CNS-Native Myeloid Cells Drive Immune Suppression in the Brain Metastatic Niche through Cxcl10.

Brain metastasis (br-met) develops in an immunologically unique br-met niche. Central nervous system-native myeloid cells (CNS-myeloids) and bone-marrow-derived myeloid cells (BMDMs) cooperatively regulate brain immunity. The phenotypic heterogeneity and specific roles of these myeloid subsets in shaping the br-met niche to regulate br-met outgrowth have not been fully revealed. Applying multimodal single-cell analyses, we elucidated a heterogeneous but spatially defined CNS-myeloid response during br-met outgrowth. We found Ccr2+ BMDMs minimally influenced br-met while CNS-myeloid promoted br-met outgrowth. Additionally, br-met-associated CNS-myeloid exhibited downregulation of Cx3cr1. Cx3cr1 knockout in CNS-myeloid increased br-met incidence, leading to an enriched interferon response signature and Cxcl10 upregulation. Significantly, neutralization of Cxcl10 reduced br-met, while rCxcl10 increased br-met and recruited VISTAHi PD-L1+ CNS-myeloid to br-met lesions. Inhibiting VISTA- and PD-L1-signaling relieved immune suppression and reduced br-met burden. Our results demonstrate that loss of Cx3cr1 in CNS-myeloid triggers a Cxcl10-mediated vicious cycle, cultivating a br-met-promoting, immune-suppressive niche.

Role of the androgen receptor in breast cancer and preclinical analysis of enzalutamide.

INTRODUCTION: The androgen receptor (AR) is widely expressed in breast cancers and has been proposed as a therapeutic target in estrogen receptor alpha (ER) negative breast cancers that retain AR. However, controversy exists regarding the role of AR, particularly in ER + tumors. Enzalutamide, an AR inhibitor that impairs nuclear localization of AR, was used to elucidate the role of AR in preclinical models of ER positive and negative breast cancer. METHODS: We examined nuclear AR to ER protein ratios in primary breast cancers in relation to response to endocrine therapy. The effects of AR inhibition with enzalutamide were examined in vitro and in preclinical models of ER positive and negative breast cancer that express AR. RESULTS: In a cohort of 192 women with ER + breast cancers, a high ratio of AR:ER (≥2.0) indicated an over four fold increased risk for failure while on tamoxifen (HR = 4.43). The AR:ER ratio had an independent effect on risk for failure above ER % staining alone. AR:ER ratio is also an independent predictor of disease-free survival (HR = 4.04, 95% CI: 1.68, 9.69; p = 0.002) and disease specific survival (HR = 2.75, 95% CI: 1.11, 6.86; p = 0.03). Both enzalutamide and bicalutamide inhibited 5-alpha-dihydrotestosterone (DHT)-mediated proliferation of breast cancer lines in vitro; however, enzalutamide uniquely inhibited estradiol (E2)-mediated proliferation of ER+/AR + breast cancer cells. In MCF7 xenografts (ER+/AR+) enzalutamide inhibited E2-driven tumor growth as effectively as tamoxifen by decreasing proliferation. Enzalutamide also inhibited DHT- driven tumor growth in both ER positive (MCF7) and negative (MDA-MB-453) xenografts, but did so by increasing apoptosis. CONCLUSIONS: AR to ER ratio may influence breast cancer response to traditional endocrine therapy. Enzalutamide elicits different effects on E2-mediated breast cancer cell proliferation than bicalutamide. This preclinical study supports the initiation of clinical studies evaluating enzalutamide for treatment of AR+ tumors regardless of ER status, since it blocks both androgen- and estrogen- mediated tumor growth.

The miR-200 and miR-221/222 microRNA families: opposing effects on epithelial identity.

Carcinogenesis is a complex process during which cells undergo genetic and epigenetic alterations. These changes can lead tumor cells to acquire characteristics that enable movement from the primary site of origin when conditions become unfavorable. Such characteristics include gain of front-rear polarity, increased migration/invasion, and resistance to anoikis, which facilitate tumor survival during metastasis. An epithelial to mesenchymal transition (EMT) constitutes one way that cancer cells can gain traits that promote tumor progression and metastasis. Two microRNA (miRNA) families, the miR-200 and miR-221 families, play crucial opposing roles that affect the differentiation state of breast cancers. These two families are differentially expressed between the luminal A subtype of breast cancer as compared to the less well-differentiated triple negative breast cancers (TNBCs) that exhibit markers indicative of an EMT. The miR-200 family promotes a well-differentiated epithelial phenotype, while high miR-221/222 results in a poorly differentiated, mesenchymal-like phenotype. This review focuses on the mechanisms (specific proven targets) by which these two miRNA families exert opposing effects on cellular plasticity during breast tumorigenesis and metastasis.

Targeting CXCL16 and STAT1 augments immune checkpoint blockade therapy in triple-negative breast cancer.

Chemotherapy prior to immune checkpoint blockade (ICB) treatment appears to improve ICB efficacy but resistance to ICB remains a clinical challenge and is attributed to highly plastic myeloid cells associating with the tumor immune microenvironment (TIME). Here we show by CITE-seq single-cell transcriptomic and trajectory analyses that neoadjuvant low-dose metronomic chemotherapy (MCT) leads to a characteristic co-evolution of divergent myeloid cell subsets in female triple-negative breast cancer (TNBC). Specifically, we identify that the proportion of CXCL16 + myeloid cells increase and a high STAT1 regulon activity distinguishes Programmed Death Ligand 1 (PD-L1) expressing immature myeloid cells. Chemical inhibition of STAT1 signaling in MCT-primed breast cancer sensitizes TNBC to ICB treatment, which underscores the STAT1's role in modulating TIME. In summary, we leverage single-cell analyses to dissect the cellular dynamics in the tumor microenvironment (TME) following neoadjuvant chemotherapy and provide a pre-clinical rationale for modulating STAT1 in combination with anti-PD-1 for TNBC patients.

Targets of miR-200c mediate suppression of cell motility and anoikis resistance.

INTRODUCTION: miR-200c and other members of the miR-200 family promote epithelial identity by directly targeting ZEB1 and ZEB2, which repress E-cadherin and other genes involved in polarity. Loss of miR-200c is often observed in carcinoma cells that have undergone epithelial to mesenchymal transition (EMT). Restoration of miR-200c to such cells leads to a reduction in stem cell-like characteristics, reduced migration and invasion, and increased sensitivity to taxanes. Here we investigate the functional role of novel targets of miR-200c in the aggressive behavior of breast and endometrial cancer cells. METHODS: Putative target genes of miR-200c identified by microarray profiling were validated as direct targets using dual luciferase reporter assays. Following restoration of miR-200c to triple negative breast cancer and type 2 endometrial cancer cell lines that had undergone EMT, levels of endogenous target mRNA and respective protein products were measured. Migration and sensitivity to anoikis were determined using wound healing assays or cell-death ELISAs and viability assays respectively. RESULTS: We found that restoration of miR-200c suppresses anoikis resistance, a novel function for this influential miRNA. We identified novel targets of miR-200c, including genes encoding fibronectin 1 (FN1), moesin (MSN), neurotrophic tyrosine receptor kinase type 2 (NTRK2 or TrkB), leptin receptor (LEPR), and Rho GTPase activating protein 19 (ARHGAP19). These targets all encode proteins normally expressed in cells of mesenchymal or neuronal origin; however, in carcinoma cells that lack miR-200c they become aberrantly expressed and contribute to the EMT phenotype and aggressive behavior. We showed that these targets are inhibited upon restoration of miR-200c to aggressive breast and endometrial cancer cells. We demonstrated that inhibition of MSN and/or FN1 is sufficient to mediate the ability of miR-200c to suppress cell migration. Lastly, we showed that targeting of TrkB mediates the ability of miR-200c to restore anoikis sensitivity. CONCLUSIONS: miR-200c maintains the epithelial phenotype not only by targeting ZEB1/2, which usually facilitates restoration of E-cadherin expression, but also by actively repressing a program of mesenchymal and neuronal genes involved in cell motility and anoikis resistance.

Rab11b-mediated integrin recycling promotes brain metastatic adaptation and outgrowth.

Breast cancer brain metastases (BCBM) have a 5-20 year latency and account for 30% of mortality; however, mechanisms governing adaptation to the brain microenvironment remain poorly defined. We combine time-course RNA-sequencing of BCBM development with a Drosophila melanogaster genetic screen, and identify Rab11b as a functional mediator of metastatic adaptation. Proteomic analysis reveals that Rab11b controls the cell surface proteome, recycling proteins required for successful interaction with the microenvironment, including integrin ß1. Rab11b-mediated control of integrin ß1 surface expression allows efficient engagement with the brain ECM, activating mechanotransduction signaling to promote survival. Lipophilic statins prevent membrane association and activity of Rab11b, and we provide proof-of principle that these drugs prevent breast cancer adaptation to the brain microenvironment. Our results identify Rab11b-mediated recycling of integrin ß1 as regulating BCBM, and suggest that the recycleome, recycling-based control of the cell surface proteome, is a previously unknown driver of metastatic adaptation and outgrowth.

Combined Scaffold Evaluation and Systems-Level Transcriptome-Based Analysis for Accelerated Lead Optimization Reveals Ribosomal Targeting Spirooxindole Cyclopropanes.

With evolutionary drug resistance impacting efforts to treat disease, the need for small molecules that exhibit novel molecular mechanisms of action is paramount. In this study, we combined scaffold-directed synthesis with a hybrid experimental and transcriptome analysis to identify bis-spirooxindole cyclopropanes that inhibit cancer cell proliferation through disruption of ribosomal function. These findings demonstrate the value of an integrated, biologically inspired synthesis and assay strategy for the accelerated identification of first-in-class cancer therapeutic candidates.

Death effector domain-containing protein induces vulnerability to cell cycle inhibition in triple-negative breast cancer.

Lacking targetable molecular drivers, triple-negative breast cancer (TNBC) is the most clinically challenging subtype of breast cancer. In this study, we reveal that Death Effector Domain-containing DNA-binding protein (DEDD), which is overexpressed in > 60% of TNBCs, drives a mitogen-independent G1/S cell cycle transition through cytoplasm localization. The gain of cytosolic DEDD enhances cyclin D1 expression by interacting with heat shock 71 kDa protein 8 (HSC70). Concurrently, DEDD interacts with Rb family proteins and promotes their proteasome-mediated degradation. DEDD overexpression renders TNBCs vulnerable to cell cycle inhibition. Patients with TNBC have been excluded from CDK 4/6 inhibitor clinical trials due to the perceived high frequency of Rb-loss in TNBCs. Interestingly, our study demonstrated that, irrespective of Rb status, TNBCs with DEDD overexpression exhibit a DEDD-dependent vulnerability to combinatorial treatment with CDK4/6 inhibitor and EGFR inhibitor in vitro and in vivo. Thus, our study provided a rationale for the clinical application of CDK4/6 inhibitor combinatorial regimens for patients with TNBC.

Tumor-induced Stromal STAT1 Accelerates Breast Cancer via Deregulating Tissue Homeostasis.

The tumor microenvironment (TME), the dynamic tissue space in which the tumor exists, plays a significant role in tumor initiation, and is a key contributor in cancer progression; however, little is known about tumor-induced changes in the adjacent tissue stroma. Herein, tumor-induced changes in the TME were explored at the morphologic and molecular level to further understand cancer progression. Tumor-adjacent mammary glands (TAG) displayed altered branching morphology, expansion of myofibroblasts, and increased mammosphere formation, broadly suggesting a tumor-induced field effect. FACS analysis of TAGs demonstrated an increased number of Lin-CD24+/CD49+ enriched mammary gland stem cells (MaSC), suggesting deregulated tissue homeostasis in TAGs. Comparative transcriptome analysis of TAGs and contralateral control glands coupled with meta-analysis on differentially expressed genes with two breast cancer stromal patient microarray datasets identified shared upregulation of STAT1. Knockdown of STAT1 in cancer-associated fibroblast (CAF) cocultured with human breast cancer cells altered cancer cell proliferation, indicating a role for STAT1 as a stromal contributor of tumorigenesis. Furthermore, depletion of STAT1 in CAFs significantly reduced periductal reactive fibrosis and delayed early breast cancer progression in vivo Finally, cotreatment with fludarabine, a FDA-approved STAT1 activation inhibitor and DNA synthesis inhibitor, in combination with doxorubicin, showed enhanced therapeutic efficacy in treating mouse mammary gland tumors. Taken together, these results demonstrate that stromal STAT1 expression promotes tumor progression and is a potential therapeutic target for breast cancer.Implications: Tumors induce stromal STAT1-dependent cytokine secretion that promotes tumor cell proliferation and can be targeted using clinically-approved inhibitors of STAT1. Mol Cancer Res; 15(5); 585-97. ©2017 AACR.

GAD1 Upregulation Programs Aggressive Features of Cancer Cell Metabolism in the Brain Metastatic Microenvironment.

The impact of altered amino acid metabolism on cancer progression is not fully understood. We hypothesized that a metabolic transcriptome shift during metastatic evolution is crucial for brain metastasis. Here, we report a powerful impact in this setting caused by epigenetic upregulation of glutamate decarboxylase 1 (GAD1), a regulator of the GABA neurotransmitter metabolic pathway. In cell-based culture and brain metastasis models, we found that downregulation of the DNA methyltransferase DNMT1 induced by the brain microenvironment-derived clusterin resulted in decreased GAD1 promoter methylation and subsequent upregulation of GAD1 expression in brain metastatic tumor cells. In a system to dynamically visualize cellular metabolic responses mediated by GAD1, we monitored the cytosolic NADH:NAD+ equilibrium in tumor cells. Reducing GAD1 in metastatic cells by primary glia cell coculture abolished the capacity of metastatic cells to utilize extracellular glutamine, leading to cytosolic accumulation of NADH and increased oxidative status. Similarly, genetic or pharmacologic disruption of the GABA metabolic pathway decreased the incidence of brain metastasis in vivo Taken together, our results show how epigenetic changes in GAD1 expression alter local glutamate metabolism in the brain metastatic microenvironment, contributing to a metabolic adaption that facilitates metastasis outgrowth in that setting. Cancer Res; 77(11); 2844-56. ©2017 AACR.

Metformin-induced killing of triple-negative breast cancer cells is mediated by reduction in fatty acid synthase via miRNA-193b.

The anti-diabetic drug metformin (1,1-dimethylbiguanide hydrochloride) reduces both the incidence and mortality of several types of cancer. Metformin has been shown to selectively kill cancer stem cells, and triple-negative breast cancer (TNBC) cell lines are more sensitive to the effects of metformin as compared to luminal breast cancer. However, the mechanism underlying the enhanced susceptibility of TNBC to metformin has not been elucidated. Expression profiling of metformin-treated TNBC lines revealed fatty acid synthase (FASN) as one of the genes most significantly downregulated following 24 h of treatment, and a decrease in FASN protein was also observed. Since FASN is critical for de novo fatty acid synthesis and is important for the survival of TNBC, we hypothesized that FASN downregulation facilitates metformin-induced apoptosis. Profiling studies also exposed a rapid metformin-induced increase in miR-193 family members, and miR-193b directly targets the FASN 3'UTR. Addition of exogenous miR-193b mimic to untreated TNBC cells decreased FASN protein expression and increased apoptosis of TNBC cells, while spontaneously immortalized, non-transformed breast epithelial cells remained unaffected. Conversely, antagonizing miR-193 activity impaired the ability of metformin to decrease FASN and cause cell death. Further, the metformin-stimulated increase in miR-193 resulted in reduced mammosphere formation by TNBC lines. These studies provide mechanistic insight into metformin-induced killing of TNBC.

MicroRNA regulation of epithelial plasticity in cancer.

MicroRNAs (miRNAs) are small non-coding RNA molecules capable of regulating multiple target genes through repression of mRNA stability or translation. miRNAs regulate a wide variety of developmental and pathological processes, and many have oncogenic or tumor suppressor activity. Carcinoma cells can undergo changes resembling a partial or transient epithelial-mesenchymal transition (EMT) as part of the metastatic cascade. Numerous miRNAs regulate EMT by targeting genes that control epithelial or mesenchymal characteristics. The involvement of miRNAs in feedback loops with EMT-regulating transcription factors reveals a critical role in governing epithelial plasticity. In this review we summarize miRNAs involved in carcinoma cell plasticity.

miR-200c targets a NF-κB up-regulated TrkB/NTF3 autocrine signaling loop to enhance anoikis sensitivity in triple negative breast cancer.

Anoikis is apoptosis initiated upon cell detachment from the native extracellular matrix. Since survival upon detachment from basement membrane is required for metastasis, the ability to resist anoikis contributes to the metastatic potential of breast tumors. miR-200c, a potent repressor of epithelial to mesenchymal transition, is expressed in luminal breast cancers, but is lost in more aggressive basal-like, or triple negative breast cancers (TNBC). We previously demonstrated that miR-200c restores anoikis sensitivity to TNBC cells by directly targeting the neurotrophic receptor tyrosine kinase, TrkB. In this study, we identify a TrkB ligand, neurotrophin 3 (NTF3), as capable of activating TrkB to induce anoikis resistance, and show that NTF3 is also a direct target of miR-200c. We present the first evidence that anoikis resistant TNBC cells up-regulate both TrkB and NTF3 when suspended, and show that this up-regulation is necessary for survival in suspension. We further demonstrate that NF-κB activity increases 6 fold in suspended TNBC cells, and identify RelA and NF-κB1 as the transcription factors responsible for suspension-induced up-regulation of TrkB and NTF3. Consequently, inhibition of NF-κB activity represses anoikis resistance. Taken together, our findings define a critical mechanism for transcriptional and post-transcriptional control of suspension-induced up-regulation of TrkB and NTF3 in anoikis resistant breast cancer cells.

Progestin regulated miRNAs that mediate progesterone receptor action in breast cancer.

Progesterone receptors (PRs) mediate response to progestins in the normal breast and breast cancer. To determine if liganded PR regulate microRNAs (miRNAs) as a component of their action, we profiled mature miRNA levels following progestin treatment. Indeed, 28 miRNAs are significantly altered by 6h of progestin treatment. Many progestin-responsive genes are putative targets of progestin-regulated miRNAs; for example, progestin treatment decreases miR-29, thereby relieving repression of one of its direct targets, the gene encoding ATPase, Na(+)/K(+) transporting, beta 1 polypeptide (ATP1B1). Thus, liganded PR regulates ATP1B1 through sites in the promoter and the 3'UTR, to achieve maximal tight hormonal regulation of ATP1B1 protein via both transcriptional and translational control. We find that ATP1B1 serves to limit migration and invasion in breast cancer cells. Lastly, we demonstrate that PR itself is regulated by a progestin-upregulated miRNA, miR-513a-5p, providing a novel mechanism for tight control of PR protein expression.

Restoration of miR-200c to ovarian cancer reduces tumor burden and increases sensitivity to paclitaxel.

A therapeutic intervention that could decrease tumor burden and increase sensitivity to chemotherapy would have a significant impact on the high morbidity rate associated with ovarian cancer. miRNAs have emerged as potential therapeutic candidates due to their ability to downregulate multiple targets involved in tumor progression and chemoresistance. miRNA-200c (miR-200c) is downregulated in ovarian cancer cell lines and stage III ovarian tumors, and low miR-200c correlates with poor prognosis. miR-200c increases sensitivity to taxanes in vitro by targeting class III ß-tubulin gene (TUBB3), a tubulin known to mediate chemoresistance. Indeed, we find that patients with tumors having low TUBB3 had significantly prolonged survival (average survival 52.73 ± 4.08 months) as compared with those having high TUBB3 (average survival 42.56 ± 3.19 months). miR-200c also targets TrkB, a mediator of resistance to anoikis. We show that restoration of miR-200c to ovarian cancer cells results in increased anoikis sensitivity and reduced adherence to biologic substrates in vitro. Because both chemo- and anoikis-resistance are critical steps in the progression of ovarian cancer, we sought to determine how restoration of miR-200c affects tumor burden and chemosensitivity in an in vivo preclinical model of ovarian cancer. Restoration of miR-200c in an intraperitoneal xenograft model of human ovarian cancer results in decreased tumor formation and tumor burden. Furthermore, even in established tumors, restoration of miR-200c, alone or in combination with paclitaxel, results in significantly decreased tumor burden. Our study suggests that restoration of miR-200c immediately before cytotoxic chemotherapy may allow for a better response or lower effective dose.

Loss of miR-200c: A Marker of Aggressiveness and Chemoresistance in Female Reproductive Cancers.

We focus on unique roles of miR-200c in breast, ovarian, and endometrial cancers. Members of the miR-200 family target ZEB1, a transcription factor which represses E-cadherin and other genes involved in polarity. We demonstrate that the double negative feedback loop between miR-200c and ZEB1 is functional in some, but not all cell lines. Restoration of miR-200c to aggressive cancer cells causes a decrease in migration and invasion. These effects are independent of E-cadherin status. Additionally, we observe that restoration of miR-200c to ovarian cancer cells causes a decrease in adhesion to laminin. We have previously reported that reintroduction of miR-200c to aggressive cells that lack miR-200c expression restores sensitivity to paclitaxel. We now prove that this ability is a result of direct targeting of class III beta-tubulin (TUBB3). Introduction of a TUBB3 expression construct lacking the miR-200c target site into cells transfected with miR-200c mimic results in no change in sensitivity to paclitaxel. Lastly, we observe a decrease in proliferation in cells transfected with miR-200c mimic, and cells where ZEB1 is knocked down stably, demonstrating that the ability of miR-200c to enhance sensitivity to paclitaxel is not due to an increased proliferation rate.

MicroRNAs link estrogen receptor alpha status and Dicer levels in breast cancer.

To identify microRNAs (miRNAs) associated with estrogen receptor (ESR1) status, we profiled luminal A, ESR1+ breast cancer cell lines versus triple negative (TN), which lack ERα, progesterone receptor and Her2/neu. Although two thirds of the differentially expressed miRNAs are higher in ESR1+ breast cancer cells, some miRNAs, such as miR-222/221 and miR-29a, are dramatically higher in ESR1- cells (∼100- and 16-fold higher, respectively). MiR-222/221 (which target ESR1 itself) and miR-29a are predicted to target the 3' UTR of Dicer1. Addition of these miRNAs to ESR1+ cells reduces Dicer protein, whereas antagonizing miR-222 in ESR1- cells increases Dicer protein. We demonstrate via luciferase reporter assays that these miRNAs directly target the Dicer1 3' UTR. In contrast, miR-200c, which promotes an epithelial phenotype, is 58-fold higher in the more well-differentiated ERα+ cells, and restoration of miR-200c to ERα- cells causes increased Dicer protein, resulting in increased levels of other mature miRNAs typically low in ESR1- cells. Together, our findings explain why Dicer is low in ERα negative breast cancers, since such cells express high miR-221/222 and miR-29a levels (which repress Dicer) and low miR-200c (which positively affect Dicer levels). Furthermore, we find that miR-7, which is more abundant in ERα+ cells and is estrogen regulated, targets growth factor receptors and signaling intermediates such as EGFR, IGF1R, and IRS-2. In summary, miRNAs differentially expressed in ERα+ versus ERα- breast cancers actively control some of the most distinguishing characteristics of the luminal A and TN subtypes, such as ERα itself, Dicer, and growth factor receptor levels.

MicroRNA-200c mitigates invasiveness and restores sensitivity to microtubule-targeting chemotherapeutic agents.

The transcription factor ZEB1 is normally not expressed in epithelial cells. When inappropriately expressed in carcinomas, ZEB1 initiates epithelial to mesenchymal transition due to its ability to repress E-cadherin and other genes involved in polarity. Recently, ZEB1 and ZEB2 have been identified as direct targets of the microRNA-200c family. We find that miR-200c levels are high in well-differentiated endometrial, breast, and ovarian cancer cell lines, but extremely low in poorly differentiated cancer cells. Low or absent miR-200c results in aberrant expression of ZEB1 and consequent repression of E-cadherin. Reinstatement of miR-200c to such cells restores E-cadherin and dramatically reduces migration and invasion. Microarray profiling reveals that in addition to ZEB1 and ZEB2, other mesenchymal genes (such as FN1, NTRK2, and QKI), which are also predicted direct targets of miR-200c, are indeed inhibited by addition of exogenous miR-200c. One such gene, class III ß-tubulin (TUBB3), which encodes a tubulin isotype normally found only in neuronal cells, is a direct target of miR-200c. This finding is of particular significance because we show that restoration of miR-200c increases sensitivity to microtubule-targeting agents by 85%. Because expression of TUBB3 is a common mechanism of resistance to microtubule-binding chemotherapeutic agents in many types of solid tumors, the ability of miR-200c to restore chemosensitivity to such agents may be explained by its ability to reduce TUBB3. Because miR-200c is crucial for maintenance of epithelial identity, behavior, and sensitivity to chemotherapy, we propose that it warrants further investigation as a therapeutic strategy for aggressive, drug-resistant cancers.