Nancy Dumont

Sustained induction of epithelial to mesenchymal transition activates DNA methylation of genes silenced in basal-like breast cancers

The active acquisition of epigenetic changes is a poorly understood but important process in development, differentiation, and disease. Our work has shown that repression of the p16/pRb pathway in human epithelial cells, a condition common to stem cells and many tumor cells, induces dynamic epigenetic remodeling resulting in the targeted methylation of a selected group of CpG islands. We hypothesized that cells in this epigenetically plastic state could be programmed by the microenvironment to acquire epigenetic changes associated with tumorigenesis. Here, we describe an in vitro model system where epigenetically plastic cells were placed in an environment that induced epithelial to mesenchymal transition (EMT) and led to a program of acquired de novo DNA methylation at targeted sites. In this model, we found that repression of E-cadherin transcription preceded the subsequent acquisition of methylated CpG sites. Furthermore, the induction of EMT was accompanied by de novo methylation of several other gene promoters, including those of the estrogen receptor and Twist. These data demonstrate that signals from the microenvironment can induce phenotypic and gene expression changes associated with targeted de novo epigenetic alterations important in tumor progression, and that these alterations occur through a deterministic, rather than stochastic, mechanism. Given the dynamic epigenetic reprogramming that occurs in these cells, DNA methylation profiles observed in human tumors may reflect the history of environmental exposures during the genesis of a tumor.

No paradox, no progress: inverse cancer comorbidity in people with other complex diseases

In the past 5 years, several leading groups have attempted to explain why individuals with Downs syndrome have a reduced risk of many solid tumours and an increased risk of leukaemia and testicular cancer. Niels Bohr, the Danish physicist, noted that a paradox could initiate progress. We think that the paradox of a medical disorder protecting against cancer could be formalised in a new model of inverse cancer morbidity in people with other serious diseases. In this Personal View, we review evidence from epidemiological and clinical studies that supports a consistently lower than expected occurrence of cancer in patients with Downs syndrome, Parkinsons disease, schizophrenia, diabetes, Alzheimers disease, multiple sclerosis, and anorexia nervosa. Intriguingly, most comorbidities are neuropsychiatric or CNS disorders. We provide a brief overview of evidence indicating genetic and molecular connections between cancer and these complex diseases. Inverse comorbidity could be a valuable model to investigate common or related pathways or processes and test new therapies, but, most importantly, to understand why certain people are protected from the malignancy.

CD36 Repression Activates a Multicellular Stromal Program Shared by High Mammographic Density and Tumor Tissues

UNLABELLED Although high mammographic density is considered one of the strongest risk factors for invasive breast cancer, the genes involved in modulating this clinical feature are unknown. Tissues of high mammographic density share key histologic features with stromal components within malignant lesions of tumor tissues, specifically low adipocyte and high extracellular matrix (ECM) content. We show that CD36, a transmembrane receptor that coordinately modulates multiple protumorigenic phenotypes, including adipocyte differentiation, angiogenesis, cell-ECM interactions, and immune signaling, is greatly repressed in multiple cell types of disease-free stroma associated with high mammographic density and tumor stroma. Using both in vitro and in vivo assays, we show that CD36 repression is necessary and sufficient to recapitulate the above-mentioned phenotypes observed in high mammographic density and tumor tissues. Consistent with a functional role for this coordinated program in tumorigenesis, we observe that clinical outcomes are strongly associated with CD36 expression. SIGNIFICANCE CD36 simultaneously controls adipocyte content and matrix accumulation and is coordinately repressed in multiple cell types within tumor and high mammographic density stroma, suggesting that activation of this stromal program is an early event in tumorigenesis. Levels of CD36 and extent of mammographic density are both modifiable factors that provide potential for intervention.

Rare somatic cells from human breast tissue exhibit extensive lineage plasticity

We identified cell surface markers associated with repression of p16INK4a/cyclin-dependent kinase inhibitor 2A(CDKN2A), a critical determinant in the acquisition of a plastic state. These cell surface markers allowed direct isolation of rare cells from healthy human breast tissue that exhibit extensive lineage plasticity. This subpopulation is poised to transcribe plasticity markers, OCT3/4, SOX2, and NANOG, at levels similar to those measured in human embryonic stem cells and to acquire a plastic state sensitive to environmental programming. In vitro, in vivo, and teratoma assays demonstrated that either a directly sorted (uncultured) or a single-cell (clonogenic) cell population from primary tissue can differentiate into functional derivatives of each germ layer, ectodermal, endodermal, and mesodermal. In contrast to other cells that express OCT3/4, SOX2, and NANOG, these human endogenous plastic somatic cells are mortal, express low telomerase activity, expand for an extensive but finite number of population doublings, and maintain a diploid karyotype before arresting in G1.

Human mammary cancer progression model recapitulates methylation events associated with breast premalignancy

IntroductionWe have previously identified a rare subpopulation of variant human mammary epithelial cells (vHMEC) with repressed p16INK4A that exist in disease-free women yet display premalignant properties, suggesting that they have engaged the process of malignant transformation. In order to gain insight into the molecular alterations required for vHMEC to progress to malignancy, and to characterize the epigenetic events associated with early progression, we examined the effect of oncogenic stress on the behavior of these cells.MethodsHMEC that express p16INK4A and vHMEC that do not, were transduced with constitutively active Ha-rasV12 and subsequently exposed to serum to determine whether signals from the cellular microenvironment could cooperate with ras to promote the malignant transformation of vHMEC. Epigenetic alterations were assessed using methylation-specific polymerase chain reaction (PCR).ResultsvHMEC expressing Ha-rasV12 (vHMEC-ras) bypassed the classic proliferative arrest that has been previously documented in normal fibroblasts following oncogenic stress, and that we also observe here in normal HMEC. Moreover, vHMEC-ras cells exhibited many additional alterations that are observed during progression to malignancy such as the generation of chromosomal abnormalities, upregulation of telomerase activity, immortalization following exposure to serum, and anchorage-independent growth, but they did not form tumors following orthotopic injection in vivo. Associated with their early progression to malignancy was an increase in the number of genes methylated, two of which (RASSF1A and SFRP1) were also methylated in other immortalized mammary cell lines as well as in breast cancer cells and tissues.ConclusionsWe have characterized a mammary progression model that recapitulates molecular and methylation alterations observed in many breast cancers. Our data suggest that concomitant methylation of RASSF1A and SFRP1 marks an early event in mammary transformation and may thus have prognostic potential.

Reflections on miR-ing Effects in Metastasis

In a recent issue of Cell, Valastyan et al. demonstrate that miR-31 can regulate multiple steps in the metastatic cascade independent of confounding effects on primary tumor development. These data have potential to provide biomarkers for prognosis and novel targets for intervention in this most lethal aspect of malignancy.

Abstract LB-061: HER2-targeted PEGylated liposomal doxorubicin (MM-302) efficiently targets the HER2 intermediate cell population in vitro and in vivo

Introduction: MM-302 is an antibody-liposomal drug conjugate designed specifically to target doxorubicin to HER2-overexpressing tumor cells. MM-302 is currently being evaluated in a Phase II trial in HER2 positive metastatic breast cancer (NCT02213744). HER2-positive breast cancer accounts for about 15-20% of breast cancer cases and is defined as IHC 3+ or 2+ and HER2 FISH amplified. A substantial percentage (∼30%) of breast cancer patients show positive HER2 IHC (1+/2+) without HER2 gene amplification (“HER2 intermediate”). This population is not eligible for treatment with currently approved HER2-targeted therapies. The purpose of this study is to investigate the in vitro and in vivo delivery/activity of MM-302 in the HER2 intermediate population. Methods: In vitro binding and viability studies were performed with MM-302, PEGylated liposomal doxorubicin (PLD) and T-DM1 with a panel of cell lines representing a range of HER2 expression. HER2-mediated cellular delivery of MM-302 was investigated in vivo in different HER2 expressing tumor models using a novel PEG immunofluorescent assay herein described. Frozen tumor tissues were stained for PEG, HER2 and cytokeratin, side-by-side with two cell standard arrays: A PEG array, obtained by cell incubation with increasing concentrations of MM-302, and a HER2 array, built with a panel of cell lines at different HER2 expression (from ∼50,000 to over 1,000,000 HER2). Image analysis and subsequent regression of the PEG and HER2 fluorescent intensities from the respective standards allowed for the quantification of the number of liposomes in individual tumor cells at distinct HER2 receptor numbers. Tumor cell apoptosis following MM-302 cellular delivery was measured by immunofluorescence followed by image analysis. Results: MM-302 efficiently bound to, and induced, tumor cell death across a panel of cell lines, with no significant distinction between cell lines expressing 300-400,000 HER2 or above 1,000,000 HER2 (IHC 3+). Conversely, T-DM1, used as control, significantly bound to and induced cell death only to cells above 1,000,000 HER2 (IHC 3+). In vivo evaluation of HER2-mediated cellular delivery via PEG immunofluorescent staining showed that MM-302 can be equally efficiently internalized in tumor cells above 1,000,000 HER2 (IHC 3+) and in the HER2 intermediate expression range. Preliminary analysis on post-treatment patient biopsies collected during a MM-302 Phase I study (NCT01304797) support clinical translation of these preclinical observations. Conclusions: Treatment with MM-302 results in efficient HER2 binding and liposome cellular delivery across a panel of HER2 models that extend beyond the traditional HER2 positive definition. This study suggests that MM-302 may be a promising candidate for the treatment of patients with intermediate HER2 expression who represent a significant unmet medical need. Citation Format: Elena Geretti, Christopher Espelin, Bambang Adiwijaya, Nancy Dumont, Silvia Coma, Zachary Koncki, Minh Pham, Gabriela Garcia, Troy Bloom, Victoria Rimkunas, Joe Reynolds, Karen Campbell, Victor Moyo, Istvan Molnar, Patricia LoRusso, Ian Krop, Kathy Miller, Cynthia Ma, Pamela Munster, Thomas Wickham. HER2-targeted PEGylated liposomal doxorubicin (MM-302) efficiently targets the HER2 intermediate cell population in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-061.

Abstract 4134: MM-302 is more effective than PEGylated liposomal doxorubicin (PLD) at reducing pulmonary metastatic burden in breast cancer models expressing intermediate levels of HER2

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction: Despite improvements in treatment with newly approved HER2-targeted therapies, safe and effective treatments are still needed, not only for HER2-positive metastatic breast cancer (MBC), but also for MBC expressing intermediate levels of HER2 that are still considered HER2-negative (e.g. IHC 2+, FISH-negative). MM-302 is a liposomal antibody drug conjugate (ADC) designed to target doxorubicin to HER2-overexpressing cancer cells. MM-302 is currently being evaluated in HER2-positive locally advanced breast cancer (LABC)/MBC patients in the registration-directed HERMIONE trial. The objective of this study was to compare the relative efficacy of MM-302 and PLD in treating HER2-intermediate MBC (corresponding to 1+/2+ by IHC) using models that closely mimic how HER2-overexpressing metastatic tumors are established in humans. Methods: To establish metastatic disease, the murine 4T1-HER2 cell line engineered to express intermediate levels of Her2 (median of ∼1×105 HER2 receptors/cell), and the human MDA-MB-453 cells that endogenously express intermediate levels of Her2 (2+ by IHC and median of ∼3×105 HER2 receptors/cell), were inoculated orthotopically into the right and left mammary fat pads of immunocompromised mice. Primary tumors then spontaneously seed cancer cells in distant visceral organs such as the lung. When primary tumor volumes reached ∼150 mm3 (4T1-Her2) or ∼270 mm3 (MDA-MB-453), mice were randomized and treated with vehicle control, PLD or MM-302. At the end of the study, primary tumors and lungs were harvested to assess liposome delivery and quantify pulmonary metastatic burden. Results: MM-302 was more effective than PLD at reducing total pulmonary metastatic burden in both HER2-intermediate models as evidenced by the lower number of surface metastases in the 4T1-Her2 model and the lower number of human cytokeratin positive cells per lung in the MDA-MB-453 model. MM-302 and PLD were equally effective at slowing (4T1-Her2) and inhibiting (MDA-MB-453) primary tumor growth. Mechanisms responsible for differences in efficacy are being explored. To date, better distribution of liposomes in metastatic lesions than in primary tumors has been observed where liposome delivery appears to be restricted to the tumor periphery in the 4T1-Her2 model. Conclusion: MM-302 was more effective than PLD at reducing pulmonary metastatic tumor burden in both HER2-expressing models. The superiority of MM-302 over PLD was unique to metastatic lesions where there was better distribution of liposomes than in the primary tumor. These results support the rationale for evaluating MM-302 in patients with HER2-intermediate MBC. Citation Format: Nancy Dumont, Elena Geretti, Shannon Curtis Leonard, Christopher Espelin, Daniel Gaddy, Bart Hendriks, Ulrik Nielsen, Thomas Wickham. MM-302 is more effective than PEGylated liposomal doxorubicin (PLD) at reducing pulmonary metastatic burden in breast cancer models expressing intermediate levels of HER2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4134. doi:10.1158/1538-7445.AM2015-4134

Abstract P6-11-05: Tumor priming with cyclophosphamide for enhanced tumor delivery, penetration and anti-tumor activity of MM-302, HER2-targeted liposomal doxorubicin

MM-302, HER2-targeted PEGylated liposomal doxorubicin, is a liposomal antibody drug conjugate designed to target doxorubicin to HER2-overexpressing cancer cells, while limiting uptake into non-target cells. Effective tumor delivery and penetration are critical barriers to the clinical activity of nanomedicines, including liposomes. Cyclophosphamide has been successfully combined with both doxorubicin and liposomal doxorubicin in breast cancer therapy, with the two drugs being administered on the same day to patients. We have evaluated a novel sequential combination regimen of cyclophosphamide with MM-302 with the goal of improving tumor delivery and penetration of MM-302. Methods: Biodistribution studies were carried out in multiple tumor xenograft models to assess the delivery of MM-302 and free doxorubicin, either as single agents or in combination with cyclophosphamide at different dosing schedules. The total doxorubicin within tumors was quantified by HPLC and microscopically by determining the number of doxorubicin-positive nuclei within frozen tumor sections. Induction of DNA damage/repair, tumor cell apoptosis, and changes in the tumor architecture in response to drug treatment (tumor cell density and vascular parameters) were quantified by automated image analysis. Interstitial fluid pressure measurements were carried out to evaluate changes in the tumor physiology upon cyclophosphamide treatment. Anti-tumor activity studies in BT474-M3 tumor-bearing mice were performed to evaluate the ability of the different dosing regimens to inhibit tumor growth. Results: Pre-dosing of tumors with cyclophosphamide enhanced subsequent MM-302 delivery to tumor xenografts (2-3-fold) without affecting delivery to non-target tissues, such as the heart and skin. We demonstrate that this effect is critically dependent on the timing of cyclophosphamide administration. Analysis of cyclophosphamide-treated tumors suggests that the mechanism for improved MM-302 delivery involves the induction of tumor cell apoptosis, reduction of overall tumor cell density, substantial lowering of interstitial fluid pressure and increase in vascular perfusion. Finally, treatment of tumors xenografts with cyclophosphamide followed by MM-302 resulted in a significantly greater tumor growth inhibition compared to either single agent alone. Conclusions: Rational combination of MM-302 with cyclophosphamide results in an active and tolerable regimen in preclinical models that enhances the tumor delivery and activity of MM-302, without affecting doxorubicin exposure to non-target organs. This novel sequential dosing strategy represents an advance in addressing the critical challenge for tumor delivery of nanomedicines. This work provided data supporting the initiation of a clinical evaluation of the effect of cyclophosphamide on MM-302 delivery as part of an on-going Phase 1 clinical trial of MM-302 in HER2-positive metastatic breast cancer (http://clinicaltrials.gov/show/NCT01304797). Citation Format: Elena Geretti, Shannon C Leonard, Nancy Dumont, Christopher W Espelin, Daniel F Gaddy, Thomas J Wickham, Bart S Hendriks. Tumor priming with cyclophosphamide for enhanced tumor delivery, penetration and anti-tumor activity of MM-302, HER2-targeted liposomal doxorubicin [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-11-05.

Abstract LB-273: The tumor suppressor p16INK4a regulates extensive plasticity in rare somatic cells found in adult human tissue.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Extensive studies have demonstrated that repression of p16INK4a (CDKN2A) not only silences a powerful tumor suppressor activity, but also is associated with the acquisition of a plastic state, i.e. the ability of a cell to change phenotypes. In epithelial cells, repression of p16INK4a does not only inactivate cell cycle arrest in response to stress but also allows increased expression of chromatin remodeling proteins that are important for epigenetic plasticity underlying differentiation. The up-regulation of such chromatin remodeling proteins sets the expression pattern of pluripotent cells in Drosophila and inhibits differentiation and dictates the decision between progenitor and differentiated states in murine myoblasts. Furthermore, mice engineered for knock-out of BMI-1, a polycomb protein that inhibits p16INK4a transcription, fail to repress p16INK4a activity and fail to generate hematopoietic and neural stem cells. In light of these observations, we reasoned that repression of p16INK4a might also modulate expression of cell surface markers that could be used for the prospective isolation of stem or progenitor cells. We identified cell surface markers associated with repression of p16INK4a and found that they allowed direct isolation of rare cells from healthy human breast tissue that exhibit extensive lineage plasticity. This subpopulation of cells has the ability to transcribe pluripotency markers, Oct3/4, Sox2 and Nanog at levels similar to those measured in human embryonic stem cells and to acquire a plastic state sensitive to environmental programming. In vitro , in vivo and teratoma assays demonstrated that either a directly-sorted (uncultured) or a single cell (clonogenic) cell population from primary human tissue can differentiate into functional derivatives of each germ layer, ectodermal, endodermal and mesodermal. In contrast to other cells that express Oct3/4, Sox2 and Nanog, these human endogenous Plastic Somatic cells (ePS cells) are mortal, express low telomerase activity, expand for an extensive but finite number of population doublings, and maintain a diploid karyotype before arresting in G1. The observation that repressed p16INK4a, a key tumor suppressor gene, is associated with epigenetic and phenotypic plasticity suggests functional links between tumor suppressor genes and plastic states that are yet to be determined and may some day be exploited for cancer intervention. Citation Format: Somdutta Roy, Philippe Gascard, Nancy Dumont, Jianxin Zhao, Deng Pan, Sarah Petrie, Marta Margeta, Thea Dorothy Tlsty. The tumor suppressor p16INK4a regulates extensive plasticity in rare somatic cells found in adult human tissue. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-273. doi:10.1158/1538-7445.AM2013-LB-273