Gabriela Paroni

Retinoids and breast cancer: From basic studies to the clinic and back again

All-trans retinoic acid (ATRA) is the most important active metabolite of vitamin A controlling segmentation in the developing organism and the homeostasis of various tissues in the adult. ATRA as well as natural and synthetic derivatives, collectively known as retinoids, are also promising agents in the treatment and chemoprevention of different types of neoplasia including breast cancer. The major aim of the present article is to review the basic knowledge acquired on the anti-tumor activity of classic retinoids, like ATRA, in mammary tumors, focusing on the underlying cellular and molecular mechanisms and the determinants of retinoid sensitivity/resistance. In the first part, an analysis of the large number of pre-clinical studies available is provided, stressing the point that this has resulted in a limited number of clinical trials. This is followed by an overview of the knowledge acquired on the role played by the retinoid nuclear receptors in the anti-tumor responses triggered by retinoids. The body of the article emphasizes the potential of ATRA and derivatives in modulating and in being influenced by some of the most relevant cellular pathways involved in the growth and progression of breast cancer. We review the studies centering on the cross-talk between retinoids and some of the growth-factor pathways which control the homeostasis of the mammary tumor cell. In addition, we consider the cross-talk with relevant intra-cellular second messenger pathways. The information provided lays the foundation for the development of rational and retinoid-based therapeutic strategies to be used for the management of breast cancer.

Atypical retinoids ST1926 and CD437 are S-phase-specific agents causing DNA double-strand breaks: significance for the cytotoxic and antiproliferative activity

Retinoid-related molecules (RRM) are novel agents with tumor-selective cytotoxic/antiproliferative activity, a different mechanism of action from classic retinoids and no cross-resistance with other chemotherapeutics. ST1926 and CD437 are prototypic RRMs, with the former currently undergoing phase I clinical trials. We show here that ST1926, CD437, and active congeners cause DNA damage. Cellular and subcellular COMET assays, H2AX phosphorylation (γ-H2AX), and scoring of chromosome aberrations indicate that active RRMs produce DNA double-strand breaks (DSB) and chromosomal lesions in NB4, an acute myeloid leukemia (AML) cell line characterized by high sensitivity to RRMs. There is a direct quantitative correlation between the levels of DSBs and the cytotoxic/antiproliferative effects induced by RRMs. NB4.437r blasts, which are selectively resistant to RRMs, do not show any sign of DNA damage after treatment with ST1926, CD437, and analogues. DNA damage is the major mechanism underlying the antileukemic activity of RRMs in NB4 and other AML cell lines. In accordance with the S-phase specificity of the cytotoxic and antiproliferative responses of AML cells to RRMs, increases in DSBs are maximal during the S phase of the cell cycle. Induction of DSBs precedes inhibition of DNA replication and is associated with rapid activation of ataxia telangectasia mutated, ataxia telangectasia RAD3-related, and DNA-dependent protein kinases with subsequent stimulation of the p38 mitogen-activated protein kinase. Inhibition of ataxia telangectasia mutated and DNA-dependent protein kinases reduces phosphorylation of H2AX. Cells defective for homologous recombination are particularly sensitive to ST1926, indicating that this process is important for the protection of cells from the RRM-dependent DNA damage and cytotoxicity. [Mol Cancer Ther 2008;7(9):2941–54]

Cellular and molecular determinants of all-trans retinoic acid sensitivity in breast cancer: Luminal phenotype and RARα expression

Forty‐two cell lines recapitulating mammary carcinoma heterogeneity were profiled for all‐trans retinoic acid (ATRA) sensitivity. Luminal and ER+ (estrogen‐receptor‐positive) cell lines are generally sensitive to ATRA, while refractoriness/low sensitivity is associated with a Basal phenotype and HER2 positivity. Indeed, only 2 Basal cell lines (MDA‐MB157 and HCC‐1599) are highly sensitive to the retinoid. Sensitivity of HCC‐1599 cells is confirmed in xenotransplanted mice. Short‐term tissue‐slice cultures of surgical samples validate the cell‐line results and support the concept that a high proportion of Luminal/ER+ carcinomas are ATRA sensitive, while triple‐negative (Basal) and HER2‐positive tumors tend to be retinoid resistant. Pathway‐oriented analysis of the constitutive gene‐expression profiles in the cell lines identifies RARα as the member of the retinoid pathway directly associated with a Luminal phenotype, estrogen positivity and ATRA sensitivity. RARα3 is the major transcript in ATRA‐sensitive cells and tumors. Studies in selected cell lines with agonists/antagonists confirm that RARα is the principal mediator of ATRA responsiveness. RARα over‐expression sensitizes retinoid‐resistant MDA‐MB453 cells to ATRA anti‐proliferative action. Conversely, silencing of RARα in retinoid‐sensitive SKBR3 cells abrogates ATRA responsiveness. All this is paralleled by similar effects on ATRA‐dependent inhibition of cell motility, indicating that RARα may mediate also ATRA anti‐metastatic effects. We define gene sets of predictive potential which are associated with ATRA sensitivity in breast cancer cell lines and validate them in short‐term tissue cultures of Luminal/ER+ and triple‐negative tumors. In these last models, we determine the perturbations in the transcriptomic profiles afforded by ATRA. The study provides fundamental information for the development of retinoid‐based therapeutic strategies aimed at the stratified treatment of breast cancer subtypes.

All-trans-retinoic Acid Modulates the Plasticity and Inhibits the Motility of Breast Cancer Cells: ROLE OF NOTCH1 AND TRANSFORMING GROWTH FACTOR (TGFβ).

Background: All-trans-retinoic acid is a promising therapeutic agent in breast cancer. Results: All-trans-retinoic acid modulates mammary tumor cell epithelial-to-mesenchymal-transition via the TGFβ and NOTCH pathways. Conclusion: The present study unveils a new aspect of all-trans-retinoic acid activity (i.e. regulation of phenotypic cell plasticity). Significance: Our results indicate that all-trans-retinoic acid is endowed with anti-metastatic properties that could be exploited at the therapeutic level. All-trans-retinoic acid (ATRA) is a natural compound proposed for the treatment/chemoprevention of breast cancer. Increasing evidence indicates that aberrant regulation of epithelial-to-mesenchymal transition (EMT) is a determinant of the cancer cell invasive and metastatic behavior. The effects of ATRA on EMT are largely unknown. In HER2-positive SKBR3 and UACC812 cells, showing co-amplification of the ERBB2 and RARA genes, ATRA activates a RARα-dependent epithelial differentiation program. In SKBR3 cells, this causes the formation/reorganization of adherens and tight junctions. Epithelial differentiation and augmented cell-cell contacts underlie the anti-migratory action exerted by the retinoid in cells exposed to the EMT-inducing factors EGF and heregulin-β1. Down-regulation of NOTCH1, an emerging EMT modulator, is involved in the inhibition of motility by ATRA. Indeed, the retinoid blocks NOTCH1 up-regulation by EGF and/or heregulin-β1. Pharmacological inhibition of γ-secretase and NOTCH1 processing also abrogates SKBR3 cell migration. Stimulation of TGFβ contributes to the anti-migratory effect of ATRA. The retinoid switches TGFβ from an EMT-inducing and pro-migratory determinant to an anti-migratory mediator. Inhibition of the NOTCH1 pathway not only plays a role in the anti-migratory action of ATRA; it is relevant also for the anti-proliferative activity of the retinoid in HCC1599 breast cancer cells, which are addicted to NOTCH1 for growth/viability. This effect is enhanced by the combination of ATRA and the γ-secretase inhibitor N-(N-(3,5-difluorophenacetyl)-l-alanyl)-S-phenylglycine t-butyl ester, supporting the concept that the two compounds act at the transcriptional and post-translational levels along the NOTCH1 pathway.

New insights into the molecular mechanisms underlying sensitivity/resistance to the atypical retinoid ST1926 in acute myeloid leukaemia cells: the role of histone H2A.Z, cAMP-dependent protein kinase A and the proteasome.

ST1926 is an atypical retinoid and a promising anti-tumour agent with selective apoptotic activity on the leukaemic blast. The anti-tumour activity of the compound has been associated with its capacity to induce DNA double stranded breaks. Target profiling by affinity chromatography coupled to mass spectrometry led to the identification of histone H2A.Z as a protein capable of binding ST1926 specifically. The result was confirmed by studies involving Surface Plasmon Resonance (SPR). This indicates that H2A.Z is a primary target of ST1926 and links the perturbations of the histone pathway observed by microarray analysis to the DNA damage and apoptotic responses caused by the atypical retinoid. Comparison of the whole-genome gene-expression profiles of the ST1926-sensitive NB4 and the ST1926-resistant NB4.437r cell lines demonstrated differential expression of numerous genes. Network analysis of the data indicated enrichment of the cellular pathways controlling cAMP (cyclic adenosine-monophosphate)-dependent signal transduction, proteasome-dependent protein degradation and nuclear histones in NB4.437r cells. Pharmacological inhibition of cAMP-dependent protein kinase A with H89 partially reverted resistance of NB4.437r cells to ST1926. Conversely, inhibition of the proteasome with MG132 or bortezomib blocked the apoptotic response afforded by ST1926 in the NB4 cell line. This last effect was associated with a dramatic reduction in the DNA damage caused by the atypical retinoid. The results corroborate the idea that DNA damage is an important determinant of ST1926 apoptotic activity. More importantly, they demonstrate a proactive role of the proteasome in the DNA damaging and ensuing apoptotic response observed upon the challenge of acute myeloid leukaemia cells with ST1926.

RARα2 and PML-RAR similarities in the control of basal and retinoic acid induced myeloid maturation of acute myeloid leukemia cells

Treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA) is the first example of targeted therapy. In fact, the oncogenic fusion-protein (PML-RAR) typical of this leukemia contains the retinoid-nuclear-receptor RARα. PML-RAR is responsible for the differentiation block of the leukemic blast. Besides PML-RAR, two endogenous RARα proteins are present in APL blasts, i.e. RARα1 and RARα2. We developed different cell populations characterized by PML-RAR, RARα2 and RARα1 knock-down in the APL-derived NB4 cell-line. Unexpectedly, silencing of PML-RAR and RARα2 results in similar increases in the constitutive expression of several granulocytic differentiation markers. This is accompanied by enhanced expression of the same granulocytic markers upon exposure of the NB4 blasts to ATRA. Silencing of PML-RAR and RARα2 causes also similar perturbations in the whole genome gene-expression profiles of vehicle and ATRA treated NB4 cells. Unlike PML-RAR and RARα2, RARα1 knock-down blocks ATRA-dependent induction of several granulocytic differentiation markers. Many of the effects on myeloid differentiation are confirmed by over-expression of RARα2 in NB4 cells. RARα2 action on myeloid differentiation does not require the presence of PML-RAR, as it is recapitulated also upon knock-down in PML-RAR-negative HL-60 cells. Thus, relative to RARα1, PML-RAR and RARα2 exert opposite effects on APL-cell differentiation. These contrasting actions may be related to the fact that both PML-RAR and RARα2 interact with and inhibit the transcriptional activity of RARα1. The interaction surface is located in the carboxy-terminal domain containing the D/E/F regions and it is influenced by phosphorylation of Ser-369 of RARα1.

Network-guided modelling allows tumor-type independent prediction of sensitivity to all-trans retinoic acid.

BackgroundnAll-trans-retinoic acid (ATRA) is a differentiating agent used in the treatment of acute-promyelocytic-leukemia (APL) and it is under-exploited in other malignancies despite its low systemic toxicity. A rational/personalized use of ATRA requires the development of predictive tools allowing identification of sensitive cancer types and responsive individuals.nnnMaterials and methodsnRNA-sequencing data for 10xa0080 patients and 33 different tumor types were derived from the TCGA and Leucegene datasets and completely re-processed. The study was carried out using machine learning methods and network analysis.nnnResultsnWe profiled a large panel of breast-cancer cell-lines for in vitro sensitivity to ATRA and exploited the associated basal gene-expression data to initially generate a model predicting ATRA-sensitivity in this disease. Starting from these results and using a network-guided approach, we developed a generalized model (ATRA-21) whose validity extends to tumor types other than breast cancer. ATRA-21 predictions correlate with experimentally determined sensitivity in a large panel of cell-lines representative of numerous tumor types. In patients, ATRA-21 correctly identifies APL as the most sensitive acute-myelogenous-leukemia subtype and indicates that uveal-melanoma and low-grade glioma are top-ranking diseases as for average predicted responsiveness to ATRA. There is a consistent number of tumor types for which higher ATRA-21 predictions are associated with better outcomes.nnnConclusionsnIn summary, we generated a tumor-type independent ATRA-sensitivity predictor which consists of a restricted number of genes and has the potential to be applied in the clinics. Identification of the tumor types that are likely to be generally sensitive to the action of ATRA paves the way to the design of clinical studies in the context of these diseases. In addition, ATRA-21 may represent an important diagnostic tool for the selection of individual patients who may benefit from ATRA-based therapeutic strategies also in tumors characterized by lower average sensitivity.

Lipid-sensors, enigmatic-orphan and orphan nuclear receptors as therapeutic targets in breast-cancer

Breast-cancer is heterogeneous and consists of various groups with different biological characteristics. Innovative pharmacological approaches accounting for this heterogeneity are needed. The forty eight human Nuclear-Hormone-Receptors are ligand-dependent transcription-factors and are classified into Endocrine-Receptors, Adopted-Orphan-Receptors (Lipid-sensors and Enigmatic-Orphans) and Orphan-receptors. Nuclear-Receptors represent ideal targets for the design/synthesis of pharmacological ligands. We provide an overview of the literature available on the expression and potential role played by Lipid-sensors, Enigmatic-Orphans and Orphan-Receptors in breast-cancer. The data are complemented by an analysis of the expression levels of each selected Nuclear-Receptor in the PAM50 breast-cancer groups, following re-elaboration of the data publicly available. The major aim is to support the idea that some of the Nuclear-Receptors represent largely unexploited therapeutic-targets in breast-cancer treatment/chemo-prevention. On the basis of our analysis, we conclude that the Lipid-Sensors, NR1C3, NR1H2 and NR1H3 are likely to be onco-suppressors in breast-cancer. The Enigmatic-Orphans, NR1F1 NR2A1 and NR3B3 as well as the Orphan-Receptors, NR0B1, NR0B2, NR1D1, NR2F1, NR2F2 and NR4A3 exert a similar action. These Nuclear-Receptors represent candidates for the development of therapeutic strategies aimed at increasing their expression or activating them in tumor cells. The group of Nuclear-Receptors endowed with potential oncogenic properties consists of the Lipid-Sensors, NR1C2 and NR1I2, the Enigmatic-Orphans, NR1F3, NR3B1 and NR5A2, as well as the Orphan-Receptors, NR2E1, NR2E3 and NR6A1. These oncogenic Nuclear-Receptors should be targeted with selective antagonists, reverse-agonists or agents/strategies capable of reducing their expression in breast-cancer cells.

Abstract P6-04-09: Cellular and molecular determinants of breast cancer sensitivity to all-trans retinoic acid: Identification of a gene expression fingerprint predicting responsiveness

All-trans retinoic acid (ATRA) and derived natural as well as synthetic retinoids are promising agents in the treatment and chemoprevention of various types of neoplasia, including mammary tumors. ATRA is an important component of the therapeutic schemes used for the treatment of a rare form of Acute Myelogenous Leukemia known as Acute Promyelocytic Leukemia. A rational use of the paradigmatic retinoid, ATRA, in a heterogeneous disease, like breast cancer, requires the definition of the cellular and molecular determinants of sensitivity to the agent. The major aim of the study was the definition of a predictive gene expression fingerprint that can be used for the selection of patients who may benefit from treatment protocols containing ATRA. To this purpose, we selected 45 breast cancer cell lines characterized for the constitutive whole genome gene expression profiles. The sensitivity of 30 cell lines (training set) to the anti-proliferative action of ATRA was defined after challenge with increasing concentrations of the retinoid for 3, 6 and 9 days. This analysis established that Luminal and ER-positive cell lines are enriched within the ATRA sensitive group. In contrast, cell lines characterized by a Basal-like phenotype, according to the PAM50 gene expression signature, are generally refractory to the growth inhibitory action of ATRA. The sensitivity of Luminal-A and Luminal-B and the general refractoriness of Basal-like tumors to ATRA was validated in short-term tissue slice cultures of surgical breast cancer specimens. The training set was used to define a gene-expression fingerprint consisting of approximately 50 genes significantly associated with ATRA sensitivity. The fingerprint was generated by reprocessing the RNA sequencing data contained in the CCLE (Cancer Cell line Encyclopedia) of the Broad Institute and it was built from approximately 60,000 coding and non-coding loci. The approach involved the use of general linear models (machine learning algorithm). The identified gene-expression fingerprint was subsequently used to successfully predict ATRA sensitivity in a test set consisting of the remaining 15 cell lines. As a first step towards the use of the fingerprint for the stratification of patients, we evaluated the proportion of predicted ATRA sensitive breast tumors in the TCGA dataset. In accordance with the cell line and primary tumor data, approximately 30% of the Luminal tumors present with a high similarity score to the identified gene expression fingerprint associated with ATRA sensitivity. In contrast, only 5% of the Basal-like or Triple-negative mammary tumors are characterized by the same high similarity score. Curiously, the ATRA sensitivity signature seems to be tumor context independent, as it correctly identifies the 20 Acute Promyelocytic Leukemia patients present in the 198 Acute Myelogenous Leukemia patients present in the TCGA dataset. Citation Format: Garattini E, Bolis M, Paroni G, Fratelli M, Zambelli A, Terao M. Cellular and molecular determinants of breast cancer sensitivity to all-trans retinoic acid: Identification of a gene expression fingerprint predicting responsiveness. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-04-09.

Abstract 2287: A sub-population of HER2+ breast carcinomas is characterized by co-amplification of the ERBB2 and RARA genes that renders cancer cells sensitive to retinoids and combinations of these agents with lapatinib

In breast cancer, HER2-positivity is often the result of an amplification of the corresponding gene (ERBB2) mappping to chr 17 close to the locus of the retinoid receptor, RARα (RARA). We set up a PCR assay and a FISH method to evaluate the proportion of patients with co-amplification of ERBB2 and RARA using sections obtained from 76 HER2 + cases. We observed that 20-28% of the cases showed co-amplification of ERBB2 and RARA (ERBB2 + /RARA + ). To evaluate whether RARA co-amplification translated into retinoid sensitivity, we turned to a panel of 12 breast carcinoma cell lines. The growth of 3 ERBB2 + /RARA + cell lines, SKBR3, UACC-812 and AU565, was inhibited by all-trans-retinoic acid (ATRA). In response to the retinoid, growth inhibition was accompanied by cell differentiation. ERBB2 + /RARA − cell lines were totally refractory to ATRA. Interestingly, SKBR3, UACC-812, and AU565 showed IC 50 values to the anti-proliferative effects of ATRA falling in the low nanomolar range. These values were two orders of magnitude lower than the IC 50 s calculated for the control cell lines MCF-7 and T47D (ERBB2 − /RARA − ), representing popular models of the well known association between ERα-positivity and sensitivity to retinoids in breast carcinoma. We evaluated whether targeting the retinoid and HER2 pathways simultaneously has the potential to represent a viable therapeutic strategy for this group of patients. The anti-proliferative, differentiating and apoptotic effects of combinations between ATRA and lapatinib, a tyrosine kinase inhibitor used for the treatment of HER2 + breast carcinoma, was assessed in our panel of cell lines. In agreement with retinoid sensitivity, lapatinib and ATRA determined synergistic anti-proliferative effects only in the case of the cell lines, SKBR3, UACC-812 and AU565. Furthermore, apoptosis was observed only in ERBB2 + /RARA + cells treated with the combination of lapatinib and ATRA. In SKBR3 cells, this effect was preceded by a lapatinib-dependent enhancement of the lactogenic differentiation afforded by ATRA. The molecular mechanisms underlying the cross-talk between lapatinib and ATRA were evaluated focusing specifically on the RARα and HER2 signal transduction pathways, as well as performing whole-genome gene expression studies in SKBR3 cells. These last studies and functional approaches demonstrated that the combination produced perturbations of the TGFβ, FGF and IGF pathways and indicated a role for RIG-I and FOXO3A in the cross-talk. Our results constitute the rationale for the use of combinations between lapatinib and ATRA in the treatment of ERBB2 + /RARA + breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2287. doi:10.1158/1538-7445.AM2011-2287