Basal-like Cancer Research Articles

CCL3 predicts exceptional response to TGFβ inhibition in basal-like pancreatic cancer enriched in LIF-producing macrophages

The TGFβ receptor inhibitor galunisertib showed promising efficacy in patients with pancreatic ductal adenocarcinoma (PDAC) in the phase 2 H9H-MC-JBAJ study. Identifying biomarkers for this treatment remains essential. Baseline plasma levels of chemokine CCL3 were integrated with clinical outcomes in PDAC patients treated with galunisertib plus gemcitabine (n = 104) or placebo plus gemcitabine (n = 52). High CCL3 was a poor prognostic factor in the placebo group (mOS 3.6 vs. 10.1 months; p < 0.01) but a positive predictor for galunisertib (mOS 9.2 vs. 3.6 months; p < 0.01). Mechanistically, tumor-derived CCL3 activates Tgfβ signaling in macrophages, inducing their M2 phenotype and Lif secretion, sustaining a mesenchymal/basal-like ecotype. TGFβ inhibition redirects macrophage polarization to M1, reducing Lif and shifting PDAC cells to a more epithelial/classical phenotype, improving gemcitabine sensitivity. This study supports exploring TGFβ-targeting agents in PDAC with a mesenchymal/basal-like ecotype driven by high CCL3 levels.

Abstract A077: The role of RhoV in basal-like pancreatic cancer

Abstract Background Pancreatic ductal adenocarcinoma is a devastating disease lacking targeted therapy. Basal-like pancreatic cancer is a tumor subtype with a worse clinical outcome than other subtypes. These cancers are characterized by the expression of basal keratins and mesenchymal markers. However, the genes that drive the basal-like phenotype are poorly understood. Ras homolog family member V (RhoV) is one of the Rho GTPases, and it has been demonstrated to drive the progression of lung and breast cancers. The current study aims to reveal the role of RhoV in basal-like pancreatic cancer. Design RhoV, p63, and CK5/6 were stained in sequential sections by immunohistochemistry. Expression of RhoV in pancreatic cancer and cell lines was reanalyzed based on the data from ProteinAtlas and Broad Insitute DepMap Portal. Basal cell line, BxPC3, and non-basal cell line, Panc1 were selected for functional evaluation based on their differentiated RhoV expression. RhoV gene was overexpressed and knocked down using the CRISPR/gRNA-based Lentiviral system, followed by sphere formation, colony formation, migration, and invasion assays. Results RhoV, p63, and CK5/6 were expressed in the same histologic location of basal pancreatic cancer. The gene expression level of RhoV was co-expressed with p63 (a classic basal marker) in four pancreatic cancer cell lines (broadinstitute.org). Knockout of RhoV by three runs of gRNAs in BxPC3 cells all significantly attenuated tumor sphere formation with less colony formation as compared to mock knockout. Overexpression of RhoV in non-basal-like cells, Panc 1, significantly increased the size of sphere formation, numbers of colony formation, and p38 activation compared to EV control. Cell migration and invasion assays showed the same RhoV-dependent migration and invasion in corresponding cells. Panc1 cells with RhoV knockout significantly generated smaller tumor xenografts in immunodeficient mice and RhoV overexpression led to larger tumor sizes. Conclusion Finding the driver of the basal-like phenotype helps to reveal the pathophysiology of the basal-like subtype of pancreatic cancer and facilitates the identification of new targets for therapeutic purposes. Our preliminary results indicated that RhoV, a small G protein from the Rho family of GTPases, played essential roles in tumor proliferation and progression, by activating the p38 signaling pathway. Further study will be focused on revealing Rhov's downstream pathways and exploring the role of RhoV in apoptosis/cell cycle, cell metabolism, autophagy, etc. in basel-like pancreatic cancer. Citation Format: Shang Wu, Jia Feng, Selina Gao, Ting Wang, Zebang Li, Yifei Liu, Chiung-Kuei Huang, Shaolei Lu. The role of RhoV in basal-like pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A077.

Abstract A021: In silico Prediction of Proteins Interacting with a Basal-like Pancreatic Cancer marker, RhoV, Using AlphaFold

Abstract Introduction Basal-like pancreatic cancer is a subtype with a worse clinical outcome than other subtypes. Ras homolog family member V (RhoV) is one of the basal-like markers that was recently identified. RhoV is a Rho GTPase and is closely regulated by guanine exchange factors (GEFs) and GTPase activating proteins (GAPs). An in silico prediction was performed using AlphaFold to identify RhoV regulators. Methods Amino acid sequences of 120 known GEFs and GAPs were collected. AlphaFold Colab, which uses AlphaFold2 and AlphaFold2-multier, was initially used to screen GEFs and GAPs for their binding affinities to RhoV. The top hits from AlphaFold2 were further verified by AlphaFold3, the most current version of AlphaFold partially open to nonprofit users. RAC1, a well-studied Rho GTPase with known GEFs and GAPs, was used as a control for the prediction process. Results AlphaFold predicted strong interactions of RAC1 and RhoV with GTP and GDP (ipTM of 0.92 between RAC1 and GTP; 0.95 between RAC1 and GDP; 0.92 between GTP and RhoV; 0.95 between GDP and RhoV). Known RAC1 binding partners, ARHGEF5 and ARHGAP21, showed strong binding affinities with RAC1 with ipTM of 0.91 and 0.92, respectively. Out of 120 GEFs and GAPs, AlphaFold2 predicted 6 of them with high binding affinities to RhoV (ipTM greater than 0.79). They are ARHGEF5, ARHGEF3, ARHGAP21, ITSN1, and ITSN2. Their binding affinities were confirmed by AlphaFold3, showing a strong correlation with AlphaFold2 (R of 0.79). AlphaFold3 also demonstrated high binding affinities between RhoV and compounds Palmitic acid (PLM), Citric acid (CIT), and Oleic acid (OLA). Conclusion AlphaFold is a revolutionary protein structural prediction tool with the ability to predict protein-protein binding affinities with high accuracy. AlphaFold2 is open to researchers in the field and allows unlimited queries on its server. AlphaFold3 only allows 20 predictions per research account per day. AlphaFold successfully predicted known binding partners of RAC1 and that RhoV could interact with the following GEFs and GAPs, ARHGEF5, ARHGAP21, and ARHGEF3, with high affinities. AlphaFold is a reliable in silico tool to screen binding partners of any protein of interest before meaningful biochemical interaction is confirmed in vitro and in vivo. Citation Format: Zebang Mark Li, Xiaoxu Zhou, Shaolei Lu. In silico Prediction of Proteins Interacting with a Basal-like Pancreatic Cancer marker, RhoV, Using AlphaFold [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A021.

Mcam stabilizes a luminal progenitor-like breast cancer cell state via Ck2 control and Src/Akt/Stat3 attenuation

Cell state control is crucial for normal tissue development and cancer cell mimicry of stem/progenitor states, contributing to tumor heterogeneity, therapy resistance, and progression. Here, we demonstrate that the cell surface glycoprotein Mcam maintains the tumorigenic luminal progenitor (LP)-like epithelial cell state, leading to Basal-like mammary cancers. In the Py230 mouse mammary carcinoma model, Mcam knockdown (KD) destabilized the LP state by deregulating the Ck2/Stat3 axis, causing a switch to alveolar and basal states, loss of an estrogen-sensing subpopulation, and resistance to tamoxifen—an effect reversed by Ck2 and Stat3 inhibitors. In vivo, Mcam KD blocked generation of Basal-like tumors and Sox10+Krt14+ cells. In human tumors, MCAM loss was largely exclusive of the Basal-like subtype, linked instead to proliferative Luminal subtypes, including often endocrine-resistant Luminal B cancers. This study has implications for developing therapies targeting MCAM, CK2, and STAT3 and their likely effective contexts.

A YAP-centered mechanotransduction loop drives collective breast cancer cell invasion

Dense and aligned Collagen I fibers are associated with collective cancer invasion led by protrusive tumor cells, leader cells. In some breast tumors, a population of cancer cells (basal-like cells) maintain several epithelial characteristics and express the myoepithelial/basal cell marker Keratin 14 (K14). Emergence of leader cells and K14 expression are regarded as interconnected events triggered by Collagen I, however the underlying mechanisms remain unknown. Using breast carcinoma organoids, we show that Collagen I drives a force-dependent loop, specifically in basal-like cancer cells. The feed-forward loop is centered around the mechanotransducer Yap and independent of K14 expression. Yap promotes a transcriptional program that enhances Collagen I alignment and tension, which further activates Yap. Active Yap is detected in invading breast cancer cells in patients and required for collective invasion in 3D Collagen I and in the mammary fat pad of mice. Our work uncovers an essential function for Yap in leader cell selection during collective cancer invasion.

Abstract PO3-02-04: Comparison of OncotypeDX Recurrence Scores (RS) and MammaPrint (MP) scores, and chemotherapy indications in early-stage Estrogen Receptor positive/HER2 negative (ER-/HER2-) breast cancer

Abstract Background: Both RS and MP are endorsed by ASCO and NCCN practice guidelines to estimate prognosis of early-stage ER+/HER2- breast cancers and results guide adjuvant chemotherapy treatment recommendations. Earlier studies reported around 30% discordance in risk assignments by different genomic assays, but these comparisons were complicated by the 3-way (low, intermediate, high) versus 2-way (low, high) risk assignment by RS and MP respectively. More recently, RS thresholds were re-evaluated in the context of the TAILORx and RxPONDER randomized clinical trials and RS &amp;gt;25 is now considered indication for adjuvant chemotherapy. MP results are now also reported as continuous scores similar to RS, and MP high risk patients can be subdivided into MP1 (high) and MP2 (ultra-high) risk groups. The goal of this analysis was to assess correlation between RS and MP scores as continuous variables and examine concordance in chemotherapy eligibility and risk category assignments. Methods: 168 patients treated at the West Cancer Center and Research Institute had patients with both MP and RS results available for the same patient generated by Agendia as a research activity and Exact Sciences in the context of routine care. Molecular class was assigned by the MammaPrint Blueprint assay. Results and clinical information were retrieved from the medical records. Median age was 64 years (range 25-92), 77% were postmenopausal, 59% White, 37% Black and 4% other. 78% had histologic grade 1 or 2 cancers, 53%, 38%, and 8% were stage I, II, and III, respectively. 84% were invasive ductal carcinoma, 13% invasive lobular carcinoma, the rest mixed or other rare histologies. Patients were considered high-risk and chemotherapy eligible if the RS was &amp;gt;25 or the MP score was &amp;lt; 0 (i.e. any negative value), MP high-risk patients were further categorized into MP1 (MP score between 0 to -0.5699) and MP2 (MPs score -0.57 or less). Correlation between scores were assessed using Pearson correlation. Results: There was statistically significant correlation between RS and MP scores with a Pearson correlation coefficient of -0.61, (p&amp;lt; 0.0001). 51 (30%) cancers were MP low-risk (Luminal A), and 122 (73%) were RS low risk (RS≤25). Among the MP low-risk cancers, 47 (92%) had concordant low risk assignment by RS. Based on RS, 46 (27%) patients were high-risk chemotherapy eligible and 42 of these (91%) were concordantly classified as MP high-risk. MP high-risk cancers included 115 Luminal B and 1 Basal-like cancers, and 64% of these (n=74) had low-risk status by RS. When the MP high category was subdivided into MP 1 (n=105) and MP2 (n=11) subgroups, (35/105) 33% and (7/11) 64% of cases were RS &amp;gt;25, respectively. Black patients had a statistically higher mean index MP score of -0.191 compared to -0.082 for White patients (p=0.02). No statistical difference was noted by race for RS. Conclusions: There was strong concordance (92%) between low-risk MP and low-risk RS. Similar concordance was not identified when comparing chemotherapy eligible high-risk MP (MP1 and MP2) to high-risk ( &amp;gt;25) recurrence scores. Only 36% of chemotherapy eligible MP patients (MP1 and MP2) would be chemotherapy eligible by RS. Higher concordance (58%) for chemotherapy eligibility was seen when limited to the ultra-high (MP2) scores. By contrast most (92%) of chemotherapy eligible patients by RS (&amp;gt;25) were considered high risk by MP (MP1 or MP2). Table1. RS and MP risk categories Citation Format: Rishika Singh, William Barlow, Nupur Singh, James Elliott, Richard Fine, Michael Berry, Erin Cobain, Lajos Pusztai, Gregory Vidal. Comparison of OncotypeDX Recurrence Scores (RS) and MammaPrint (MP) scores, and chemotherapy indications in early-stage Estrogen Receptor positive/HER2 negative (ER-/HER2-) breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-02-04.

Abstract B094: The role of paraspeckles and splicing regulation in driving basal-like pancreatic cancer

Abstract The basal-like pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive type of mesenchymal PDAC that resists treatment and has a poor prognosis. Understanding the mechanisms driving basal-like PDAC biology will be essential for development of treatment strategies for this aggressive tumor. We have previously shown that loss of the tumor-suppressive non-coding RNA Neat1 recapitulate the phenotype of Arid1a-deficient mice (a component of the SWI/SNF chromatin remodeling complex) in a mouse model for PDAC, with loss of exocrine pancreatic identity and development of acinar- and ductal-derived lesions (Mello et al., 2017), suggesting that Neat1 and Arid1a may share a common molecular mechanism. We now demonstrate that the loss of these two divergent genes induce the emergence of basal-like PDAC. This provides an opportunity to probe for shared features specific to Neat1- and Arid1a-deficient PDAC, as compared to PDAC in which both genes are functional. Notably, our data indicate that both Neat1- and Arid1a-deficient pancreatic cells fail to form nuclear paraspeckles, i.e. structures implicated in the retention of RNA splicing factors. Consistent with this idea, we also find significantly increased exon-skipping, including a sub-set of such events shared between Neat1- and Arid1a-deficient PDAC. In addition, nucleotide sequence motifs associated with exon-skipping locations suggest involvement of RNA splicing factors known to accumulate in paraspeckles, such as Srsf1, Srsf2, Srsf10, Rbm38, Dazap1 and others. We also show that, upon genetic or pharmacological attenuation of splicing regulator function, basal-like PDAC develop epithelial features, reminiscent of mesenchymal-epithelial transition, reduce both their proliferation and migration potential and undergo cell death. Our findings provide an important opportunity to elucidate the molecular pathways that link aberrant RNA splicing and paraspeckle deficiency to basal-like PDAC, providing new insight for the development of mechanism-based novel PDAC treatment strategies to improve patient outcome. Citation Format: Zamira G. Soares, Emily Berry, Dakarai Esgdaille, Jennifer Twardowski, Stephano S. Mello. The role of paraspeckles and splicing regulation in driving basal-like pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B094.

A basal-like pancreatic cancer molecular subtype can be identified on EUS-acquired tissue and is associated to current smoking, lower Ca19.9 expression and worse prognosis

A basal-like pancreatic cancer molecular subtype can be identified on EUS-acquired tissue and is associated to current smoking, lower Ca19.9 expression and worse prognosis

EMT Features in Claudin-Low versus Claudin-Non-Suppressed Breast Cancers and the Role of Epigenetic Modifications.

Breast cancers are heterogeneous and are classified according to the expression of ER, PR and HER2 receptors to distinct groups with prognostic and therapeutic implications. Within the triple-negative group, with no expression of these three receptors, molecular heterogeneity exists but is currently not exploited in the clinic. The claudin-low phenotype is present in a subset of triple-negative breast cancers and constitutes together with basal-like cancers the most extensive groups within triple-negative breast cancers. Suppression of epithelial cell adhesion molecules in claudin-low cancers is also a hallmark of Epithelial Mesenchymal Transition (EMT). The groups of claudin-low and claudin-non-suppressed breast cancers from the extensive publicly available genomic cohorts of the METABRIC study were examined to delineate and compare their molecular landscape. Genetic and epigenetic alterations of key factors involved in EMT and potentially associated with the pathogenesis of the claudin-low phenotype were analyzed in the two groups. Claudin-low cancers displayed up-regulation of several core transcription factors of EMT at the mRNA level, compared with claudin-non-suppressed breast cancers. Global promoter DNA methylation was increased in both groups of triple-negative cancers and in claudin-low ER-positive cancers compared with the rest of ER-positive cancers. Histone modifier enzymes, including methyltransferases, demethylases, acetyltransferases and deacetylases displayed amplifications more frequently in claudin-non-suppressed triple-negative cancers than in claudin-low counterparts and the expression of some of these enzymes differed significantly between the two groups. Claudin-low and claudin-non-suppressed triple-negative breast cancers differ in their landscape of EMT core regulators and epigenetic regulators. These differences may be explored as targets for therapeutic interventions specific to the two groups of triple-negative breast cancers.

Comparison of Clinical Subtypes of Breast Cancer within the Claudin-Low Molecular Cluster Reveals Distinct Phenotypes

Background: Molecular subtyping of breast cancer has provided a new perspective on the pathogenesis of the disease and a foundation for building a clinical classification for this heterogeneous disease. The initial classification categorizing breast cancers into five groups, luminal A, luminal B, ERBB2-overexpressing, basal-like and normal-like, was later supplemented by an additional group, claudin-low tumors. However, the claudin-low group has been more difficult to align with clinically used immunohistochemical categories. The identity of this group among clinical cases remains ill defined. Methods: The METABRIC cohort comprising more than 1700 breast cancers and providing information for classifying them in both clinical groups and the genomic PAM50/claudin-low groups was analyzed to derive relationships and clarify potential pathogenic ramifications. Comparisons of the claudin-low cases bearing different clinical group classifications and of the respective cases with the same clinical non-claudin-low classifications were performed. Results: ER-negative/HER2-negative breast cancers are predominantly (88.4%) basal-like and claudin low. Conversely, most basal-like cancers (83.6%) are ER negative/HER2 negative. However, claudin-low breast cancers are only in 68.4% of cases ER negative/HER2 negative and the other clinical phenotypes, mostly ER positive/HER2 negative/low proliferation, are also represented in more than 30% of claudin-low cancers. These claudin-low non-ER-negative/HER2-negative breast cancers differ from claudin-low ER-negative/HER2-negative cases in grade, prevalence of integrative clusters, and prevalence of common mutations and common amplifications. Differences also exist between the two groups classified clinically as ER negative/HER2 negative, that are genomically basal-like or claudin-low, including in menopause status, grade, histology, prevalence of high tumor mutation burden, distribution of integrative clusters, prevalence of TP53 mutations and of amplifications in the MYC and MCL1 loci. Furthermore, distinct characteristics are observed between the luminal A and claudin-low groups within the clinical ER-positive/HER2-negative/low proliferation group. Conclusion: Within genomically claudin-low breast cancers, the ER-negative/HER2-negative group is distinct from the group with either ER or HER2 positivity. Conversely, within clinical phenotypes, claudin-low and non-claudin-low breast cancers differ in clinical characteristics and molecular attributes.

YBX1 integration of oncogenic PI3K/mTOR signalling regulates the fitness of malignant epithelial cells

In heterogeneous head and neck cancer (HNC), subtype-specific treatment regimens are currently missing. An integrated analysis of patient HNC subtypes using single-cell sequencing and proteome profiles reveals an epithelial-mesenchymal transition (EMT) signature within the epithelial cancer-cell population. The EMT signature coincides with PI3K/mTOR inactivation in the mesenchymal subtype. Conversely, the signature is suppressed in epithelial cells of the basal subtype which exhibits hyperactive PI3K/mTOR signalling. We further identify YBX1 phosphorylation, downstream of the PI3K/mTOR pathway, restraining basal-like cancer cell proliferation. In contrast, YBX1 acts as a safeguard against the proliferation-to-invasion switch in mesenchymal-like epithelial cancer cells, and its loss accentuates partial-EMT and in vivo invasion. Interestingly, phospho-YBX1 that is mutually exclusive to partial-EMT, emerges as a prognostic marker for overall patient outcomes. These findings create a unique opportunity to sensitise mesenchymal cancer cells to PI3K/mTOR inhibitors by shifting them towards a basal-like subtype as a promising therapeutic approach against HNC.

Abstract P1-10-01: Postmastectomy electron beams radiation therapy (PMERT) : long term results

Abstract Background/Purpose: This study evaluates the toxicity after radiotherapy after mastectomy without reconstruction in patients irradiated to the chest wall using previously reported technique of PMERT. Materials/Methods: We included all women irradiated after mastectomy for not metastatic breast cancer with PMERT between 2007 and 2011 in the Department of Radiation Oncology of the Institut Curie. Previously reported technique using mostly electrons was evaluated in terms of efficacy and toxicity. Acute and late toxicities were assessed retrospectively using CTCAE v.4.0. A clinical exam was weekly performed during radiotherapy and one and three months following the completion of radiotherapy. Patients were then followed as recommended in Institut Curie guidelines (Senorif). Quantitative and qualitative data were described respectively as means and proportions. Statistical comparisons were computed using X² or Fischer’s exact test for categorical data. Recurrence free survival (RFS) was defined as the time between the end of treatment and the date of recurrence or death. Overall survival was the same but recurrences were not taken into account. Patients who did not experienced any event were censored at the date of last news. Results: Among the 796 women included, 51.3% had multifocal lesions, 10.1% a triple negative (TN) status and 18.8% a HER2+ positive status; 196 (24.6%) received a neoadjuvant chemotherapy, and 208 (26.1 %) a systemic therapy during radiotherapy (chemotherapy and/or targeted therapy); 514 (64.6%) had at least one positive lymph node (LN). Internal mammary chain (IMC) was treated in 85.6% of cases, supraclavicular LN (L4) in 88.3% of cases, infraclavicular (L3-2-IP+/- L1) LN in 77.9% of cases and low axilla (L1) in 14.9% of cases. With a median follow up of 113 months [range : 2-164] locoregional recurrence-free survival and overall survival at 10 years were respectively 94.02 (IC95% : 92.13-98.94) and 79.84 (IC95% : 76.83-82.97). The median survival was not reached. In the long term, 29.6% of patients had telangiectasia (grade 1: 23.3%, grade 2: 5.2% Grade 3: 1.1%). Totally 279 patients (35.1%) had breast reconstruction, on average 21 months after the end of radiotherapy. Twenty-five patients (3%) had early esophageal toxicity, not exceeding the grade 1. Of these patients, 21 had had chemotherapy and all were irradiated on the lymph nodes (24 including the IMC). Irradiation of the IMC was not associated with an increased chronic lung toxicity (OR=1.03 [0.98-1.09]). There were 35 patients who developed heart disease after the end of the treatment. Of them, 30 patients had received anthracyclines (p=1.05) and 9 trastuzumab (p =1.09). Four patients developed ischemic heart disease, 3/4 irradiated on the left chest wall, all on the CMI and supra and infraclavicular LN, but all of them presented multiple cardiovascular risk factors (2 to 4). Conclusions: Our series have shown that the PMERT using the Institut Curie technique is effective and well tolerated. Table 1. patients and tumor characteristics (n=796). HR+ : hormonal receptor positive HER2+ : HER-2 overexpressed Cardiovascular risk factor : (smoking, hypertension, dyslipidemia, diabetes, obesity, family history of coronary heart disease or vascular events) as described by the French Health Authority (HAS, Haute Autorité de Santé). Table 2. Efficiency (n=796, median follow-up: 113 months; range : [2-164]). HR+ : hormonal receptor positive HER 2+ : Her-2 overexpressed TNBC : Triple negative or basal-like cancer, OS : overall survival LRFS : local recurrence free survival LRRFS : locoregional recurrence free survival MFS : metastasis free survival Multivariate analysis : long term toxicity IMC irradiation : Irradiation of the internal mammary chain, OR : Odd ratio Citation Format: Souidi Sami, Pierre Loap, Alain Fourquet, Youlia M. Kirova. Postmastectomy electron beams radiation therapy (PMERT) : long term results [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-10-01.

Single-cell RNA sequencing reveals the effects of chemotherapy on human pancreatic adenocarcinoma and its tumor microenvironment

The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) is a complex ecosystem that drives tumor progression; however, in-depth single cell characterization of the PDAC TME and its role in response to therapy is lacking. Here, we perform single-cell RNA sequencing on freshly collected human PDAC samples either before or after chemotherapy. Overall, we find a heterogeneous mixture of basal and classical cancer cell subtypes, along with distinct cancer-associated fibroblast and macrophage subpopulations. Strikingly, classical and basal-like cancer cells exhibit similar transcriptional responses to chemotherapy and do not demonstrate a shift towards a basal-like transcriptional program among treated samples. We observe decreased ligand-receptor interactions in treated samples, particularly between TIGIT on CD8 + T cells and its receptor on cancer cells, and identify TIGIT as the major inhibitory checkpoint molecule of CD8 + T cells. Our results suggest that chemotherapy profoundly impacts the PDAC TME and may promote resistance to immunotherapy.

Overexpression of KMT9α Is Associated with Aggressive Basal-like Muscle-Invasive Bladder Cancer.

Muscle-invasive bladder cancer (MIBC) is associated with limited response rates to systemic therapy leading to a significant risk of recurrence and death. A recently discovered histone methyltransferase KMT9, acts as an epigenetic regulator of carcinogenesis in different tumor entities. In this study, we investigated the presence and association of histological and molecular subtypes and their impact on the survival of KMT9α in MIBC. We performed an immunohistochemical (IHC) analysis of KMT9α in 135 MIBC patients undergoing radical cystectomy. KMT9α was significantly overexpressed in the nucleus in MIBC compared to normal urothelium and low-grade urothelial cancer. Using the HTG transcriptome panel, we assessed mRNA expression profiles to determine molecular subtypes and identify differentially expressed genes. Patients with higher nuclear and nucleolar KMT9α expression showed basal/squamous urothelial cancer characteristics confirmed by IHC and differentially upregulated KRT14 expression. We identified a subset of patients with nucleolar expression of KMT9α, which was associated with an increased risk of death in uni- and multivariate analyses (HR 2.28, 95%CI 1.28-4.03, p = 0.005). In conclusion, basal-like MIBC and the squamous histological subtype are associated with high nuclear KMT9α expression. The association with poor survival makes it a potential target for the treatment of bladder cancer.

NURR1 Is Differentially Expressed in Breast Cancer According to Patient Racial Identity and Tumor Subtype.

Breast carcinoma (BCa) remains the second most common cause of cancer-related death among American women. Whereas estrogen receptor (ER) expression is typically regarded as a favorable prognostic indicator, a significant proportion of ER(+) patients still experience either de novo or acquired endocrine resistance. Previously, we have shown that the loss of orphan nuclear receptor NURR1 expression is associated with neoplastic transformation of the breast epithelium and shorter relapse-free survival (RFS) among systemically treated breast cancer (BCa) patients. Here, we further ascertain the prognostic value of NURR1 in BCa, and its differential expression among Black and White female BCa patients. We assessed the expression of NURR1 mRNA in BCa patients using the Cancer Genome Atlas (TGCA) and compared the occurrence of basal-like cancer and luminal A breast cancer subtypes. Expression levels were further stratified according to racial identity of the patient. We next assessed the correlation of NURR1 expression with Oncotype DX prognostic markers, and the association of NURR1 expression with relapse free survival in patients treated with endocrine therapy. Our study shows that NURR1 mRNA expression is differentially correlated with luminal A vs. basal-like cancer BCa and is predictive of poor relapse-free survival, confirming a similar trend observed in our previous studies using microarray data. NURR1 expression was positively correlated with expression of Oncotype DX biomarkers associated with estrogen responsiveness, while being inversely correlated with biomarkers associated with cell proliferation. Furthermore, we observed that NURR1 expression was positively associated with greater relapse-free survival at 5 years among patients treated with endocrine therapy. Interestingly, we found that among Black women with luminal A BCa, NURR1 expression was repressed in comparison to White women with the same subtype.

Abstract PR010: Single-cell sequencing elucidates the effects of chemotherapy on cancer cell heterogeneity and the tumor microenvironment of human pancreatic adenocarcinoma

Abstract The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) is a complex ecosystem that drives tumor progression. Improving our understanding of the PDAC TME and its role in response to therapy via in-depth single cell characterization will have broad clinical implications for biomarker development and therapeutic design. In this study, we performed single-cell RNA sequencing on freshly collected human PDAC samples of primary (n=16) or metastatic (n=11) origin, either before (n=20) or after (n=7) chemotherapy. We found a heterogeneous mixture of basal and classical Moffitt cancer cell subtypes in all samples, along with distinct cancer-associated fibroblast (CAF) and tumor-associated macrophage (TAM) subpopulations. We identified the major CAF subpopulations as inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs); within these subpopulations were a very few cells expressing immunogenic features which have previously been associated with antigen presenting CAFs (apCAFs). Tumor-associated macrophages (TAMs) could be categorized into two major subpopulations, C1QC+ TAMs or SPP1+ TAMs, each with distinct functional characteristics. For example, phagocytosis-associated gene sets were enriched in C1QC+ TAMs, while angiogenesis-associated gene sets were enriched in SPP1+ TAMs. Comparison of naïve and chemotherapy treated primary PDAC samples revealed that classical and basal-like cancer cells exhibited similar transcriptional responses to chemotherapy; this contrasts with some previous reports which posited a shift towards a basal-like transcriptional program among treated samples. We further noted that treated samples evinced fewer ligand-receptor interactions, particularly between TIGIT on CD8+ T cells and its ligand on cancer cells. We also identified TIGIT, not PD1, as the major inhibitory checkpoint molecule of CD8+ T cells in the PDAC TME. Altogether, our results suggest that chemotherapy impacts the PDAC TME and may further reduce response to immunotherapy. Citation Format: Daniel Weissinger, Emily A. Kawaler, Gregor Werba, Ende Zhao, Despoina Kalfakakou, Surajit Dhara, Grace Oh, Xiaohong Jing, Nina Beri, Lauren Khanna, Tamas Gonda, Paul E. Oberstein, Cristina Hajdu, Cynthia Loomis, Adriana Heguy, Mara H. Sherman, Amanda W. Lund, Theodore H. Welling, Igor Dolgalev, Aristotelis Tsirigos, Diane M. Simeone. Single-cell sequencing elucidates the effects of chemotherapy on cancer cell heterogeneity and the tumor microenvironment of human pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR010.

High Expression of CD58 and ALDH1A3 Predicts a Poor Prognosis in Basal-like Breast Cancer.

CD58 is an immune adhesion molecule on the cellular surface. It was previously found that a high expression of CD58 predicted a poor prognosis of patients with lower-grade gliomas. Therefore, the aim of this paper was to investigate the association between CD58 and breast cancer. CD58 gene expression data downloaded from cBioPortal was compared between the different subtypes of breast cancer. Clinical prognosis was examined using Kaplan-Meier analysis and multivariable Cox regression analysis. The association between CD58 expression and immune cell infiltration was estimated using the TIMER 2.0 web platform. Finally, the tumour sphere formation of aldehyde dehydrogenase 1 (ALDH1)high basal-like breast cancer stem cells in which CD58 was knocked down using siRNA was measured. CD58 mRNA was mainly enriched in claudin-low and basal-like subtypes. The high expression of CD58 predicted a good prognosis in patients with luminal A and luminal B breast cancer. This prediction may be due to the association of immune cell infiltration with CD58. Notably, patients with luminal A breast cancer with a high expression of CD58 in association with ALDH1A3 exhibited a good prognosis; however, this did not apply to patients with basal-like breast cancer. The in vitro experiments revealed that knockdown of CD58 inhibited the tumour sphere formation ability of ALDH1high basal-like cancer cells. CD58 may function as a potential prognostic biomarker and therapeutic target in ALDH-positive basal-like cancer stem cells.

Induction of alternative NF-κB within TAg-induced basal mammary tumors in activation-resistant inhibitor of κ-B kinase (IKKα) mutant mice.

The alternative NF-κB pathway is activated by the NF-κB-inducing kinase (NIK) mediated phosphorylation of the inhibitor of κ-B kinase α (IKKα). IKKα then phosphorylates p100/NFKB2 to result in its processing to the active p52 subunit. Evidence suggests that basal breast cancers originate within a subpopulation of luminal progenitor cells which is expanded by signaling to IKKα. To determine the role of IKKα in the development of basal tumors. Kinase dead IkkαAA/AA mice were crossed with the C3(1)-TAg mouse model of basal mammary cancer. Tumor growth and tumor numbers in WT and IkkαAA/AA mice were assessed and immunopathology, p52 expression and stem/progenitor 3D colony forming assays were performed. Nik-/- mammary glands were isolated and mammary colonies were characterized. While tumor growth was slower than in WT mice, IkkαAA/AA tumor numbers and pathology were indistinguishable from WT tumors. Both WT and IkkαAA/AA tumors expressed p52 except those IkkαAA/AA tumors where NIK, IKKαAA/AA and ErbB2 were undetectable. Colonies formed by WT and IkkαAA/AA mammary cells were nearly all luminal/acinar however, colony numbers and sizes derived from IkkαAA/AA cells were reduced. In contrast to IkkαAA/AA mice, virgin Nik-/- mammary glands were poorly developed and colonies were primarily derived from undifferentiated bipotent progenitor cells. C3(1)-TAg induced mammary tumors express p100/p52 even without functional IKKα. Therefore the development of basal-like mammary cancer does not strictly rely on IKKα activation. Signal-induced stabilization of NIK may be sufficient to mediate processing of p100NFKB2 which can then support basal-like mammary tumor formation. Lastly, in contrast to the pregnancy specific role of IKKα in lobuloalveogenesis, NIK is obligatory for normal mammary gland development.

1722P The subtype-specific cell fitness of head and neck cancer is regulated by the YBX1 integration of oncogenic PI3K/mTOR signalling

Molecular subtyping of heterogeneous cancers has gained substantial biological interest although with limited translation to the clinic. In heterogeneous head and neck cancer (HNC), subtype-specific treatment regimens are currently missing. A multi-omic approach integrating whole-genome and single-cell transcriptome sequencing with reverse-phase protein array was employed in patient primary and metastatic tumours and human cell lines to investigate the subtype-specific molecular mechanisms as the therapeutic vulnerabilities in HNC. An integrated analysis of HNC subtypes using single-cell sequencing and proteome profiles revealed a hallmark epithelial-mesenchymal transition (EMT) signature within the epithelial cancer-cell population. The EMT signature was evident in the invasive mesenchymal subtype and surprisingly coincided with PI3K/mTOR pathway inactivation. Conversely, the EMT signature was suppressed in epithelial cancer cells of the basal subtype which exhibited hyperactive PI3K/mTOR oncogenic signalling. We further identified that phosphorylation of Y Box binding protein 1 (YBX1), downstream of the PI3K/mTOR pathway, restrained the basal-like cancer cell proliferation. In contrast, YBX1 acted as a safeguard against the proliferation to invasion switch in mesenchymal-like epithelial cancer cells, and its loss accentuated partial EMT acquisition and in vivo invasion. Interestingly, the proteomic analysis of patient HNC identified phospho-YBX1 as a prognostic marker for overall patient outcomes, the loss of which correlated with HNC metastasis and poor prognosis. Our results indicate that HNC cells adopt a PI3K-phospho-YBX1-dependent proliferation program that is mutually exclusive to partial EMT involvement which relies on the lack of phosphorylation of YBX1 in PI3K-inactive cancer cells. These findings create a unique opportunity to sensitise mesenchymal cancer cells to PI3K/mTOR inhibitors by shifting them towards a basal-like subtype, constituting a promising therapeutic approach against HNC.

Stat3 Tyrosine 705 and Serine 727 Phosphorylation Associate With Clinicopathological Characteristics and Distinct Tumor Cell Phenotypes in Triple-Negative Breast Cancer.

Signal transducer and activator of transcription 3 (Stat3) is responsible for many aspects of normal development and contributes to the development and progression of cancer through regulating epithelial cell identity and cancer stem cells. In breast cancer, Stat3 is associated with triple-negative breast cancers (TNBC) and its function has been related to the activation of p63, itself a marker of basal-like TNBC and a master regulator of stem cell activities. Stat3 activation is controlled by dual phosphorylation at tyrosine 705 (pTyr705) and serine 727 (pSer727), although it is unclear whether these have equivalent effects, and whether they are related or independent events. To address these issues, we investigated Stat3 phosphorylation at the two sites by immunohistochemistry in 173 patients with TNBC. Stat3 phosphorylation was assessed by automated quantitative measurements of digitized scanned images and classified into four categories based on histoscore. The results were analyzed for associations with multiple markers of tumor phenotype, proliferation, BRCA status, and clinicopathological characteristics. We show that the levels of pTyr705- and pSer727-Stat3 were independent in 34% of tumors. High pTyr705-Stat3 levels were associated with the luminal differentiation markers ERβ/AR and MUC1, whereas tumors with high levels of pSer727-Stat3 were more likely to be positive for the basal marker CK5/6, but were independent of p63 and were EGFR negative. Combined high pSer727- and low Tyr705-Stat3 phosphorylation associated with basal-like cancer. Although high Stat3 phosphorylation levels were associated with less aggressive tumor characteristics, they did not associate with improved survival, indicating that Stat3 phosphorylation is an unfavorable indicator for tumors with an otherwise good prognosis according to clinicopathological characteristics. These findings also show that pTyr705-Stat3 and pSer727-Stat3 associate with specific breast tumor phenotypes, implying that they exert distinct functional activities in breast cancer.