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Microarray gene expression analyses for MDA-MB-468, MDA-MB-231, and SUM159 cells after decitabine treatment

Gene Set Enrichment Analysis for genes upregulated by decitabine treatment in MDA-MB-468, MDA-MB-231, or SUM159

HM450 DNA methylation B-values for genes up- or down-regulated by decitabine in MDA-MB-468, MDA-MB-231, or SUM159

Additional figures, tables, and methods

Genes up-regulated by decitabine treatment in MDA-MB-468, MDA-MB-231, or SUM159 with hypermethylated promoters (TSS200)

Purpose:Stem-like cancer cells, with characteristic self-renewal abilities, remain highly refractory to various clinical interventions. As such, stemness-inhibiting entities, such as tumor suppressor p53, are therapeutically pursued for their anticancer activities. Interestingly, similar implications for tumor suppressor TAp73 in regulating stemness features within stem-like cancer cells remain unknown.Experimental Design: This study utilizes various in vitro molecular biology techniques, including immunoblotting, qRT-PCR, and mass spectrometry–based proteomics, and metabolomics approaches to study the role of TAp73 in human and murine embryonal carcinoma stem-like cells (ECSLC) as well as human breast cancer stem-like cells (BCSLC). These findings were confirmed using patient-derived brain tumor–initiating cells (BTIC) and in vivo xenograft models.Results:TAp73 inhibition decreases the expression of stem cell transcription factors Oct4, Nanog, and Sox-2, as well as tumorsphere formation capacity in ECSLCs. In vivo, TAp73-deficient ECSLCs and BCSLCs demonstrate decreased tumorigenic potential when xenografted in mice. Mechanistically, TAp73 modifies the proline regulatory axis through regulation of enzymes GLS, OAT, and PYCR1 involved in the interconversion of proline–glutamine–ornithine. Further, TAp73 deficiency exacerbates glutamine dependency, enhances accumulation of reactive oxygen species through reduced superoxide dismutase 1 (SOD1) expression, and promotes differentiation by arresting cell cycle and elevating autophagy. Most importantly, the knockdown of TAp73 in CD133HI BTICs, separated from three different glioblastoma patients, strongly decreases the expression of prosurvival factors Sox-2, BMI-1, and SOD1, and profoundly decreases their self-renewal capacity as evidenced through their reduced tumorsphere formation ability.Conclusions:Collectively, we reveal a clinically relevant aspect of cancer cell growth and stemness regulation through TAp73-mediated redox-sensitive metabolic reprogramming.

Supplementary Figures

Supplementary Tables

Paclitaxel is a common breast cancer drug; however, some tumors are resistant. The identification of biomarkers for paclitaxel resistance or sensitivity would enable the development of strategies to improve treatment efficacy. A genome‐wide in vivo shRNA screen was performed on paclitaxel‐treated mice with MDA‐MB‐231 tumors to identify genes associated with paclitaxel sensitivity or resistance. Gene expression of the top screen hits was associated with tumor response (resistance or sensitivity) among patients who received neoadjuvant chemotherapy containing paclitaxel. We focused our validation on screen hit B‐cell lymphoma 6 (BCL6), which is a therapeutic target in cancer but for which no effects on drug response have been reported. Knockdown of BCL6 resulted in increased tumor regression in mice treated with paclitaxel. Similarly, inhibiting BCL6 using a small molecule inhibitor enhanced paclitaxel treatment efficacy both in vitro and in vivo in breast cancer models. Mechanism studies revealed that reduced BCL6 enhances the efficacy of paclitaxel by inducing sustained G1/S arrest, concurrent with increased apoptosis and expression of target gene cyclin‐dependent kinase inhibitor 1A. In summary, the genome‐wide shRNA knockdown screen has identified BCL6 as a potential targetable resistance biomarker of paclitaxel response in breast cancer., An in vivo genome‐wide shRNA screen identified several potential biomarkers of paclitaxel response in breast cancer, including transcriptional repressor BCL6 as a biomarker of paclitaxel resistance. Inhibition of BCL6 in breast cancer cells and tumors resulted in increased response to paclitaxel. Paclitaxel with BCL6 inhibition resulted in increased expression of BCL6 target gene CDKN1A, sustained G1/S arrest, and increased apoptosis.

Eccrine porocarcinoma is a rare aggressive cutaneous malignancy. Complete surgical excision is the standard of care, although there are high rates of local and distant recurrence. We present a unique case of locally recurrent and metastatic subungal porocarcinoma successfully treated with intralesional interleukin-2.

Non-melanoma skin cancers are the most prevalent form of cancer, with cutaneous squamous cell carcinoma (cSCC) being the 2nd most common type. Patients presenting with high-risk lesions associated with locally advanced or metastatic cSCC face high rates of recurrence and mortality. Accurate staging and risk stratification for patients can be challenging because no system is universally accepted, and no Canadian guidelines currently exist. Patients with advanced cSCC are often deemed ineligible for either or both of curative surgery and radiation therapy (RT) and, until recently, were limited to off-label systemic cisplatin–fluorouracil or cetuximab therapy, which offers modest clinical benefits and potentially severe toxicity. A new systemic therapy, cemiplimab, has been approved for the treatment of locally advanced and metastatic cSCC. In the present review, we provide recommendations for patient classification and staging based on current guidelines, direction for determining patient eligibility for surgery and RT, and an overview of the available systemic treatment options for advanced cSCC and of the benefits of a multidisciplinary approach to patient management.

Mast cells are important sentinel cells in host defense against infection and major effector cells in allergic disease. The role of these cells in cancer settings has been widely debated. The diverse range of mast cell functions in both immunity and tissue remodeling events, such as angiogenesis, provides multiple opportunities for mast cells to modify the tumor microenvironment. In this review, we consider both skin and breast cancer settings to address the controversy surrounding the importance of mast cells in the host response to tumors. We specifically address the key mediators produced by mast cells which impact tumor development. The role of environmental challenges in modifying mast cell responses and opportunities to modify mast cell responses to enhance anti-tumor immunity are also considered. While the mast cell’s role in many cancer contexts is complicated and poorly understood, the activities of these tissue resident and radioresistant cells can provide important opportunities to enhance anti-cancer responses and limit cancer development.

Modern management of colorectal cancer (CRC) with peritoneal metastasis (PM) is based on a combination of cytoreductive surgery (CRS), systemic chemotherapy, and hyperthermic intraperitoneal chemotherapy (HIPEC). Although the role of hipec has recently been questioned with respect to results from the PRODIGE 7 trial, the role and benefit of a complete CRS were confirmed, as observed with a 41-month gain in median survival in that study, and 15% of patients remaining disease-free at 5 years. Still, CRC with PM is associated with a poor prognosis, and good patient selection is essential. Many questions about the optimal management approach for such patients remain, but all patients with PM from CRC should be referred to, or discussed with, a PM surgical oncologist, because cure is possible. The objective of the present guideline is to offer a practical approach to the management of PM from CRC and to reflect on the new practice standards set by recent publications on the topic.

Background: The COVID-19 pandemic has put enormous pressure on hospital resources, and has affected all aspects of patient care. As operative volumes decrease, cancer surgeries must be triaged and prioritized with careful thought and attention to ensure maximal benefit for the maximum number of patients. Peritoneal malignancies present a unique challenge, as surgical management can be resource intensive, but patients have limited non-surgical treatment options. This review summarizes current data on outcomes and resource utilization to help inform decision-making and case prioritization in times of constrained health care resources. Methods: A rapid literature review was performed, examining surgical and non-surgical outcomes data for peritoneal malignancies. Narrative data synthesis was cross-referenced with relevant societal guidelines. Peritoneal malignancy surgeons and medical oncologists reviewed recommendations to establish a national perspective on case triage and mitigating treatment strategies. Results and Conclusions: Triage of peritoneal malignancies during this time of restricted health care resource is nuanced and requires multidisciplinary discussion with consideration of individual patient factors. Prioritization should be given to patients where delay may compromise resectability of disease, and where alternative treatment options are lacking. Mitigating strategies such as systemic chemotherapy and/or surgical deferral may be utilized with close surveillance for disease stability or progression, which may affect surgical urgency. Unique hospital capacity, and ability to manage the complex post-operative course for these patients must also be considered to ensure patient and system needs are aligned.

Purpose: Stem-like cancer cells, with characteristic self-renewal abilities, remain highly refractory to various clinical interventions. As such, stemness-inhibiting entities, such as tumor suppressor p53, are therapeutically pursued for their anticancer activities. Interestingly, similar implications for tumor suppressor TAp73 in regulating stemness features within stem-like cancer cells remain unknown. Experimental Design: This study utilizes various in vitro molecular biology techniques, including immunoblotting, qRT-PCR, and mass spectrometry–based proteomics, and metabolomics approaches to study the role of TAp73 in human and murine embryonal carcinoma stem-like cells (ECSLC) as well as human breast cancer stem-like cells (BCSLC). These findings were confirmed using patient-derived brain tumor–initiating cells (BTIC) and in vivo xenograft models. Results: TAp73 inhibition decreases the expression of stem cell transcription factors Oct4, Nanog, and Sox-2, as well as tumorsphere formation capacity in ECSLCs. In vivo, TAp73-deficient ECSLCs and BCSLCs demonstrate decreased tumorigenic potential when xenografted in mice. Mechanistically, TAp73 modifies the proline regulatory axis through regulation of enzymes GLS, OAT, and PYCR1 involved in the interconversion of proline–glutamine–ornithine. Further, TAp73 deficiency exacerbates glutamine dependency, enhances accumulation of reactive oxygen species through reduced superoxide dismutase 1 (SOD1) expression, and promotes differentiation by arresting cell cycle and elevating autophagy. Most importantly, the knockdown of TAp73 in CD133HI BTICs, separated from three different glioblastoma patients, strongly decreases the expression of prosurvival factors Sox-2, BMI-1, and SOD1, and profoundly decreases their self-renewal capacity as evidenced through their reduced tumorsphere formation ability. Conclusions: Collectively, we reveal a clinically relevant aspect of cancer cell growth and stemness regulation through TAp73-mediated redox-sensitive metabolic reprogramming.

The incidence of cutaneous melanoma, a highly malignant skin cancer, is increasing yearly. While surgical removal of the tumor is the mainstay of treatment for patients with locally confined disease, those with metastases face uncertainty when it comes to their treatment. As melanoma is a relatively immunogenic cancer, current guidelines suggest using immunotherapies that can rewire the host immune response to target melanoma tumor cells. Intralesional therapy, where immunomodulatory agents are injected directly into the tumor, are an emerging aspect of treatment for in-transit melanoma because of their ability to mitigate severe off-target immune-related adverse events. However, their immunomodulatory mechanisms are poorly understood. In this review, we will summarize and discuss the different intralesional therapies for metastatic melanoma with respect to their clinical outcomes and immune molecular mechanisms.

Cancer stem-like cells (CSCs), a small population of pluripotent cells residing within heterogeneous tumor mass, remain highly resistant to various chemotherapies as compared to the differentiated cancer cells. It is being postulated that CSCs possess unique molecular mechanisms, such as autophagic homeostasis, that allow CSCs to withstand the therapeutic assaults. Here we demonstrate that HDAC6 inhibition differentially modulates macroautophagy/autophagy in CSCs as compared to that of differentiated cancer cells. Using human and murine CSC models and differentiated cells, we show that the inhibition or knockdown (KD) of HDAC6 decreases CSC pluripotency by downregulating major pluripotency factors POU5F1, NANOG and SOX2. This decreased HDAC6 expression increases ACTB, TUBB3 and CSN2 expression and promotes differentiation in CSCs in an apoptosis-independent manner. Mechanistically, HDAC6 KD in CSCs decreases pluripotency by promoting autophagy, whereas the inhibition of pluripotency via retinoic acid treatment, POU5F1 or autophagy-related gene (ATG7 and ATG12) KD in CSCs decreases HDAC6 expression and promotes differentiation. Interestingly, HDAC6 KD-mediated CSC growth inhibition is further enhanced in the presence of autophagy inducers Tat-Beclin 1 peptide and rapamycin. In contrast to the results observed in CSCs, HDAC6 KD in differentiated breast cancer cells downregulates autophagy and increases apoptosis. Furthermore, the autophagy regulator p-MTOR, upstream negative regulators of p-MTOR (TSC1 and TSC2) and downstream effectors of p-MTOR (p-RPS6KB and p-EIF4EBP1) are differentially regulated in CSCs versus differentiated cancer cells following HDAC6 KD. Overall these data identify the differential regulation of autophagy as a molecular link behind the differing chemo-susceptibility of CSCs and differentiated cancer cells.

The efficacy of oncolytic viruses (OVs), such as reovirus, is dictated by host immune responses, including those mediated by the pro- versus anti-inflammatory macrophages. As such, a detailed understanding of the interaction between reovirus and different macrophage types is critical for therapeutic efficacy. To explore reovirus-macrophage interactions, we performed tandem mass tag (TMT)-based quantitative temporal proteomics on mouse bone marrow-derived macrophages (BMMs) generated with two cytokines, macrophage colony stimulating factor (M-CSF) and granulocytic-macrophage colony stimulating factor (GM-CSF), representing anti- and proinflammatory macrophages, respectively. We quantified 6863 proteins across five time points in duplicate, comparing M-CSF (M-BMM) and GM-CSF (GM-BMM) in response to OV. We find that GM-BMMs have lower expression of key intrinsic proteins that facilitate an antiviral immune response, express higher levels of reovirus receptor protein JAM-A, and are more susceptible to oncolytic reovirus infection compared to M-BMMs. Interestingly, although M-BMMs are less susceptible to reovirus infection and subsequent cell death, they initiate an antireovirus adaptive T cell immune response comparable to that of GM-BMMs. Taken together, these data describe distinct proteome differences between these two macrophage populations in terms of their ability to mount antiviral immune responses.

Dysregulation of DNA methylation is an established feature of breast cancers. DNA demethylating therapies like decitabine are proposed for the treatment of triple-negative breast cancers (TNBC) and indicators of response need to be identified. For this purpose, we characterized the effects of decitabine in a panel of 10 breast cancer cell lines and observed a range of sensitivity to decitabine that was not subtype specific. Knockdown of potential key effectors demonstrated the requirement of deoxycytidine kinase (DCK) for decitabine response in breast cancer cells. In treatment-naïve breast tumors, DCK was higher in TNBCs, and DCK levels were sustained or increased post chemotherapy treatment. This suggests that limited DCK levels will not be a barrier to response in patients with TNBC treated with decitabine as a second-line treatment or in a clinical trial. Methylome analysis revealed that genome-wide, region-specific, tumor suppressor gene–specific methylation, and decitabine-induced demethylation did not predict response to decitabine. Gene set enrichment analysis of transcriptome data demonstrated that decitabine induced genes within apoptosis, cell cycle, stress, and immune pathways. Induced genes included those characterized by the viral mimicry response; however, knockdown of key effectors of the pathway did not affect decitabine sensitivity suggesting that breast cancer growth suppression by decitabine is independent of viral mimicry. Finally, taxol-resistant breast cancer cells expressing high levels of multidrug resistance transporter ABCB1 remained sensitive to decitabine, suggesting that the drug could be used as second-line treatment for chemoresistant patients.

Breast cancer stem cells (CSCs) can be identified by increased Aldefluor fluorescence caused by increased expression of aldehyde dehydrogenase 1A3 (ALDH1A3), as well as ALDH1A1 and ALDH2. In addition to being a CSC marker, ALDH1A3 regulates gene expression via retinoic acid (RA) signaling and plays a key role in the progression and chemotherapy resistance of cancer. Therefore, ALDH1A3 represents a druggable anti-cancer target of interest. Since to date, there are no characterized ALDH1A3 isoform inhibitors, drugs that were previously described as inhibiting the activity of other ALDH isoforms were tested for anti-ALDH1A3 activity. Twelve drugs (3-hydroxy-dl-kynurenine, benomyl, citral, chloral hydrate, cyanamide, daidzin, DEAB, disulfiram, gossypol, kynurenic acid, molinate, and pargyline) were compared for their efficacy in inducing apoptosis and reducing ALDH1A3, ALDH1A1 and ALDH2-associated Aldefluor fluorescence in breast cancer cells. Citral was identified as the best inhibitor of ALDH1A3, reducing the Aldefluor fluorescence in breast cancer cell lines and in a patient-derived tumor xenograft. Nanoparticle encapsulated citral specifically reduced the enhanced tumor growth of MDA-MB-231 cells overexpressing ALDH1A3. To determine the potential mechanisms of citral-mediated tumor growth inhibition, we performed cell proliferation, clonogenic, and gene expression assays. Citral reduced ALDH1A3-mediated colony formation and expression of ALDH1A3-inducible genes. In conclusion, citral is an effective ALDH1A3 inhibitor and is able to block ALDH1A3-mediated breast tumor growth, potentially via blocking its colony forming and gene expression regulation activity. The promise of ALDH1A3 inhibitors as adjuvant therapies for patients with tumors that have a large population of high-ALDH1A3 CSCs is discussed.

Hydrogen peroxide (H2O2) is a main second messenger in oncogenic signaling networks including the Ras and the growth factor receptor pathways. This is achieved predominantly through the oxidation of redox-sensitive cysteine (Cys) residues in proteins resulting in changes to their structure and function. We previously identified annexin A2 (ANXA2) as a redox regulatory protein that plays an important cellular role during oxidative stress and also promoting tumorigenesis. Here we investigated the role of ANXA2 in the regulation of H2O2-dependent signaling that drives tumor progression. We show that depletion of ANXA2 leads to the enhanced activation of AKT following either EGF/EGFR stimulation or oncogenic Ras transformation. The phosphatase and tensin homologue (PTEN) protein negatively regulates the PI3K/AKT pathway. We demonstrate that ANXA2 via its reactive Cys-8 residue, binds to PTEN and that the co-expression of PTEN and ANXA2, but not ANXA2 Cys-8-Ala mutant, inhibits AKT phosphorylation on Ser 473. These results indicate that ANXA2 is important for PTEN regulation within the PI3K/AKT signaling cascade. Furthermore, we also reveal that ANXA2 inversely regulates the expression of the peroxidase, peroxiredoxin 2, in a reactive oxygen species dependent manner.

Changes in gene expression are required for the progression of breast cancer; and DNA methylation, which silences tumor suppressor genes, is often responsible for these changes. Several de-methylating/DNA synthesis inhibiting drugs (such as decitabine) are potential breast cancer therapies; however, for their application to be successful, we need to identify which patients would most benefit from these treatments. To accomplish this, the critical genes, which must be silenced by hypermethylation in order for breast cancer to progress, need to be identified. For this purpose, we have performed an in vivo total genome knockdown screen. A genome-wide lentiviral-based shRNA screen was performed with breast cancer cell line MDA-MB-231 tumors implanted in the mammary fat pads of female NOD/SCID mice, with or without decitabine treatment (50mg/kg). The resulting tumors were harvested and shRNA sequences retrieved and hybridized to a Decode microarray, allowing for the identification of shRNA sequences that were enriched or depleted under decitabine treatment. The enriched shRNA sequences likely target genes of two categories: 1) they are methylated in the tumor and when expressed have tumor suppressive function, or 2) they are required for decitabine-mediated DNA synthesis inhibition and apoptosis. To determine if the shRNA-targeted genes identified in the screen fall within category 1 or 2, further analysis was done using GEO datasets for methylation in breast cancer cell lines, primary tumors, and normal breast tissues. In replicates of 6 mice, 111 shRNA sequences were enriched more than 2-fold in 5/6 mice. Of the 111 genes, 29 genes showed significant enrichment (p We have identified several genes that are hypermethylated in breast cancer and are potentially involved in tumor progression. Confirmation experiments will reveal a list of genes that when found hypermethylated in patient tumors would identify candidate breast cancer patients who would benefit from decitabine treatment. Citation Format: Margaret Thomas, Krysta Coyle, Mohammad Sultan, Luzhe Pan, Dae-Gyun Ahn, Patrick Lee, Carman Giacomantonio, Paola Marcato. Identifying hypermethylated tumor suppressor genes in breast cancer with an in vivo total genome knockdown screen [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 P1-06-02.

INTRODUCTION: Incisional biopsies, including the diagnostic core needle biopsy (CNB), routinely performed before surgical excision of breast cancer tumors are hypothesized to increase the risk of metastatic disease. In this study, we experimentally determined whether CNB of breast cancer tumors results in increased distant metastases and examine important resultant changes in the primary tumor and tumor microenvironment associated with this outcome. METHOD: To evaluate the effect of CNB on metastasis development, we implanted murine mammary 4T1 tumor cells in BALB/c mice and performed CNB on palpable tumors in half the mice. Subsequently, emulating the human scenario, all mice underwent complete tumor excision and were allowed to recover, with attendant metastasis development. Tumor growth, lung metastasis, circulating tumor cell (CTC) levels, variation in gene expression, composition of the tumor microenvironment, and changes in immunologic markers were compared in biopsied and non-biopsied mice. RESULTS: Mice with biopsied tumors developed significantly more lung metastases compared to non-biopsied mice. Tumors from biopsied mice contained a higher frequency of myeloid-derived suppressor cells (MDSCs) accompanied by reduced CD4+ T cells, CD8+ T cells, and macrophages, suggesting biopsy-mediated development of an increasingly immunosuppressive tumor microenvironment. We also observed a CNB-dependent up-regulation in the expression of SOX4, Ezh2, and other key epithelial-mesenchymal transition (EMT) genes, as well as increased CTC levels among the biopsy group. CONCLUSION: CNB creates an immunosuppressive tumor microenvironment, increases EMT, and facilitates release of CTCs, all of which likely contribute to the observed increase in development of distant metastases.

Aldehyde dehydrogenase (ALDH) 1A enzymes produce retinoic acid (RA), a transcription induction molecule. To investigate if ALDH1A1 or ALDH1A3‐mediated RA signaling has an active role in breast cancer tumorigenesis, we performed gene expression and tumor xenograft studies. Analysis of breast patient tumors revealed that high levels of ALDH1A3 correlated with expression of RA‐inducible genes with retinoic acid response elements (RAREs), poorer patient survival and triple‐negative breast cancers. This suggests a potential link between ALDH1A3 expression and RA signaling especially in aggressive and/or triple‐negative breast cancers. In MDA‐MB‐231, MDA‐MB‐468 and MDA‐MB‐435 cells, ALDH1A3 and RA increased expression of RA‐inducible genes. Interestingly, ALDH1A3 had opposing effects in tumor xenografts, increasing tumor growth and metastasis of MDA‐MB‐231 and MDA‐MB‐435 cells, but decreasing tumor growth of MDA‐MB‐468 cells. Exogenous RA replaced ALDH1A3 in inducing the same opposing tumor growth and metastasis effects, suggesting that ALDH1A3 mediates these effects by promoting RA signaling. Genome expression analysis revealed that ALDH1A3 induced largely divergent gene expression in MDA‐MB‐231 and MDA‐MB‐468 cells which likely resulted in the opposing tumor growth effects. Treatment with DNA methylation inhibitor 5‐aza‐2'deoxycytidine restored uniform RA‐inducibility of RARE‐containing HOXA1 and MUC4 in MDA‐MB‐231 and MDA‐MB‐468 cells, suggesting that differences in epigenetic modifications contribute to differential ALDH1A3/RA‐induced gene expression in breast cancer. In summary, ALDH1A3 induces differential RA signaling in breast cancer cells which affects the rate of breast cancer progression.

Background Malignant triton tumors (MTT) are a rare form of peripheral nerve sheath tumors that follows a particularly aggressive course. Given its rarity, only case reports and small series of patients have been published. Methods A Pubmed search was conducted (1966–2009) using the terms “triton tumor,” “rhabdomyosarcoma,” and “malignant peripheral nerve sheath tumor.” The reference lists of retrieved articles were searched. Cases were included when the diagnosis was clear, the patient underwent surgery, and follow-up data were available. Univariate and multivariate analyses were conducted for predictors of positive resection margin, local recurrence/progression, development of metastases, and mortality. Results A total of 124 cases were included. The overall 5-year survival was 14% and the median time to death was 13 months. The overall local recurrence/progression rate was 50% and the median time to recurrence/progression was 6 months. On multivariate Cox proportional hazards analysis, positive margin status (HR 2.2, P = 0.01), local recurrence/progression (HR 3.1, P = 0.003), and development of metastases (HR 2.6, P = 0.003) were associated with mortality. Adjuvant radiotherapy was associated with improved survival (HR 0.4, P = 0.005). Conclusion Complete surgical resection and adjuvant radiotherapy should be the cornerstones of treatment for MTT. J. Surg. Oncol. 2012; 106:51–56. © 2012 Wiley Periodicals, Inc.

Annexins are a structurally related family of calcium and phospholipid-binding proteins that are involved in the regulation of a wide range of molecular and cellular processes. Annexin A2 is unique among the annexins in that it possesses redox sensitive cysteine(s). The ubiquitous and abundant expression of ANXA2 in cells and its reactivity with hydrogen peroxide led us to hypothesize that this protein could play a role in cellular redox regulation. Here we show that ANXA2 protein levels are induced by hydrogen peroxide. Furthermore, depletion of ANXA2 resulted in the elevation of cellular reactive oxygen species (ROS) upon oxidative stress, increased activation of the ROS-induced pro-apoptotic kinases, JNK, p38 and Akt and elevated sensitivity to ROS-mediated cellular damage/death. ANXA2-null mice showed significantly elevated protein oxidation in the liver and lung tissues compared to WT mice. ANXA2 depleted cancer cells showed enhanced cellular protein oxidationconcomitant with decreased tumor growth compared to control cancer cells andboth the oxidation of cellular proteins and tumor growth deficit werereversed by the antioxidant N-acetyl cysteine, indicating that ANXA2 plays akey role in the regulation of cellular redox during tumorigenesis. Ex-vivo human cancer studies showed that up-regulation of the reduced form of ANXA2 is associated with protection of the tumor proteins from oxidation. In summary, our results indicate that ANXA2 plays an important role incellular redox regulation by protecting cells from oxidative stress, aneffect that is particularly important during tumorigenesis.

Cancer stem cells (CSCs) are proposed to initiate cancer and propagate metastasis. Breast CSCs identified by aldehyde dehydrogenase (ALDH) activity are highly tumorigenic in xenograft models. However, in patient breast tumor immunohistological studies, where CSCs are identified by expression of ALDH isoform ALDH1A1, CSC prevalence is not correlative with metastasis, raising some doubt as to the role of CSCs in cancer. We characterized the expression of all 19 ALDH isoforms in patient breast tumor CSCs and breast cancer cell lines by total genome microarray expression analysis, immunofluorescence protein expression studies, and quantitative polymerase chain reaction. These studies revealed that ALDH activity of patient breast tumor CSCs and cell lines correlates best with expression of another isoform, ALDH1A3, not ALDH1A1. We performed shRNA knockdown experiments of the various ALDH isoforms and found that only ALDH1A3 knockdown uniformly reduced ALDH activity of breast cancer cells. Immunohistological studies with fixed patient breast tumor samples revealed that ALDH1A3 expression in patient breast tumors correlates significantly with tumor grade, metastasis, and cancer stage. Our results, therefore, identify ALDH1A3 as a novel CSC marker with potential clinical prognostic applicability, and demonstrate a clear correlation between CSC prevalence and the development of metastatic breast cancer.

All-trans retinoic acid (atRA) regulates gene expression and is used to treat acute promyelocytic leukemia. Attempts to use atRA in breast cancer without a stratification strategy have resulted in limited overall effectiveness. To identify biomarkers for the treatment of triple-negative breast cancer (TNBC) with atRA, we characterized the effects of atRA on the tumor growth of 13 TNBC cell lines. This resulted in a range of effects that was not predictable based on previously hypothesized predictors of response, such as the levels of atRA nuclear shuttling proteins fatty acid binding protein 5 (FABP5) and cellular retinoic acid binding protein 2 (CRABP2). Transcriptional profiling revealed that atRA induced distinct gene expression changes in the sensitive versus resistant cell lines that were mostly independent of the presence of retinoic acid response elements (RAREs) or peroxisome proliferator response elements (PPREs). Given the importance of DNA methylation in regulating gene expression, we hypothesized that differential DNA methylation could predict the response of TNBCs to atRA. We identified over 1400 sites that were differentially methylated between atRA resistant and sensitive cell lines. These CpG sites predicted the response of four TNBC patient-derived xenografts to atRA, and we utilized these xenografts to refine the profile and identified that as many as 17% of TNBC patients could benefit from atRA treatment. These data illustrate that differential methylation of specific CpGs may be useful biomarkers for predicting the response of patient tumors to atRA treatment.

We have identified a novel, epigenetic-based strategy to identify breast cancer patients who will benefit from an existing anticancer agent, all-trans retinoic acid (atRA). Triple-negative breast cancer (TNBC) is among the most aggressive breast cancers and lacks targeted therapies. TNBCs are a heterogeneous group of breast cancers with a wide range of gene expression profiles and drug sensitivities. Profiling these differences will lead to the development of more effective treatment options for TNBC. We hypothesize that atRA can be an effective therapy for a subset of TNBC patients. To test our hypothesis, we profiled the response of 12 TNBC cell lines to atRA using in vivo tumor growth assays. We found that atRA treatment had a range of effects on the tumor growth of TNBC cell lines: significantly decreasing tumor growth in HCC70, SUM149, and HCC1937, while increasing the growth of MDA-MB-231, MDA-MB-436, and Du4475 tumors. Gene expression and methylation analysis of these 12 cell lines revealed subtype-specific expression of atRA-inducible genes due to silencing by DNA methylation, e.g., of the atRA-inducible tumor-suppressor gene RARRES1. RARRES1 is silenced by methylation in cell lines where atRA promotes tumor growth, but is hypomethylated and expressed in cells that can be effectively treated with atRA. Use of the distinct gene expression and methylation profiles allowed us to accurately predict the response of 4 patient-derived xenografts (PDXs) to RA treatment. Continued classification of TNBCs by gene expression and DNA methylation can predict the response of patient tumors to RA treatment, thus identifying a novel targeted therapy strategy for TNBCs. Citation Format: Krysta M. Coyle, Cheryl A. Dean, Dejan Vidovic, Ian Weaver, Carman Giacomantonio, Lucy Helyer, Paola Marcato. DNA methylation predicts response of triple-negative breast cancer to all-trans retinoic acid treatment [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A07.

Curcumin, the principal curcuminoid of tumeric, has potent anticancer activity. To determine the mechanism of curcumin-induced cytotoxicity in prostate cancer cells, we exposed PC3 prostate carcinoma cells to 25 to 100 microM curcumin for 24 to 72 h. Curcumin treatment of PC3 cells caused time- and dose-dependent induction of apoptosis and depletion of cellular reduced glutathione (GSH). Exogenous GSH and its precursor N-acetyl-cysteine, but not ascorbic acid (AA) or ebselen, decreased curcumin accumulation in PC3 cells and also prevented curcumin-induced DNA fragmentation. The failure of AA and ebselen to protect PC3 cells from curcumin-induced apoptosis argued against the involvement of reactive oxygen species; rather, GSH-mediated inhibition of curcumin-induced cytotoxicity was due to reduced curcumin accumulation in PC3 cells. Curcumin-treated PC3 cells showed apoptosis-inducing cellular ceramide accumulation and activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase (JNK). Caspase-3, caspase-8, and caspase-9 were activated, and cytochrome c and apoptosis-inducing factor (AIF) were released from mitochondria following curcumin treatment. Interestingly, curcumin-induced apoptosis was not prevented by p38 MAPK, JNK, or caspase inhibition. We conclude that curcumin-induced cytotoxicity was due to cellular ceramide accumulation and damage to mitochondria that resulted in apoptosis mediated by AIF and other caspase-independent processes.

New cytotoxic agents are urgently needed for the treatment of advanced ovarian cancer because of the poor long-term response of this disease to conventional chemotherapy. Curcumin, obtained from the rhizome of Curcuma longa, has potent anticancer activity; however, the mechanism of curcumin-induced cytotoxicity in ovarian cancer cells remains a mystery. In this study we show that curcumin exhibited time- and dose-dependent cytotoxicity against monolayer cultures of ovarian carcinoma cell lines with differing p53 status (wild-type p53: HEY, OVCA429; mutant p53: OCC1; null p53: SKOV3). In addition, p53 knockdown or p53 inhibition did not diminish curcumin killing of HEY cells, confirming p53-independent cytotoxicity. Curcumin also killed OVCA429, and SKOV3 cells grown as multicellular spheroids. Nuclear condensation and fragmentation, as well as DNA fragmentation and poly (ADP-ribose) polymerase-1 cleavage in curcumin-treated HEY cells, indicated cell death by apoptosis. Procaspase-3, procaspase-8, and procaspase-9 cleavage, in addition to cytochrome c release and Bid cleavage into truncated Bid, revealed that curcumin activated both the extrinsic and intrinsic pathways of apoptosis. Bax expression was unchanged but Bcl-2, survivin, phosphorylated Akt (on serine 473), and total Akt were downregulated in curcumin-treated HEY cells. Curcumin also activated p38 mitogen-activated protein kinase (MAPK) without altering extracellular signal-regulated kinase 1/2 activity. We conclude that p53-independent curcumin-induced apoptosis in ovarian carcinoma cells involves p38 MAPK activation, ablation of prosurvival Akt signaling, and reduced expression of the antiapoptotic proteins Bcl-2 and survivin. These data provide a mechanistic rationale for the potential use of curcumin in the treatment of ovarian cancer.

Breast cancer subtyping, based on the expression of hormone receptors and other genes, can determine patient prognosis and potential options for targeted therapy. Among breast cancer subtypes, tumors of basal-like and claudin-low subtypes are typically associated with worse patient outcomes, are primarily classified as triple-negative breast cancers (TNBC), and cannot be treated with existing hormone-receptor-targeted therapies. Understanding the molecular basis of these subtypes will lead to the development of more effective treatment options for TNBC. In this study, we focus on retinoic acid receptor responder 1 (RARRES1) as a paradigm to determine if breast cancer subtype dictates protein function and gene expression regulation. Patient tumor dataset analysis and gene expression studies of a 26 cell-line panel, representing the five breast cancer subtypes, demonstrate that RARRES1 expression is greatest in basal-like TNBCs. Cell proliferation and tumor growth assays reveal that RARRES1 is a tumor suppressor in TNBC. Furthermore, gene expression studies, Illumina HumanMethylation450 arrays, and chromatin immunoprecipitation demonstrate that expression of RARRES1 is retained in basal-like breast cancers due to hypomethylation of the promoter. Additionally, expression of the cancer stem cell marker, aldehyde dehydrogenase 1A3, which provides the required ligand (retinoic acid) for RARRES1 transcription, is also specific to the basal-like subtype. We functionally demonstrate that the combination of promoter methylation and retinoic acid signaling dictates expression of tumor suppressor RARRES1 in a subtype-specific manner. These findings provide a precedent for a therapeutically-inducible tumor suppressor and suggest novel avenues of therapeutic intervention for patients with basal-like breast cancer.

To meet the needs of patients, Canadian surgical and medical oncology leaders in the treatment of peritoneal surface malignancies (PSMS), together with patient representatives, formed the Canadian HIPEC Collaborative Group (CHICG). The group is dedicated to standardizing and improving the treatment of PSM in Canada so that access to treatment and, ultimately, the prognosis of Canadian patients with PSM are improved. Patients with resectable PSM arising from colorectal or appendiceal neoplasms should be reviewed by a multidisciplinary team including surgeons and medical oncologists with experience in treating patients with PSM. Cytoreductive surgery and hyperthermic intraperitoneal chemotherapy should be offered to appropriately selected patients and performed at experienced centres. The aim of this publication is to present guidelines that we recommend be applied across the country for the treatment of PSM.

We have identified a novel strategy to identify breast cancer patients who will benefit from an existing anti-cancer agent, retinoic acid (RA). While RA has not yet achieved success in the treatment of breast cancers, we hypothesized that it can be an effective therapy for a subset of triple-negative breast cancer (TNBC) patients. TNBC is among the most aggressive breast cancers, and lacks targeted therapies. TNBCs can be further subtyped into basal-like and claudin-low, which differ in gene expression and drug sensitivities. Understanding the molecular basis of these subtypes will lead to the development of more effective treatment options for TNBC. To test this hypothesis, we performed tumor growth assays on TNBC cell lines and patient-derived xenografts (PDXs). We found that RA treatment decreased the tumor growth of four basal-like TNBC cell lines (MDA-MB-468, HCC70, SUM149, HCC1937). In contrast, RA increased the tumor growth of two claudin-low TNBC cell lines (MDA-MB-231, MDA-MB-436). Gene expression and methylation analysis of these affected cell lines revealed subtype-specific expression of RA-inducible genes due to silencing by DNA methylation, e.g. of the RA-inducible tumor-suppressor gene RARRES1. RARRES1 is silenced by methylation in claudin-low cell lines, but is hypomethylated and expressed in basal-like cells. Use of the subtype-specific expression and methylation profiles allowed us to accurately predict the response of 4 PDXs to RA treatment. Continued classification of TNBCs into these two subtypes will enable clinical use of RA, in part due to the subtype-specific hypomethylation of RA-inducible tumor suppressor genes including RARRES1. We have identified additional subtype-specific biomarkers which can predict the response of patient tumors to RA treatment, thus identifying a novel targeted therapy strategy for TNBCs. Citation Format: Krysta Mila Coyle, Cheryl A. Dean, Dejan Vidovic, Ian C. Weaver, Carman A. Giacomantonio, Paola Marcato. Retinoic acid: An effective therapy for basal-like breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3064. doi:10.1158/1538-7445.AM2017-3064

Colorectal cancer (CRC) is the third most common form of cancer and a leading cause of cancer-related mortality in both men and women, particularly in Western and developed nations. The high mortality rate has been attributed to the fact that colon cancer is often diagnosed at a late stage. Three primary subtypes of CRC have been described based on their molecular pathology and their underlying genetics: chromosomal instability (CIN), microsatellite instability (MSI), and CpG island hypermethylation. Over the last 30 years, molecular and genetic studies have determined a number of key genetic pathways that are subverted in CRC, including those involving APC, KRAS, and the p53 tumor suppressor. More recently, high-throughput, genome-wide studies have begun to characterize the broader genomic features of CRC. These high-throughput studies provide an ever expanding, and increasingly complex view of the molecular underpinnings of CRC. The chief goal of these studies being the identification of new therapeutic targets, as well as the definition of prognostic and diagnostic biological markers of CRC. Here, we highlight the key genetic and molecular pathways underlying CRC, as well as more recent insights into this disease uncovered by genomic studies.

Breast cancer stem cells (CSCs) can be identified by increased Aldefluor activity which is primarily due to aldehyde dehydrogenase 1A3 (ALDH1A3) expression. In addition to being a CSC marker, ALDH1A3 regulates genes expression via inducing retinoic acid (RA) signaling and plays an important role in mediating progression of cancers, including breast, melanoma, lung and glioblastoma. Therefore, ALDH1A3 represents a novel druggable anti-cancer target of interest. There are no currently characterized inhibitors of the ALDH1A3 isoform, so twelve general ALDH inhibitors were tested for their effectiveness in targeting ALDH1A3 activity. Ability to directly inhibit ALDH activity was quantified with the Aldefluor assay on a patient tumor xenograft and on breast cancer cell lines with known ALDH isoform activity. Inhibition of RA signaling was quantified by measuring expression of the RA-inducible genes RARRES1 and RARβ. To investigate the anti-cancer properties of these compounds, apoptosis was quantified with the annexin V assay. Citral significantly reduced ALDH1A3- and ALDH2-associated Aldefluor activity in breast cancer cell lines and also reduced Aldefluor activity of a patient-derived xenograft. Citral reduced expression of RA-inducible genes through reducing ALDH1A3 activity. Citral induced apoptosis in vitro in an ALDH-independent manner in breast cancer cell lines and reduced proliferation in the MDA-MB-231 breast cancer cell line. Nanoparticle (NP)-encapsulated citral was generated for in vivo use and administered intravenously to female NOD/SCID mice harbouring MDA-MB-231 ALDH1A3-overexpression tumors. 10mg/kg NP-citral significantly decreased the growth of ALDH1A3-driven MDA-MB-231 tumors. Nanoparticle-encapsulated citral shows promise as an adjuvant therapy for patients with tumors that have a large population of high-ALDH1A3 CSCs. Citation Format: Margaret L. Thomas, Roberto De Antueno, Krysta Coyle, Brianne Cruickshank, Michael Giacomantonio, Roy Duncan, Carman Giacomantonio, Paola Marcato. Citral reduces breast tumor growth by inhibiting cancer stem cell marker ALDH1A3. [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 2506.

The retinoic acid (RA) signalling pathway plays an important role in breast cancer progression and has either a pro-tumorigenic or tumor-suppressive role depending upon the effector function of RA-inducible genes that are expressed or epigenetically silenced. To study this paradigm in breast cancer, we focused on a controversial RA-inducible gene, the retinoic acid receptor responder 1 (RARRES1) protein that is often hypermethylated in cancer and has been reported to have tumor-suppressive function in prostate and nasopharyngeal carcinomas. However, in a study focused on a rare subtype of breast cancer, inflammatory breast cancer, RARRES1 is pro-tumorigenic. This functional discrepancy requires further investigation to determine its role in breast cancer in general. First, analysis of patient data sets revealed that RARRES1 is predominantly expressed in triple-negative breast cancers (TNBCs). Knockdown of RARRES1 in claudin-low MDA-MB-231 and basal-like MDA-MB-468 and HCC1937 significantly increased tumor growth and cell proliferation, suggesting RARRES1 has a tumor suppressive function in (TNBC), regardless of position on the differentiation hierarchy. Expression analyses of 24 breast cancer cell lines (including 18 TNBC and 2 normal-like cell lines) revealed that RARRES1 is predominantly expressed in basal-like TNBC cells We found that RARRES1 expression is dependent on, and strongly correlates with, the cancer stem cell marker, and RA-producing, ALDH1A3 in fixed breast cancer patient samples. Immunohistochemistry of the same patient tumor samples revealed RARRES1 expression is localized to the endoplasmic reticulum. Importantly, however, the presence of ALDH1A3 or RA is not sufficient to induce RARRES1 expression. RARRES1 is hypermethylated in claudin-low breast cancer cell lines, and release of this silencing is required for full induction of RARRES1 expression. We have identified sites of regulation by methylation in RARRES1 using Illumina 450K methylation arrays and 5-methylcytosine ChIP. We conclude that RARRES1 is an ALDH1A3/RA-inducible tumor suppressor in TNBC with methylation and expression profiles distinct to the differentiation hierarchy observed in breast cancer. Citation Format: Krysta M. Coyle, Patrick Murphy, Dejan Vidovic, Cheryl A. Dean, Margaret L. Thomas, Derek Clements, Mohammad Sultan, Ahmad Vaghar-Kashani, Carman Giacomantonio, Lucy Helyer, Ian Weaver, Shashi Gujar, Patrick WK Lee, Paola Marcato. ALDH1A3-inducible RARRES1 is a tumor suppressor in triple-negative breast cancer and is methylated in claudin-low breast cancers. [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 3661.

Curcumin from the rhizome of theCurcuma longa plant has chemopreventative activity and inhibits the growth of neoplastic cells. Since p53 has been suggested to be important for anticancer activity by curcumin, we investigated curcumin-induced cytotoxicity in cultures of p53(+/+) and p53(-/-) HCT-116 colon cancer cells, as well as mutant p53 HT-29 colon cancer cells. Curcumin killed wild-type p53 HCT-116 cells and mutant p53 HT-29 cells in a dose- and time-dependent manner. In addition, curcumin-treated p53(+/+) HCT-116 cells and mutant p53 HT-29 cells showed upregulation of total and activated p53, as well as increased expression of p53-regulated p21, PUMA (p53 upregulated modulator of apoptosis), and Bax; however, an equivalent cytotoxic effect by curcumin was observed in p53(+/+) and p53(-/-) HCT-116 cells, demonstrating that curcumin-induced cytotoxicity was independent of p53 status. Similar results were obtained when the cytotoxic effect of curcumin was assessed in wild-type p53 HCT-116 cells after siRNA-mediated p53 knockdown. Chromatin condensation, poly (ADP-ribose) polymerase-1 cleavage and reduced pro-caspase-3 levels in curcumin-treated p53(+/+) and p53(-/-) HCT-116 cells suggested that curcumin caused apoptosis. In addition, exposure to curcumin resulted in superoxide anion production and phosphorylation of oxidative stress proteins in p53(+/+) and p53(-/-) HCT-116 cells. Collectively, our results indicate that, despite p53 upregulation and activation, curcumin-induced apoptosis in colon cancer cells was independent of p53 status and involved oxidative stress. Curcumin may therefore have therapeutic potential in the management of colon cancer, especially in tumorsthatare resistant to conventional chemotherapydue todefects inp53 expression or function.

New cytotoxic agents are urgently needed for the treatment of advanced ovarian cancer because of the poor long-term response of this disease to conventional chemotherapy. Curcumin, obtained from the rhizome of Curcuma longa, has potent anticancer activity; however, the mechanism of curcumin-induced cytotoxicity in ovarian cancer cells remains a mystery. In this study we show that curcumin exhibited time- and dose-dependent cytotoxicity against monolayer cultures of ovarian carcinoma cell lines with differing p53 status (wild-type p53: HEY, OVCA429; mutant p53: OCC1; null p53: SKOV3). In addition, p53 knockdown or p53 inhibition did not diminish curcumin killing of HEY cells, confirming p53-independent cytotoxicity. Curcumin also killed OVCA429, and SKOV3 cells grown as multicellular spheroids. Nuclear condensation and fragmentation, as well as DNA fragmentation and poly (ADP-ribose) polymerase-1 cleavage in curcumin-treated HEY cells, indicated cell death by apoptosis. Procaspase-3, procaspase-8, and procaspase-9 cleavage, in addition to cytochrome c release and Bid cleavage into truncated Bid, revealed that curcumin activated both the extrinsic and intrinsic pathways of apoptosis. Bax expression was unchanged but Bcl-2, survivin, phosphorylated Akt (on serine 473), and total Akt were downregulated in curcumin-treated HEY cells. Curcumin also activated p38 mitogen-activated protein kinase (MAPK) without altering extracellular signal-regulated kinase 1/2 activity. We conclude that p53-independent curcumin-induced apoptosis in ovarian carcinoma cells involves p38 MAPK activation, ablation of prosurvival Akt signaling, and reduced expression of the antiapoptotic proteins Bcl-2 and survivin. These data provide a mechanistic rationale for the potential use of curcumin in the treatment of ovarian cancer.

Recent evidence suggests that cancer stem cells (CSCs) play an important role in cancer, as these cells possess enhanced tumor-forming capabilities and are resistant to current anticancer therapies. Hence, novel cancer therapies will need to be tested for both tumor regression and CSC targeting. Herein we show that oncolytic reovirus that induces regression of human breast cancer primary tumor samples xenografted in immunocompromised mice also effectively targets and kills CSCs in these tumors. CSCs were identified based on CD24(-)CD44(+) cell surface expression and overexpression of aldehyde dehydrogenase. Upon reovirus treatment, the CSC population was reduced at the same rate as non-CSCs within the tumor. Immunofluorescence of breast tumor tissue samples from the reovirus- and mock-treated mice confirmed that both CSCs and non-CSCs were infectible by reovirus, and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay showed that both populations died by apoptosis. Ras, which has been shown to mediate reovirus oncolysis, was found to be present at similar levels in all cell types, and this is consistent with their comparable sensitivity to reovirus. These experiments indicate that oncolytic reovirus has the potential to induce tumor regression in breast cancer patients. More important, the CSC population was equally reduced and was as susceptible to reovirus treatment as the non-CSC population.