cell differentiation markers p63, calponin, and α-smooth muscle actin was observed in the mouse myoepithelium surrounding DCIS-involved ducts. p63 loss was an early indicator, calponin loss intermediate, and α-smooth muscle actin a later indicator of compromised myoepithelium. Loss of myoepithelial calponin was specifically associated with gain of the basal marker p63 in adjacent tumor cells. In single time point biopsies obtained from 16 women diagnosed with pure DCIS, a similar loss in myoepithelial cell markers was observed. These results suggest that further research is warranted into the role of myoepithelial cell p63 and calponin expression on DCIS progression to invasive disease.

Mammary Gland Involution as an Immunotherapeutic Target for Postpartum Breast Cancer

Postpartum mammary gland involution has been identified as tumor-promotional and is proposed to contribute to the increased rates of metastasis and poor survival observed in postpartum breast cancer patients. In rodent models, the involuting mammary gland microenvironment is sufficient to induce enhanced tumor cell growth, local invasion, and metastasis. Postpartum involution shares many attributes with wound healing, including upregulation of genes involved in immune responsiveness and infiltration of tissue by immune cells. In rodent models, treatment with non-steroidal anti-inflammatory drugs (NSAIDs) ameliorates the tumor-promotional effects of involution, consistent with the immune milieu of the involuting gland contributing to tumor promotion. Currently, immunotherapy is being investigated as a means of breast cancer treatment with the purpose of identifying ways to enhance anti-tumor immune responses. Here we review evidence for postpartum mammary gland involution being a uniquely defined ‘hot-spot’ of pro-tumorigenic immune cell infiltration, and propose that immunotherapy should be explored for prevention and treatment of breast cancers that arise in this environment.

P2-01-04: The Post-Partum Diagnosis of Pregnancy Associated Breast Cancer Confers an Increased Risk for Metastasis without Increased Incidence of Poorer Prognosis Biologic Subtype.

Background: All women who complete a childbirth have an increased risk for the subsequent breast cancer regardless of age at their first birth. In younger women, this period truncates 10 years later and pregnancy then becomes protective, whereas in older mothers the risk persists. Moreover, breast cancers diagnosed subsequent to a recent childbirth have an increased risk for metastasis and death. However much of the existing data is confounded by inclusion of cases diagnosed during pregnancy. Hypothesis: Our preclinical models of pregnancy associated breast cancer (PABC) has identified the “involution hypothesis”, where the normal physiologic post-partum breast involution is the pregnancy-related event promotional of breast cancer growth, invasion and metastasis. We have performed a retrospective cohort study, restricted to postpartum PABC and controls, to fully characterize post-partum PABC by biologic subtype and known predictors for metastasis. Also we identify if the specific period of post-partum PABC has a higher risk for metastasis then non-PABC, as predicted by our animal model studies. Methods: We performed a retrospective cohort of 527 cases of breast cancer diagnosed at age 45 or younger from 1981–2011. Pregnancy status was identified from medical records and defined as nulliparous (n=107), PABC (up to 5 years post-partum of last childbirth, n=114), and Later Parous (>10 years post-partum, n=239). Clinicopathologic characteristics were obtained from pathology records. Outcomes data obtained through the University of Colorado Tumor Registrar. Hazard ratios of risk of metastatic recurrence of nulliparous and PABC groups were analyzed using a Cox Proportional Hazards Model and time to metastatic recurrence was analyzed using standard Kaplan Meier curves. Results: Compared to nulliparous cases, PABC cases were more frequently Stage IV(11.4% v. 4.9%), T3 tumor size(17.3% v. 13.1%), poorly differentiated(56.6% v. 49.0%), lobular histology(4.5% v.1.0%), with lymphovascular invasion(30.4% v. 24.5%) and with involved lymphnodes(57.6% v. 48.6%). No differences were seen between Luminal A, Luminal B, Her 2 and Triple Negative subtypes. Average follow-up was 3.4 years for nulliparous, 3.5 for PABC and 4.6 for Later Parous. Survival analysis revealed a cumulative 5-year metastatic recurrence free probability of 80.6% for nulliparous, 68.5% for PABC and 80.5% for Later Parous. Compared to nulliparous, PABC had greater than a three times higher risk for subsequent metastatic recurrence (HR 3.29, 95% CI: 1.25−8.63). No significant difference in the risk of metastatic recurrence for Later Parous compared to nulliparous was observed (HR: 1.079, 95% CI: 0.46−2.55). Conclusion: Post-partum PABC is characterized by higher stage at diagnosis but without enrichment for poorer prognosis biologic subtypes. Post-Partum PABC has an increased risk for metastasis within five years of diagnosis, with an overall 3.29 times higher risk of a metastatic recurrence. These data support our hypothesis that the post-partum window, and the associated role of naturally occurring involution, may be promotional of tumor growth, invasion and metastasis as previously identified in our animal models. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-04.

Myoepithelial cells in lobular carcinoma in situ: distribution and immunophenotype.

Myoepithelial cells have important physical and paracrine roles in breast tissue development, maintenance, and tumor suppression. Recent molecular and immunohistochemical studies have demonstrated phenotypic alterations in ductal carcinoma in situ-associated myoepithelial cells. Although the relationship of lobular carcinoma in situ (LCIS) and myoepithelial cells was described in 1980, further characterization of LCIS-associated myoepithelial cells is lacking. We stained 27 breast specimens harboring abundant LCIS with antibodies to smooth muscle myosin heavy chain, smooth muscle actin, and calponin. Dual stains for E-cadherin/smooth muscle myosin heavy chain and CK7/p63 were also performed. In each case, the intensity and distribution of staining in LCIS-associated myoepithelial cells were compared with normal breast tissue on the same slide. In 78% of the cases, LCIS-associated myoepithelial cells demonstrated decreased staining intensity for one or more myoepithelial markers. The normal localization of myoepithelial cells (flat against the basement membrane, pattern N) was seen in 96% of LCIS, yet 85% of cases had areas with myoepithelial cell cytoplasm oriented perpendicular to the basement membrane (pattern P), and in 30% of cases, myoepithelial cells appeared focally admixed with LCIS cells (pattern C). This study characterizes detailed architectural and immunophenotypic alterations of LCIS-associated myoepithelial cells. The finding of variably diminished staining favors application of several myoepithelial immunostains in clinical practice. The interaction of LCIS with myoepithelial cells, especially in light of the perpendicular and central architectural arrangements, deserves further mechanistic investigation.

Metformin inhibits stromal aromatase expression and tumor progression in a rodent model of postmenopausal breast cancer

BackgroundObesity and type II diabetes are linked to increased breast cancer risk in postmenopausal women. Patients treated with the antidiabetic drug metformin for diabetes or metabolic syndrome have reduced breast cancer risk, a greater pathologic complete response to neoadjuvant therapy, and improved breast cancer survival. We hypothesized that metformin may be especially effective when targeted to the menopausal transition, as this is a lifecycle window when weight gain and metabolic syndrome increase, and is also when the risk for obesity-related breast cancer increases.MethodsHere, we used an 1-methyl-1-nitrosourea (MNU)-induced mammary tumor rat model of estrogen receptor (ER)-positive postmenopausal breast cancer to evaluate the long-term effects of metformin administration on metabolic and tumor endpoints. In this model, ovariectomy (OVX) induces rapid weight gain, and an impaired whole-body response to excess calories contributes to increased tumor glucose uptake and increased tumor proliferation. Metformin treatment was initiated in tumor-bearing animals immediately prior to OVX and maintained for the duration of the study.ResultsMetformin decreased the size of existing mammary tumors and inhibited new tumor formation without changing body weight or adiposity. Decreased lipid accumulation in the livers of metformin-treated animals supports the ability of metformin to improve overall metabolic health. We also found a decrease in the number of aromatase-positive, CD68-positive macrophages within the tumor microenvironment, suggesting that metformin targets the immune microenvironment in addition to improving whole-body metabolism.ConclusionsThese findings suggest that peri-menopause/menopause represents a unique window of time during which metformin may be highly effective in women with established, or at high risk for developing, breast cancer.