Drive Cancer Growth Research Articles

PU.1-driven Th9 Cells Promote Colorectal Cancer in Experimental Colitis Models Through Il-6 Effects in Intestinal Epithelial Cells.

Colorectal cancer [CRC] is one of the most frequent malignancies, but the molecular mechanisms driving cancer growth are incompletely understood. We characterised the roles of the cytokine IL-9 and Th9 cells in regulating CRC development. CRC patient samples and samples from AOM/DSS treated mice were analysed for expression of IL-9, CD3, and PU.1 by FACS analysis and immunohistochemistry. IL-9 citrine reporter mice, IL-9 knockout mice, and PU.1 and GATA3 CD4-Cre conditional knockout mice were studied in the AOM/DSS model. DNA minicircles or hyper-IL-6 were used for overexpression of cytokines in vivo. Effects of IL-6 and IL-9 were determined in organoid and T cell cultures. Claudin2/3 expression was studied by western blotting and bacterial translocation by FISH. We uncovered a significant expansion of IL-9- and PU.1-expressing mucosal Th9 cells in CRC patients, with particularly high levels in patients with colitis-associated neoplasias. PU.1+ Th9 cells accumulated in experimental colorectal neoplasias. Deficiency of IL-9 or inactivation of PU.1 in T cells led to impaired tumour growth in vivo, suggesting a protumoral role of Th9 cells. In contrast, GATA3 inactivation did not affect Th9-mediated tumour growth. Mechanistically, IL-9 controls claudin2/3 expression and T cell-derived IL-6 production in colorectal tumours. IL-6 abrogated the anti-proliferative effects of IL-9 in epithelial organoids in vivo. IL-9-producing Th9 cells expand in CRC and control IL-6 production by T cells. IL-9 is a crucial regulator of tumour growth in colitis-associated neoplasias and emerges as potential target for therapy.

Discovery of new therapeutic targets in ovarian cancer through identifying significantly non-mutated genes

BackgroundMutated and non-mutated genes interact to drive cancer growth and metastasis. While research has focused on understanding the impact of mutated genes on cancer biology, understanding non-mutated genes that are essential to tumor development could lead to new therapeutic strategies. The recent advent of high-throughput whole genome sequencing being applied to many different samples has made it possible to calculate if genes are significantly non-mutated in a specific cancer patient cohort.MethodsWe carried out random mutagenesis simulations of the human genome approximating the regions sequenced in the publicly available Cancer Growth Atlas Project for ovarian cancer (TCGA-OV). Simulated mutations were compared to the observed mutations in the TCGA-OV cohort and genes with the largest deviations from simulation were identified. Pathway analysis was performed on the non-mutated genes to better understand their biological function. We then compared gene expression, methylation and copy number distributions of non-mutated and mutated genes in cell lines and patient data from the TCGA-OV project. To directly test if non-mutated genes can affect cell proliferation, we carried out proof-of-concept RNAi silencing experiments of a panel of nine selected non-mutated genes in three ovarian cancer cell lines and one primary ovarian epithelial cell line.ResultsWe identified a set of genes that were mutated less than expected (non-mutated genes) and mutated more than expected (mutated genes). Pathway analysis revealed that non-mutated genes interact in cancer associated pathways. We found that non-mutated genes are expressed significantly more than mutated genes while also having lower methylation and higher copy number states indicating that they could be functionally important. RNAi silencing of the panel of non-mutated genes resulted in a greater significant reduction of cell viability in the cancer cell lines than in the non-cancer cell line. Finally, as a test case, silencing ANKLE2, a significantly non-mutated gene, affected the morphology, reduced migration, and increased the chemotherapeutic response of SKOV3 cells.ConclusionWe show that we can identify significantly non-mutated genes in a large ovarian cancer cohort that are well-expressed in patient and cell line data and whose RNAi-induced silencing reduces viability in three ovarian cancer cell lines. Targeting non-mutated genes that are important for tumor growth and metastasis is a promising approach to expand cancer therapeutic options.

PIK3CA-related overgrowth: silver bullets from the cancer arsenal?

Mutations that activate growth factor signaling often drive cancer growth. Many also arise in isolation, causing developmental growth disorders. PIK3CA, that encodes a catalytic subunit of phosphatidylinositol 3-kinase (PI3K), is a cardinal example of this paradigm. Recent exciting progress towards the key goal of cancer drug repurposing for PIK3CA-driven overgrowth is discussed.

MiRNAs as Modulators of Cholesterol in Breast Cancer Stem Cells: An Approach to Overcome Drug Resistance in Cancer.

It has been postulated that a small number of Cancer Stem Cells (CSCs) buried in tumour mass drive cancer growth and impart cancer drug resistance. However, their eradication has not been achieved so far as the mechanistic understanding of CSCs' role in cancer development and growth is limited. The cholesterol accumulation and efflux processes have been shown to play an important role in maintaining cell's integrity and its sensitivity towards drugs, as altered cholesterol pathways contribute to cancer drug resistance. Emerging pieces of evidence have indicated miRNAs as regulators of CSCs, and also as regulators of cholesterol pathways in cancer cells, but a link between the two has not been fully established so far. In this review, we have collated key signalling pathways and published evidence emphasising the involvement of miRNAs and cholesterol in CSCs related drug resistance. Additionally, we have used bioinformatics analysis to identify miRNAs that may modulate cholesterol pathways in CSCs at a molecular level to contribute to cancer drug resistance. Our results show that two miRNAs (hsa-miR-34a-5p and hsa-miR-373-3p) interact and bind to two known Breast CSC markers (CD44 and CD24) and mediate the expression of several cholesterol-related genes (INSIG2, APOL2, CYP51A1, HDLB, and DHCR7). Furthermore, survival analysis of the breast cancer patients' gene expression data revealed that higher expression of these genes is associated with poor disease-free survival. We, therefore, propose that targeting these two miRNAs could possibly provide a way to alter cell's response to drugs via modulating cholesterol pathways in CSCs.

Computational approaches for evaluation of isobavachin as potential inhibitor against t877a and w741l mutations in prostate cancer

Prostate cancer is the World’s second most common cancer, with the fifth-highest male mortality rate. Point mutations such as T877A and W741L are frequently seen in advanced prostate cancer patients, conferring drug-resistance and hence driving cancer growth. Such occurrence of drug resistance in prostate cancer necessitates designing of suitable ligands to ensure better interactions with the receptors which can block the progression of the disease. The present study focus on the modification of plant-derived flavonoids that might act as inhibitors against such point mutations namely, T877A and W741L. In T877A mutation threonine is substituted by alanine at the 877 codon and W741L mutation, tryptophan is substituted by lysine at the 741 codon in prostate cancer. The study revolved on the aspect of the evaluation of Isobavachin and its derivatives as a potential agent to tackle such point mutations by using the in silico approach. A total of 98 molecular dockings were performed to find the ligand-receptor complexes with the lowest binding energy employing Autodock Software to conduct the blind and site-specific docking. Additionally, ligands were screened for Drug-likeness and toxicity using several tools yielding eight possible drug candidates. Based on the results of Molecular Docking, Drug-likeness, and ADMET testing, ten structures, including six complexes and three receptors were subjected to molecular dynamics simulation of 100 ns covering RMSD, RMSF, Rg, and MM/PBSA. Based on the simulation results, Isobavachin, IsoMod4, and IsoMod7 were concluded to be stable and exhibited potential properties for developing a novel drug to combat prostate cancer and its associated drug-resistance. Communicated by Ramaswamy H. Sarma

The KRAS-regulated kinome identifies WEE1 and ERK coinhibition as a potential therapeutic strategy in KRAS-mutant pancreatic cancer

Oncogenic KRAS drives cancer growth by activating diverse signaling networks, not all of which have been fully delineated. We set out to establish a system-wide profile of the KRAS-regulated kinase signaling network (kinome) in KRAS-mutant pancreatic ductal adenocarcinoma (PDAC). We knocked down KRAS expression in a panel of six cell lines and then applied multiplexed inhibitor bead/MS to monitor changes in kinase activity and/or expression. We hypothesized that depletion of KRAS would result in downregulation of kinases required for KRAS-mediated transformation and in upregulation of other kinases that could potentially compensate for the deleterious consequences of the loss of KRAS. We identified 15 upregulated and 13 downregulated kinases in common across the panel of cell lines. In agreement with our hypothesis, all 15 of the upregulated kinases have established roles as cancer drivers (e.g., SRC, TGF-β1, ILK), and pharmacological inhibition of one of these upregulated kinases, DDR1, suppressed PDAC growth. Interestingly, 11 of the 13 downregulated kinases have established driver roles in cell cycle progression, particularly in mitosis (e.g., WEE1, Aurora A, PLK1). Consistent with a crucial role for the downregulated kinases in promoting KRAS-driven proliferation, we found that pharmacological inhibition of WEE1 also suppressed PDAC growth. The unexpected paradoxical activation of ERK upon WEE1 inhibition led us to inhibit both WEE1 and ERK concurrently, which caused further potent growth suppression and enhanced apoptotic death compared with WEE1 inhibition alone. We conclude that system-wide delineation of the KRAS-regulated kinome can identify potential therapeutic targets for KRAS-mutant pancreatic cancer.

N-acylsphingosine amidohydrolase 1 promotes melanoma growth and metastasis by suppressing peroxisome biogenesis-induced ROS production

ObjectiveMetabolic deregulation is a key hallmark of cancer cells and has been shown to drive cancer growth and metastasis. However, not all metabolic drivers of melanoma are known. Based on our finding that N-acylsphingosine amidohydrolase 1 (ASAH1) is overexpressed in melanoma, the objective of these studies was to establish its role in melanoma tumor growth and metastasis, understand its mechanism of action, and evaluate ASAH1 targeting for melanoma therapy. MethodsWe used publicly available melanoma datasets and patient-derived samples of melanoma and normal skin tissue and analyzed them for ASAH1 mRNA expression and ASAH1 protein expression using immunohistochemistry. ASAH1 was knocked down using short-hairpin RNAs in multiple melanoma cell lines that were tested in a series of cell culture-based assays and mouse-based melanoma xenograft assays to monitor the effect of ASAH1 knockdown on melanoma tumor growth and metastasis. An unbiased metabolomics analysis was performed to identify the mechanism of ASAH1 action. Based on the metabolomics findings, the role of peroxisome-mediated reactive oxygen species (ROS) production was explored in regard to mediating the effect of ASAH1. The ASAH1 inhibitor was used alone or in combination with a BRAFV600E inhibitor to evaluate the therapeutic value of ASAH1 targeting for melanoma therapy. ResultsWe determined that ASAH1 was overexpressed in a large percentage of melanoma cells and regulated by transcription factor E2F1 in a mitogen-activated protein (MAP) kinase pathway-dependent manner. ASAH1 expression was necessary to maintain melanoma tumor growth and metastatic attributes in cell cultures and mouse models of melanoma tumor growth and metastasis. To identify the mechanism by which ASAH1 facilitates melanoma tumor growth and metastasis, we performed a large-scale and unbiased metabolomics analysis of melanoma cells expressing ASAH1 short-hairpin RNAs (shRNAs). We found that ASAH1 inhibition increased peroxisome biogenesis through ceramide-mediated PPARγ activation. ASAH1 loss increased ceramide and peroxisome-derived ROS, which in turn inhibited melanoma growth. Pharmacological inhibition of ASAH1 also attenuated melanoma growth and enhanced the effectiveness of BRAF kinase inhibitor in the cell cultures and mice. ConclusionsCollectively, these results demonstrate that ASAH1 is a druggable driver of melanoma tumor growth and metastasis that functions by suppressing peroxisome biogenesis, thereby inhibiting peroxisome-derived ROS production. These studies also highlight the therapeutic utility of ASAH1 inhibitors for melanoma therapy.

Centyrin ligands for extrahepatic delivery of siRNA

RNA interference (RNAi) offers the potential to treat disease at the earliest onset by selectively turning off the expression of target genes, such as intracellular oncogenes that drive cancer growth. However, the development of RNAi therapeutics as anti-cancer drugs has been limited by both a lack of efficient and target cell-specific delivery systems and the necessity to overcome numerous intracellular barriers, including serum/lysosomal instability, cell membrane impermeability, and limited endosomal escape. Here, we combine two technologies to achieve posttranscriptional gene silencing in tumor cells: Centyrins, alternative scaffold proteins binding plasma membrane receptors for targeted delivery, and small interfering RNAs (siRNAs), chemically modified for high metabolic stability and potency. An EGFR Centyrin known to internalize in EGFR-positive tumor cells was site-specifically conjugated to a beta-catenin (CTNNb1) siRNA and found to drive potent and specific target knockdown by free uptake in cell culture and in mice inoculated with A431 tumor xenografts (EGFR amplified). The generalizability of this approach was further demonstrated with Centyrins targeting multiple receptors (e.g., BCMA, PSMA, and EpCAM) and siRNAs targeting multiple genes (e.g., CD68, KLKb1, and SSB1). Moreover, by installing multiple conjugation handles, two different siRNAs were fused to a single Centyrin, and the conjugate was shown to simultaneously silence two different targets. Finally, by specifically pairing EpCAM-binding Centyrins that exhibited optimized internalization profiles, we present data showing that an EpCAM Centyrin CTNNb1 siRNA conjugate suppressed tumor cell growth of a colorectal cancer cell line containing an APC mutation but not cells with normal CTNNb1 signaling. Overall, these data demonstrate the potential of Centyrin-siRNA conjugates to target cancer cells and silence oncogenes, paving the way to a new class of anticancer drugs.

European society for medical oncology virtual congress: Molecular analysis for precision oncology, October 9th - October 10th, 2020.

European society for medical oncology virtual congress: Molecular analysis for precision oncology, October 9th - October 10th, 2020.

Casein Kinase 1α as a Regulator of Wnt-Driven Cancer.

Wnt signaling regulates numerous cellular processes during embryonic development and adult tissue homeostasis. Underscoring this physiological importance, deregulation of the Wnt signaling pathway is associated with many disease states, including cancer. Here, we review pivotal regulatory events in the Wnt signaling pathway that drive cancer growth. We then discuss the roles of the established negative Wnt regulator, casein kinase 1α (CK1α), in Wnt signaling. Although the study of CK1α has been ongoing for several decades, the bulk of such research has focused on how it phosphorylates and regulates its various substrates. We focus here on what is known about the mechanisms controlling CK1α, including its putative regulatory proteins and alternative splicing variants. Finally, we describe the discovery and validation of a family of pharmacological CK1α activators capable of inhibiting Wnt pathway activity. One of the important advantages of CK1α activators, relative to other classes of Wnt inhibitors, is their reduced on-target toxicity, overcoming one of the major impediments to developing a clinically relevant Wnt inhibitor. Therefore, we also discuss mechanisms that regulate CK1α steady-state homeostasis, which may contribute to the deregulation of Wnt pathway activity in cancer and underlie the enhanced therapeutic index of CK1α activators.

Abstract 1424: Her3 up regulation in response to AKT and PI3K inhibition causes differential responses dependent on the presence of neuregulin isoforms in the microenvironmental milieu

Abstract Introduction: Triple Negative Breast Cancer (TNBC) is an aggressive form of Breast Cancer (BC) affecting ~20% of BC patients. TNBC is a highly heterogenous disease and numerous tyrosine kinase inhibitors (TKI) targeting different pathways including the PI3K, AKT and MAPK pathway have shown limited benefit in the clinical setting. Intrinsic and extrinsic resistance mechanisms to TKI are the main reason of therapeutic failure. Signalling from the human epidermal growth factor receptor (HER) family of proteins is dependent on a well-orchestrated series of interactions between family members to form either homo- or heterodimers. The heterodimeric complex formed with HER3 is a particularly potent oncogenic signalling unit that can act as a driver of cancer growth. HER3 upregulation in response to small molecule inhibitors has been shown to rescue a large number of cancers including TNBC. In this study we aim to characterize the extent of HER3 reliance in TNBC and to define the effect of neuregulin isoforms in TNBC cancers. Method and Materials: Basal and Claudin type Breast cancer cell lines were treated with a range of small molecule inhibitors, known to exhibit limited efficacy as single agents, including the AKT inhibitor (GDC0068), the PI3K inhibitor (GDC0077) and the EGFR inhibitors (Gefitinib and Lapatinib) or a combination of these drugs in the presence or absence of the HER3 ligand neuregulin. The effect these inhibitors on Akt/ MAPK signalling was characterized using specific biomarkers of the respective pathways. Combination therapy was evaluated in human cancer cell lines through Annexin V staining. Results: Basal (HCC-70 and MDA-MB-468) and Caudin type (MDA-MB-231) TNBC cell lines displayed differential reliance on the HER family of receptors. Dynamic HER3 upregulation was predominant in the TNBC cells that show HER signaling reliance coinciding with the basal subtype. Furthermore, the presence of the natural ligand neuregulin showed potent signaling through the HER3-AKT pathway and significantly diminished the efficacy of the inhibitors tested. We report that neuregulin augments the HER3 feedback mechanism for continued proliferation in TNBC. Interestingly, we show that neuregulin mediated resistance is dependent on the type and dose of the neuregulin isoform. Neuregulin 1β showed potent proliferative effects and overcoming this effect required a 10 fold increase in the drug concentration. In contrast, the presence of neuregulin 1α isoform may paradoxically confer sensitivity to small molecule inhibitors. We show that cellular proliferation is maintained through both the PI3K and MAPK pathway and single targeting is inefficient in overcoming resistance. We demonstrate that combination strategies that block compensatory mechanisms are effective strategies to neuregulin dependent and independent resistance mechanisms. Conclusion: The highly dynamic nature of HER3 expression and signalling combined with redundant HER signalling and downstream mechanisms allow for continued signalling in a subtype of TNBC patients. Targeted therapy to ensure complete abrogation of HER signalling is essential for effective therapeutic response. In conclusion, TNBC cancers display differential mechanism of resistance that should be adequately addressed for therapeutic strategies. Citation Format: Nicoleta Sinevici, Pedram Heidari, Umar Mahmood. Her3 up regulation in response to AKT and PI3K inhibition causes differential responses dependent on the presence of neuregulin isoforms in the microenvironmental milieu [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1424.

Abstract 4879: Identification of degron motifs and mutations by analyzing a deep neural network

Abstract Degrons, short amino acid sequences on protein substrates, play a critical role in the ubiquitin-proteasome system (UPS), which regulates over 80 percent of protein degradation in cells. When recognized by E3 ubiquitin ligases, degrons signal the destruction of its substrate protein, determining the regulatory specificity of the UPS. While a few degrons have been reported to be affected by mutations driving cancer growth, the effect of specific mutations on degrons remains largely unidentified. In this study, we analyze a deep neural network model that predicts the likelihood of an input protein sequence to contain a degron. To characterize the important C-terminal degron motifs learned by the model, we ran computational methods including sequence ranking and selection, enrichment analyses, and sequence logo visualization. This revealed the discovery of 101 significant degron motifs across lengths 2, 3, and 4. While previous studies suggested that di-amino acid motifs might be the primary source of c-end degron, the new motifs suggest that additional complexity might exist with a longer extended degron. Using these putative motifs as targets for mutations, we performed in silico mutagenesis to identify high-impact amino acid mutations that cause degron loss or gain. Our discovery of degron motif-mutation pairs could inform the development of targeted small-molecule drugs for cancer treatments and identification mutational biomarkers used for early detection of cancers. Citation Format: Cynthia Chen, Collin Tokheim, Shirley Liu. Identification of degron motifs and mutations by analyzing a deep neural network [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4879.

The Power of Metabolism in Driving Cancer Growth

The Power of Metabolism in Driving Cancer Growth

Use of oncology electronic learning resources to learn about geriatric oncology.

11033 Background: Despite the aging population driving cancer growth, oncology trainees receive little training in geriatrics. While electronic resources, such as ASCO University, may help meet this gap, use of available geriatric oncology (GO) modules is low. We sought to understand why by exploring how oncology trainees currently learn about GO, their preferred methods for learning about GO, and their attitudes towards e-learning and geriatrics. Methods: Canadian medical oncology residents and recent graduates were electronically surveyed about the following domains: demographics, self-directed learning practices, use of electronic resources, perceived facilitators and barriers to e-learning, and geriatric oncology teaching. Descriptive statistics were used to analyze the data. Results: Respondents (n = 47) were mostly aged < 35 (66%). Respondents felt that learning about older adults was important (mean 4.3±1.0 out of 5) and generally felt comfortable caring for them (mean 3.9±0.9 out of 5) despite minimal training in geriatrics.Almost half (48.9%) received 0-2 hours of teaching in GO during residency, with the majority (59.6%) receiving teaching in clinic, 36.2% through lectures and 21.3% via seminars. Respondents also learned about GO through reading journal articles (42.6%), modelling in clinic (36.2%), reading a textbook (19.2%) or attending a conference (19.2%). Respondents preferred to learn about GO through on-site lectures (46.8%), dinner meetings (42.6%), case discussion (42.6%) and attending conferences (38.3%). Although overall respondents highly valued electronic learning (mean 4.3±0.75 out of 5), only a minority (8.5%) had received GO teaching electronically using e-modules and only 23.4% respondents were aware of e-learning resources in GO. In contrast most respondents (83%) had used an e-learning resource to learn about oncology. The most common oncology e-resources used were ASCO University (61.7%), Oncology Education (61.7%), and ASCO meeting videos (44.7%). Conclusions: Although oncology trainees value and commonly use e-learning resources, e-learning is not a common or preferred way to learn about GO, potentially due to lack of awareness about these resources. Future research will explore whether the current methods of educating oncology learners about older adults are appropriate and sufficient, as well as how trainees value and prioritize learning about topics that are not included in the formal curricula.

PIM protein kinases regulate the level of the long noncoding RNA H19 to control stem cell gene transcription and modulate tumor growth.

The proviral integration site for Moloney murine leukemia virus (PIM) serine/threonine kinases have an oncogenic and prosurvival role in hematological and solid cancers. However, the mechanism by which these kinases drive tumor growth has not been completely elucidated. To determine the genes controlled by these protein kinases, we carried out a microarray analysis in T‐cell acute lymphoblastic leukemia (T‐ALL) comparing early progenitor (ETP‐ALL) cell lines whose growth is driven by PIM kinases to more mature T‐ALL cells that have low PIM levels. This analysis demonstrated that the long noncoding RNA (lncRNA) H19 was associated with increased PIM levels in ETP‐ALL. Overexpression or knockdown of PIM in these T‐ALL cell lines controlled the level of H19 and regulated the methylation of the H19 promoter, suggesting a mechanism by which PIM controls H19 transcription. In these T‐ALL cells, the expression of PIM1 induced stem cell gene expression (SOX2, OCT‐4, and NANOG) through H19. Identical results were found in prostate cancer (PCa) cell lines where PIM kinases drive cancer growth, and both H19 and stem cell gene levels. Small molecule pan‐PIM inhibitors (PIM‐i) currently in clinical trials reduced H19 expression in both of these tumor types. Importantly, the knockdown of H19 blocked the ability of PIM to induce stem cell genes in T‐ALL cells, suggesting a novel signal transduction cascade. In PCa, increases in SOX2 levels have been shown to cause both resistance to the androgen deprivation therapy (ADT) and the induction of neuroendocrine PCa, a highly metastatic form of this disease. Treatment of PCa cells with a small molecule pan‐PIM‐i reduced stem cell gene transcription and enhanced ADT, while overexpression of H19 suppressed the ability of pan‐PIM‐i to regulate hormone blockade. Together, these results demonstrate that the PIM kinases control the level of lncRNA H19, which in turn modifies stem cell gene transcription regulating tumor growth.

Cancer-associated fibroblasts in cholangiocarcinoma.

To give a state-of-art knowledge regarding cancer-associated fibroblasts (CAF) in cholangiocarcinoma (CCA) based both on direct evidence and studies on other desmoplastic cancers. High contingency of CAF characterizes CCA, a tumor with a biliary epithelial phenotype that can emerge anywhere in the biliary tree. Current treatments are very limited, the surgical resection being the only effective treatment but restricted to a minority of patients, whereas the remaining patients undergo palliative chemotherapy regimens. In cancer, CAF shape the tumor microenvironment, drive cancer growth and progression, and contribute to drug resistance. All these functions are accomplished through an interplay network between CAF and surrounding cells including tumor and other stromal cells, i.e. immune and endothelial cells. Several studies have pointed out the existence of CAF sub-populations carrying out several and opposite functions, cancer-promoting or cancer-restraining as shown in pancreatic cancer, another prototypic desmoplastic tumor in which heterogeneity of CAF is well demonstrated. New CAF functions are now emerging in pancreatic and breast cancers like the modulation of immune responses or tumor metabolism, opening new area for treatments.

Abstract P5-02-14: The MAPK pathway is variably active across breast cancer subtypes, and increased activity may be associated with hormonal therapy resistance

Abstract Background: Based on preclinical research, the MAPK growth factor pathway is thought to be an oncogenic pathway driving breast cancer, potentially in association with the ER pathway. In patients it is unclear how functional activity of the MAPK pathway activity relates to activity of the ER pathway to drive cancer growth. We have reported before on the development of assays, e.g. for the ER pathway, that enable quantitative measurement of activity of signal transduction pathways based on Bayesian computational model inference of pathway activity from measurements of mRNA levels of target genes of the ER transcription factor (Cancer Res., 2014, vol. 74, no. 11, pp. 2936-2945). We now present (1) development of the MAPK-AP1 pathway model, (2) MAPK-AP1 pathway activity and its relation to ER pathway activity in breast cancer; (3) emergence of MAPK pathway activity associated with hormonal therapy resistance. Methods: The MAPK-AP1 pathway model was developed and biologically validated on Affymetrix mRNA data from samples (different cell types) with a known MAPK pathway activity. MAPK-AP1 and ER pathway activity scores were measured on Affymetrix expression data of primary untreated breast cancer samples (GSE45827, n=141) and of fulvestrant sensitive and resistant MCF7 cell line clones (GSE74391). Results: In clinical breast cancer samples, average MAPK pathway activity was increased in all subtypes compared to normal breast tissue, with a large dynamic range indicating that in each subtype there are samples with high and with low pathway activity; ER pathway activity in Luminal A and B subtypes ranged from high to low, and was low in all other subtypes. MAPK-AP1 pathway activity did not correlate with ER pathway activity. Fulvestrant-sensitive MCF7 cells had high ER and low MAPK pathway activity scores, while resistant MCF7 clones exhibited increased MAPK pathway activity and complete loss of ER pathway activity, explaining therapy resistance. Discussion: The MAPK-AP1 pathway is frequently activated in all breast cancer subtypes, independent of ER pathway activity. We hypothesize that in case of resistance to hormonal therapy measurement of both pathway activities may be informative as to the underlying resistance mechanism, i.e. loss of ER pathway activity and/or gain of MAPK pathway activity. Future clinical studies are needed to confirm the role of these signaling pathways in hormonal resistance. Citation Format: Anja van de Stolpe, Laurent Holtzer, Yvonne Wesseling-Rozendaal, Wim Verhaegh. The MAPK pathway is variably active across breast cancer subtypes, and increased activity may be associated with hormonal therapy resistance [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-02-14.

A TFAP2C Gene Signature Is Predictive of Outcome in HER2-Positive Breast Cancer.

The AP-2γ transcription factor, encoded by the TFAP2C gene, regulates the expression of estrogen receptor-alpha (ERα) and other genes associated with hormone response in luminal breast cancer. Little is known about the role of AP-2γ in other breast cancer subtypes. A subset of HER2+ breast cancers with amplification of the TFAP2C gene locus becomes addicted to AP-2γ. Herein, we sought to define AP-2γ gene targets in HER2+ breast cancer and identify genes accounting for physiologic effects of growth and invasiveness regulated by AP-2γ. Comparing HER2+ cell lines that demonstrated differential response to growth and invasiveness with knockdown of TFAP2C, we identified a set of 68 differentially expressed target genes. CDH5 and CDKN1A were among the genes differentially regulated by AP-2γ and that contributed to growth and invasiveness. Pathway analysis implicated the MAPK13/p38δ and retinoic acid regulatory nodes, which were confirmed to display divergent responses in different HER2+ cancer lines. To confirm the clinical relevance of the genes identified, the AP-2γ gene signature was found to be highly predictive of outcome in patients with HER2+ breast cancer. We conclude that AP-2γ regulates a set of genes in HER2+ breast cancer that drive cancer growth and invasiveness. The AP-2γ gene signature predicts outcome of patients with HER2+ breast cancer and pathway analysis predicts that subsets of patients will respond to drugs that target the MAPK or retinoic acid pathways. IMPLICATIONS: A set of genes regulated by AP-2γ in HER2+ breast cancer that drive proliferation and invasion were identified and provided a gene signature that is predictive of outcome in HER2+ breast cancer.

Abstract A131: Fulvestrant resistance in an MCF-7 model for breast cancer is associated with complete loss of ER pathway activity and gain of MAPK-AP1 pathway activity

Abstract Background: 10-15 signal transduction pathways govern major cellular processes, e.g. cell division, differentiation and migration, both in physiology and pathophysiology. They are frequently abnormally active in cancer and can drive cancer growth and metastasis. Resistance to targeted drugs directed towards specific signaling pathways like the ER pathway, can be mediated by induction of activity of other signaling pathways. The past decade we developed a new method to quantitatively measure functional activity status of signal transduction pathways in individual cell and tissue samples, based on Bayesian computational model inference of pathway activity from measurements of mRNA levels of target genes of the pathway-associated transcription factor1,2,3. Assays have been developed for androgen (AR) and estrogen receptor (ER), Hedgehog (HH), Wnt, TGFb, Notch, NFkB, PI3K, JAK-STAT 1/2 and 3, and MAPK-AP1 pathways. They can be performed simultaneously on a single cell or tissue sample using Affymetrix HG-U133 Plus 2.0 microarray or qPCR, and provide quantitative pathway activity scores expressed on a log2odds scale. Method. Pathway analysis using these assays was performed on public Affymetrix expression microarray data (GSE74391,4) from ER-positive MCF-7 cells that were either sensitive or resistant to fulvestrant (n=26). Results. Resistance to fulvestrant was associated with complete loss of functional ER pathway activity (difference 16 log2odds score, p=0.00029) and gain of MAPK-AP1 pathway activity (difference 5.8 log2odds, p=0.00029) in ER positive MCF-7 cells. In the resistant MCF-7 cells, PI3K and MAPK-AP1 pathway activity were negatively correlated (Pearson 0.652, p=0.002), suggesting mutually exclusive pathway activation. Conclusion. Our pathway assays enabled identification of the previously unidentified mechanism of resistance to fulvestrant in a cell culture model system for breast cancer, demonstrating that: (1) ER positivity is a prerequisite, but not sufficient, for ER pathway activity, in agreement with our earlier clinical studies1,5,6;; (2) MAPK pathway activity may confer resistance to hormonal therapy; (3) in the absence of MAPK-AP1 pathway activity, the PI3K pathway may be an alternative resistance pathway; (3) signalling pathway assays are well suited to (quantitatively) investigate activity of signal transduction pathways which may confer resistance to a (targeted) drug treatment.

OS5.3 Quantitative signaling pathway analysis of Diffuse Intrinsic Pontine Glioma identifies two subtypes, respectively high TGFβ/MAPK-AP1 and high PI3K/HH pathway activity, which are potentially clinically actionable

Abstract BACKGROUND Diffuse Intrinsic Pontine Glioma (DIPG) is a pediatric brain tumor (glioma), resistant to chemotherapy, with only a temporary response to radiotherapy and an extremely bad prognosis. Genomic abnormalities have been found, indicating abnormal activation of certain growth factor signaling pathways, while expression analysis suggests involvement of developmental signaling pathways.10–15 signal transduction pathways can drive cancer growth and metastasis. We have developed, and biologically validated, a method which enables quantitative measurements of functional activity of signal transduction pathways in individual cell/tissue samples, based on Bayesian computational model inference of pathway activity from measurements of mRNA levels of target genes of the transcription factor associated with the respective signalling pathway. A major envisioned clinical utility is prediction of therapy response. MATERIAL AND METHODS For signaling pathway analysis, Affymetrix expression microarray data were available (GEO dataset GSE26576) from 2 normal brain stem samples and from 6 low grade glioma and 26 DIPG samples (post-mortem after therapy). Of one DIPG patient samples were available before and after therapy. Signaling pathway activity scores were calculated for estrogen and androgen receptor, PI3K-FOXO, MAPK-AP1, JAK-STAT, NFκB, Hedgehog (HH), TGFβ, NOTCH and Wnt pathways. PI3K pathway activity is the reverse of FOXO activity, in the absence of oxidative stress (measured by SOD2 expression). Pathway activity scores were compared between normal tissue and low grade glioma samples and DIPG, and k-means cluster analysis was performed on the DIPG pathway activity scores. RESULTS After treatment, HH pathway activity was increased in DIPG compared to low grade glioma (p=0.0003), PI3K pathway activity scores showed large variations in activity in the DIPG group. Tumors with cell cycle (CDK4/6, CCND1-3) or Receptor Tyrosine Kinase-related gene amplifications had higher PI3K and HH pathway activity compared to tumors without identified amplifications (p<0.05) which, in contrast, had higher MAPK-AP1 pathway activity (p<0.002). Pathway-based clustering analysis revealed two DIPG clusters, C1: high TGFβ/MAPK-AP1 and low PI3K/HH pathway activity; C2: low TGFβ/MAPK-AP1, high PI3K/HH pathway activity. C1 best resembled low grade glioma. In the patient with pre/post treatment samples, a C1 pathway profile switched to a C2 profile after treatment. CONCLUSION Using our quantitative analysis of signaling pathway activity in post-treatment DIPG, two pathway activity subtypes were identified, of which the HH/PI3K high, TGFβ low activity subtype was associated with defined gene amplifications, and may have been induced by chemoradiation therapy. Clusters are supported by a clear biological rationale. Identified signaling pathways are potentially drug targetable.