Aggressive Metastatic Phenotype Research Articles

The emerging role of the exosomal proteins in neuroblastoma.

Exosomes are a subclass of extracellular vesicles shown to promote the cancer growth and support metastatic progression. The proteomic analysis of neuroblastoma-derived exosomes has revealed proteins involved in cell migration, proliferation, metastasis, and in the modulation of tumor microenvironment - thus contributing to the tumor development and an aggressive metastatic phenotype. This review gives an overview of the current understanding of the exosomal proteins in neuroblastoma and of their potential as diagnostic/prognostic biomarker of disease and therapeutics.

Three-dimensional environment sensitizes pancreatic cancer cells to the anti-proliferative effect of budesonide by reprogramming energy metabolism

BackgroundPancreatic ductal adenocarcinoma (PDAC) is the most lethal cancer with an aggressive metastatic phenotype and very poor clinical prognosis. Interestingly, a lower occurrence of PDAC has been described in individuals with severe and long-standing asthma. Here we explored the potential link between PDAC and the glucocorticoid (GC) budesonide, a first-line therapy to treat asthma.MethodsWe tested the effect of budesonide and the classical GCs on the morphology, proliferation, migration and invasiveness of patient-derived PDAC cells and pancreatic cancer cell lines, using 2D and 3D cultures in vitro. Furthermore, a xenograft model was used to investigate the effect of budesonide on PDAC tumor growth in vivo. Finally, we combined genome-wide transcriptome analysis with genetic and pharmacological approaches to explore the mechanisms underlying budesonide activities in the different environmental conditions.ResultsWe found that in 2D culture settings, high micromolar concentrations of budesonide reduced the mesenchymal invasive/migrating features of PDAC cells, without affecting proliferation or survival. This activity was specific and independent of the Glucocorticoid Receptor (GR). Conversely, in a more physiological 3D environment, low nanomolar concentrations of budesonide strongly reduced PDAC cell proliferation in a GR-dependent manner. Accordingly, we found that budesonide reduced PDAC tumor growth in vivo. Mechanistically, we demonstrated that the 3D environment drives the cells towards a general metabolic reprogramming involving protein, lipid, and energy metabolism (e.g., increased glycolysis dependency). This metabolic change sensitizes PDAC cells to the anti-proliferative effect of budesonide, which instead induces opposite changes (e.g., increased mitochondrial oxidative phosphorylation). Finally, we provide evidence that budesonide inhibits PDAC growth, at least in part, through the tumor suppressor CDKN1C/p57Kip2.ConclusionsCollectively, our study reveals that the microenvironment influences the susceptibility of PDAC cells to GCs and provides unprecedented evidence for the anti-proliferative activity of budesonide on PDAC cells in 3D conditions, in vitro and in vivo. Our findings may explain, at least in part, the reason for the lower occurrence of pancreatic cancer in asthmatic patients and suggest a potential suitability of budesonide for clinical trials as a therapeutic approach to fight pancreatic cancer.

Abstract 4633: Differentiation driver gene HOXD4 as a potential prognostic indicator and therapeutic target in anaplastic thyroid cancer

Abstract Anaplastic thyroid cancer (ATC) is always diagnosed as stage IV with a 5 year survival rate of less than 3% due to its aggressive metastatic phenotype. ATC lacks differentiation and loses its thyroid-like functions making it a difficult cancer to treat due to its lack of targetable genetic lesions and unresponsiveness to conventional treatments. Therefore, we investigated genes that had aberrant expression in ATC in relation to differentiation. Using NCBI Gene expression omnibus (GSE33630, GSE85457), which provides publicly available transcriptomic datasets, and bioinformatics software, GEO2R, we found that Homeobox D4 (HOXD4) has 12.5FC higher expression in ATC vs. normal tissue (p=0.000552) and 4.36FC higher in ATC vs. papillary thyroid cancer (PTC) in human patients (p=6.69e-08). HOXD4 is a transcription factor that plays a crucial role in cell differentiation and has been associated with a worse prognosis in cancers such as ovarian serous carcinoma. As loss of differentiation is essential to ATC’s refractory nature and therapeutic resistance, we are exploring modulation of differentiation by all-trans retinoic acid (ATRA). ATRA was used to induce differentiation in ATC cell line T238 therefore abrogating the aggressive phenotype of this cancer. T238 treated with ATRA had a 26% decrease in proliferation at 24 hours, 46% at 48 hours, and 87% at 72 hours measured using a trypan blue exclusion assay. T238 treated with ATRA also had a 44% decrease in migratory capacity at 24 hours and 24% at 48 hours measured by a cell scratch wound assay. Immortalized normal thyroid cell line NTHY-ori-3-1 did not have a significant decrease in either proliferation or migratory capacity when treated with ATRA. T238 cells treated with ATRA for 24 hours had a 30% decrease in HOXD4 protein levels. Lastly, immunohistochemistry staining of HOXD4 in human patient tissue samples confirmed the over-expression of HOXD4 in ATC (19.5% staining) vs. normal tissue (2.5%) and PTC (7.5%). Overall, we hypothesize that the aberrant expression of HOXD4 in ATC plays a crucial role in the aggressive metastatic phenotype and therefore differentiation agents such as ATRA can target this biomarker to modulate the ATC phenotype. Therefore, HOXD4 can be used as a potential prognostic indicator for early detection of ATC. Future studies will involve CRISPRi based knockdown of HOXD4 in vitro and validation of these results in animal in vivo models. Citation Format: Kaci Kopec, Tara Jarboe, Nicole R. DeSouza, Humayun K. Islam, Jan Geliebter, Raj K. Tiwari. Differentiation driver gene HOXD4 as a potential prognostic indicator and therapeutic target in anaplastic thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4633.

Abstract 3171: The effect of estrogen and phytoestrogens in Inflammatory breast cancer

Abstract Inflammatory breast cancer is an aggressive and lethal type of breast cancer. Frequently, this cancer is diagnosed in an advanced stage with metastasis. IBC diagnosis is associated with a worse survival rate than other types of breast cancer. The presence of dermal and stromal tumor emboli blocking the lymphatic vessels under the skin is considered a hallmark of IBC and assumed to be responsible for the high metastatic behavior and aggressiveness of IBC disease. To this date, there are no effective targeted therapeutics, especially for those patients that account for approximately 20-40% of IBC cases with triple-negative breast cancer (TNBC) classification. Several studies have shown that estrogen can exert non-genomic effects in IBC and non-IBC TNBC, mediated by the expression of alternate estrogen receptors including estrogen receptor alpha-36, and GPR30. In this context, estrogen can activate non-genomic signaling pathways involved in the acquisition of oncogenic phenotypes such as increased motility and invasion in IBC cells. Phytoestrogens, naturally derived structural analogs to 17β-estradiol (E2), were evaluated as an alternative treatment option using in vitro IBC cell models (SUM149 and SUM190). Given the estrogenic properties of phytoestrogens, the drugs were hypothesized to interact with targets in pathways similar to E2 in non-genomic signaling. Several phytoestrogens including coumestrol (Cou) can induce a cytotoxic effect in TNBC. We hypothesize (1) that estrogen non-genomic signaling has an active role in the aggressive metastatic phenotype of IBC, and (2) that Cou has anticancer activity by inhibiting estrogen non-genomic signaling in IBC. To test these hypotheses, IBC cells were treated with E2 or Cou to determine their effects on the activation of downstream kinases by western blot and phospho-kinase array assays. In addition, transcriptional effects upon treatment with Cou were identified using RNA-seq. A dose-response curve in 2D and 3D models was generated to determine the half-maximal inhibitory concentration (IC50) of Cou. Finally, treatment using the IC50 of Cou caused a decrease in cell viability in the TNBC cell line. Also, in comparison to E2, Cou decreases migration, proliferation, and tumor emboli formation in IBC cell lines. In addition, IBC cells were treated using monotherapy and combination therapy with phytoestrogens including Cou, genistein (GN), and/or 8-prenylnaringenin (8-PN). Observed reductions of cell viability in IBC cell lines treated with the different phytoestrogens and combinations make them worthy of further characterization as anti-cancer therapies in in vitro and in vivo models. In summary, IBC cells are responsive to E2 making this signaling pathway attractive for the development of innovative therapeutic strategies with phytoestrogens as potential candidates for treatment. Citation Format: Esther A. Peterson-Peguero, Keishla Rodriguez-Martir, Madeline Ganshert, Dalissa Negron-Figueroa, Ayeisha N. Colon-Ortiz. The effect of estrogen and phytoestrogens in Inflammatory breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3171.

Abstract 5597: Clinical significance of RNA polymerase I inhibitor in pancreatic cancer therapy

Abstract Pancreatic cancer (PanCa) is the third leading cause of cancer-related deaths in the United States with limited therapeutic options available. Gemcitabine (GEM), a deoxycytidine nucleoside analog is currently considered the most effective therapy for PanCa. However, it shows only a marginal survival benefit of six months. Aberrant ribosome biogenesis occurs in most tumor types. We observed that PanCa cells are addicted to ribosome biogenesis (RiBi), which supports their highly aggressive metastatic phenotypes. Thus, strategically targeting RiBi process could be one of the ideal strategies for the prevention and treatment of PanCa. In this study, we elucidated the molecular mechanisms of POLR1A (RPA194) overexpression and how its targeting along with p53 status impacts RNA polymerase I inhibitor therapy against PanCa. The expression level of RPA194 was significantly elevated in pancreatic tumor tissues when compared with adjacent normal pancreatic tissues. BMH-21 is a potent pharmacological inhibitor of RNA Pol I, which is known to degrade RPA194 protein. Our results demonstrated that BMH-21 can selectively induce apoptosis in various PanCa cells but not in HPNE cells. We also found that the cytotoxic effect of BMH-21 was dependent on the expression pattern of RPA-194 and p53 status. We further examined the therapeutic efficacy of BMH-21 in orthotopic xenograft mouse models by using two different PanCa cells, AsPC1 which contains non-functional p53 and MIA PaCa-2 which contains functional mutant p53. We observed that BMH-21 significantly inhibited the growth of tumors derived from both cancer cell lines. Interestingly, BMH-21-mediated inhibition of tumor growth was more significant in tumors derived from MIA PaCa-2 cells compared to AsPC1 cells. Further examination revealed that the inhibition of tumor growth was correlated with RPA194 degradation followed by inhibition of cell proliferation. Overall, our results strongly suggest that BMH-21 is a promising non-toxic agent for the treatment of advanced PanCa and its therapeutic potential depends on RPA194 expression and p53 status in PanCa cells. Citation Format: Mudassier Ahmad. Clinical significance of RNA polymerase I inhibitor in pancreatic cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5597.

Novel lncRNA Gm33149 modulates metastatic heterogeneity in melanoma by regulating the miR-5623-3p/Wnt axis via exosomal transfer.

The high mortality rate associated with melanoma primarily results from metastasis and recurrence. However, the precise mechanisms driving these processes remain poorly understood. Intercellular communication between cancer cells and non-cancer cells significantly influences the tumor microenvironment and plays a crucial role in metastasis. Therefore, our current study aims to investigate the role and mechanism of long non-coding RNAs (lncRNAs) in regulating the interaction between melanoma cancer stem cells (CSCs) and non-CSCs during the metastatic colonization process. This study has characterized a novel lncRNA called Gm33149. Importantly, we provide evidence for the first time that Gm33149, originating from highly metastatic melanoma stem cells (OL-SD), can be packaged into exosomes and transferred to low-metastatic nonstem cells (OL). Once internalized by OL cells, Gm33149 exerts its function through a competitive endogenous RNA mechanism (ceRNA) involving miR-5623-3p. Specifically, Gm33149 competitively binds to miR-5623-3p, thereby activating the Wnt signaling pathway and promoting the acquisition of a more aggressive metastatic phenotype by OL cells. In summary, our findings suggest that targeting lncRNA Gm33149 within extracellular vesicles could potentially serve as a therapeutic strategy for the treatment of metastatic melanoma. Schematic representation of the mechanisms underlying the pro-metastatic activity of lncRNA Gm33149 mediated by exosomal transfer. The figure illustrates the key mechanisms involved in the pro-metastatic activity of lncRNA Gm33149 through exosomal transfer. Melanoma stem cells (OLSD) release exosomes containing lncRNA Gm33149. These exosomes are taken up by non-stem melanoma cells (OL), delivering lncRNA Gm33149 to the recipient cells. Within OL cells, lncRNA Gm33149 functions as a competitive endogenous RNA (ceRNA), sequestering miR-5623-3p. This sequestration prevents miR-5623-3p from binding to its target genes, thereby activating the Wnt signaling pathway. The activated Wnt signaling pathway enhances the migration, invasion, and metastatic colonization capabilities of OL cells. The transfer of lncRNA Gm33149 via exosomes contributes to OL cells acquiring "metastatic competency" while promoting their metastatic colonization. These findings underscore the importance of lncRNA Gm33149 in intercellular communication and the metastatic progression of melanoma.

Plasma-Derived Exosome Proteins as Novel Diagnostic and Prognostic Biomarkers in Neuroblastoma Patients.

Neuroblastoma (NB) is the most common extracranial solid tumor during infancy, causing up to 10% of mortality in children; thus, identifying novel early and accurate diagnostic and prognostic biomarkers is mandatory. NB-derived exosomes carry proteins (Exo-prots) reflecting the status of the tumor cell of origin. The purpose of this study was to characterize, for the first time, the Exo-prots specifically expressed in NB patients associated with tumor phenotype and disease stage. We isolated exosomes from plasma specimens of 24 HR-NB patients and 24 low-risk (LR-NB) patients at diagnosis and of 24 age-matched healthy controls (CTRL). Exo-prot expression was measured by liquid chromatography-mass spectrometry. The data are available via ProteomeXchange (PXD042422). The NB patients had a different Exo-prot expression profile compared to the CTRL. The deregulated Exo-prots in the NB specimens acted mainly in the tumor-associated pathways. The HR-NB patients showed a different Exo-prot expression profile compared to the LR-NB patients, with the modulation of proteins involved in cell migration, proliferation and metastasis. NCAM, NCL, LUM and VASP demonstrated a diagnostic value in discriminating the NB patients from the CTRL; meanwhile, MYH9, FN1, CALR, AKAP12 and LTBP1 were able to differentiate between the HR-NB and LR-NB patients with high accuracy. Therefore, Exo-prots contribute to NB tumor development and to the aggressive metastatic NB phenotype.

High-throughput functional screen identifies YWHAZ as a key regulator of pancreatic cancer metastasis

Pancreatic cancer is a leading cause of cancer death due to its early metastasis and limited response to the current therapies. Metastasis is a complicated multistep process, which is determined by complex genetic alterations. Despite the identification of many metastasis-related genes, distinguishing the drivers from numerous passengers and establishing the causality in cancer pathophysiology remains challenging. Here, we established a high-throughput and piggyBac transposon-based genetic screening platform, which enables either reduced or increased expression of chromosomal genes near the incorporation site of the gene search vector cassette that contains a doxycycline-regulated promoter. Using this strategy, we identified YWHAZ as a key regulator of pancreatic cancer metastasis. We demonstrated that functional activation of Ywhaz by the gene search vector led to enhanced metastatic capability in mouse pancreatic cancer cells. The metastasis-promoting role of YWHAZ was further validated in human pancreatic cancer cells. Overexpression of YWHAZ resulted in more aggressive metastatic phenotypes in vitro and a shorter survival rate in vivo by modulating epithelial-to-mesenchymal transition. Hence, our study established a high-throughput screening method to investigate the functional relevance of novel genes and validated YWHAZ as a key regulator of pancreatic cancer metastasis.

Abstract 3915: RNAseq analysis reveals differentiation gene drivers as potential therapeutic targets in anaplastic thyroid cancer

Abstract Papillary thyroid cancer (PTC) is a well-differentiated and highly treatable cancer with a 5-year survival rate of almost 99%. In contrast, anaplastic thyroid cancer (ATC) lacks differentiation and loses its thyroid-like functions, including lack of response to radioiodine therapy. Due to ATC’s aggressive metastatic phenotype, it is always diagnosed as stage IV with a 5 year survival rate of less than 3%. ATC lacks targetable genetic lesions making it a difficult cancer to treat, therefore RNAseq analysis was performed in an effort to investigate the differentially expressed genes between ATC to PTC. Exploration of differential gene expression between ATC and PTC was undertaken to further elucidate the differences in expression that drive the aggressive nature of ATC phenotype. RNA was isolated from human thyroid cancer cell lines T238 (ATC) and K1 (PTC) and RNA sequencing was conducted by Azenta Life Sciences. Our analysis resulted in 4,931 significant differentially expressed genes between ATC and PTC. Looking at the gene ontology analysis, pathways involving cell differentiation, proliferation, and migration revealed 90, 70, and 47 genes differentially expressed in ATC vs PTC respectively. These pathways putatively give rise to ATC’s aggressive phenotype when compared to PTC, therefore, we narrowed down the focus from 4,931 genes to 207. Through RNAseq analysis, Homeobox D4 (HOXD4) was observed to significantly increase in ATC (7.69 log2 fold change) compared to PTC. HOXD4 is a transcription factor that plays a crucial role in cell differentiation and has been associated with a worse prognosis in cancers such as ovarian serous carcinoma. As loss of differentiation is essential to ATC’s refractory nature and therapeutic resistance, targeting pathways involved in differentiation can be crucial in making ATC amenable to therapy. Other differentiation regulatory genes identified include BLK and LAMA5. BLK (3.95 log2 fold change) is a Src family tyrosine kinase proto-oncogene that is not only involved in cell proliferation but also differentiation. LAMA5 (4.76 log2 fold change) is an extracellular glycoprotein that has been linked to differentiation, migration and metastasis. Validation of the RNAseq results was completed via western blot analysis of multiple ATC and PTC cells lines. We found that HOXD4 expression was higher in ATC cell lines T238, 8505C, and SW1736 than PTC cell lines K1, TPC1, and BCPAP. Further validation was carried out by immunohistochemistry of human thyroid tissue. Preliminary IHC on ATC, PTC, and normal thyroid patient samples showed ATC has significantly increased HOXD4 expression (27.5%) compared to PTC (8.7%) and normal thyroid tissue (2.3%). Ongoing studies in the laboratory to identify novel genetic drivers of cell differentiation and proliferation so as to discover a therapeutic target to fulfill an unmet clinical need is underway. Citation Format: Kaci Kopec, Tara Jarboe, Michael Guo, Nicole R. DeSouza, Sarnath Singh, Augustine Moscatello, Humayun K. Islam, Jan Geliebter, Raj K. Tiwari. RNAseq analysis reveals differentiation gene drivers as potential therapeutic targets in anaplastic thyroid cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3915.

Fully human monoclonal antibody targeting activated ADAM10 on colorectal cancer cells

Metastasis and chemoresistance in colorectal cancer are mediated by certain poorly differentiated cancer cells, known as cancer stem cells, that are maintained by Notch downstream signaling initiated upon Notch cleavage by the metalloprotease ADAM10. It has been shown that ADAM10 overexpression correlates with aberrant signaling from Notch, erbBs, and other receptors, as well as a more aggressive metastatic phenotype, in a range of cancers including colon, gastric, prostate, breast, ovarian, uterine, and leukemia. ADAM10 inhibition, therefore, stands out as an important and new approach to deter the progression of advanced CRC. For targeting the ADAM10 substrate-binding region, which is located outside of the catalytic domain of the protease, we generated a human anti-ADAM10 monoclonal antibody named 1H5. Structural and functional characterization of 1H5 reveals that it binds to the substrate-binding cysteine-rich domain and recognizes an activated ADAM10 conformation present on tumor cells. The mAb inhibits Notch cleavage and proliferation of colon cancer cell lines in vitro and in mouse models. Consistent with its binding to activated ADAM10, the mAb augments the catalytic activity of ADAM10 towards small peptide substrates in vitro. Most importantly, in a mouse model of colon cancer, when administered in combination with the therapeutic agent Irinotecan, 1H5 causes highly effective tumor growth inhibition without any discernible toxicity effects. Our singular approach to target the ADAM10 substrate-binding region with therapeutic antibodies could overcome the shortcomings of previous intervention strategies of targeting the protease active site with small molecule inhibitors that exhibit musculoskeletal toxicity.

Chronic nitric oxide exposure induces prostate cell carcinogenesis, involving genetic instability and a pro-tumorigenic secretory phenotype

Prostate cancer is a leading cause of cancer death in men. Inflammation and overexpression of inducible nitric oxide synthase (NOS2) have been implicated in prostate carcinogenesis. We aimed to explore the hypothesis that nitric oxide NO exerts pro-tumorigenic effects on prostate cells at physiologically relevant levels contributing to carcinogenesis. We investigated the impact of acute exposure of normal immortalised prostate cells (RWPE-1) to NO on cell proliferation and activation of DNA damage repair pathways. Furthermore we investigated the long term effects of chronic NO exposure on RWPE-1 cell migration and invasion potential and hallmarks of transformation. Our results demonstrate that NO induces DNA damage as indicated by γH2AX foci and p53 activation resulting in a G1/S phase block and activation of 53BP1 DNA damage repair protein. Long term adaption to NO results in increased migration and invasion potential, acquisition of anchorage independent growth and increased resistance to chemotherapy. This was recapitulated in PC3 and DU145 prostate cancer cells which upon chronic exposure to NO displayed increased cell migration, colony formation and increased resistance to chemotherapeutics. These findings indicate that NO may play a key role in the development of prostate cancer and the acquisition of an aggressive metastatic phenotype.

AXL/PLCγ1/PKCα as a Central Axis for PD‐L1 Regulation in Triple‐Negative Breast Cancer

Breast cancer is the most prevalent malignancy in women globally, accounting for 12% of all new annual cancer cases worldwide. Triple-negative breast cancer (TNBC), which is ER, PR and HER2 negative, represents 10-15% of breast cancers and displays an aggressive metastatic phenotype, with limited treatment options and poor patient prognosis. TNBC is associated with high expression levels of immune checkpoint programmed-death ligand 1 (PD-L1) compared with other breast cancer subtypes, and it is therefore amenable to immune checkpoint therapy. In this study, we analyzed the role of diacylglycerol (DAG)-regulated protein kinase C alpha (PKCα) in the control of PD-L1 expression in TNBC. Dataset analysis of 50 breast cancer cell lines revealed a prominent up-regulation of PKCα in TNBC, particularly in TNBC-B subtype, with a concomitant down-regulation of protein kinase C delta (PKCδ). This finding was authenticated in the TCGA-BRCA database. A significant correlation was found between PKCα expression and EMT markers, i.e., up-regulation of vimentin, Zeb1, TWIST1, SNAI2 and AXL, and E-cadherin down-regulation. High PKCα expression was also observed in TNBC patient-derived xenografts (PDXs). Immunofluorescence analysis revealed significant PKCα endogenous levels in the TNBC B cell line BT549, with strong "peripheral staining", a hallmark of its activated state. Conversely, a tenuous PKCα staining was detected in luminal breast cancer cells. Notably, TNBC-B cell lines express high levels of phosphorylated (active)-phospholipase C gamma 1 (PLCγ1), an enzyme responsible for DAG generation and PKCα activation. We also found a significant positive correlation between PKCα and PD-L1 expression in breast cancer cell lines. Notably, both RNAi silencing and pharmacological inhibition of PKCα or PLCγ1 reduced PD-L1 mRNA and protein levels in TNBC cells. We also show that AXL and PKCα mutually and positively control their expression in addition to up-regulating PD-L1, and they share a common gene expression signature that is highly enriched in cytokine, extracellular matrix, and motility pathways. Mechanistic analysis showed that inhibition of AXL, PLCγ1 or PKCα significantly reduced PD-L1 promoter activity in TNBC cells, suggesting a transcriptional control of the PDL1 (CD274) gene by this pathway. In summary, our results indicate that aberrant overactivation of AXL/PLCγ1/PKCα represents a cancer cell intrinsic mechanism for PD-L1 up-regulation in TNBC, suggesting a potential role for this axis in the control of tumor immunosurveillance. PKCα and AXL may be promising candidates for integrated targeted therapy in TNBC treatment.

Intracellular nanovesicles mediate α5β1 integrin trafficking during cell migration.

Membrane traffic is an important regulator of cell migration through the endocytosis and recycling of cell surface receptors such as integrin heterodimers. Intracellular nanovesicles (INVs) are transport vesicles that are involved in multiple membrane trafficking steps, including the recycling pathway. The only known marker for INVs is tumor protein D54 (TPD54/TPD52L2), a member of the TPD52-like protein family. Overexpression of TPD52-like family proteins in cancer has been linked to poor prognosis and an aggressive metastatic phenotype, which suggests cell migration may be altered under these conditions. Here, we show that TPD54 directly binds membrane and associates with INVs via a conserved positively charged motif in its C terminus. We describe how other TPD52-like proteins are also associated with INVs, and we document the Rab GTPase complement of all INVs. Depletion of TPD52-like proteins inhibits cell migration and invasion, while their overexpression boosts motility. We show that inhibition of migration is likely due to altered recycling of α5β1 integrins in INVs.

Abstract 414: Efficacy of Balixafortide (POL6326) and Paclitaxel alone and in combination in humanized breast cancer PDX

Abstract Despite availability of new therapies, outcomes for patients with relapsed metastatic breast cancer is poor with moderate median overall survival. High CXCR4 levels correlate with aggressive metastatic phenotypes and poor prognosis in breast cancer (BC). Anti-cancer mechanisms of CXCR4 antagonists include inhibition of angiogenesis and metastasis, activation of immunosurveillance and chemo sensitization. Balixafortide (POL6326) is a potent, selective inhibitor of the chemokine receptor CXCR4 in PhIII for metastatic HER2-negative breast cancer (BC) in combination with tubulin-binding eribulin (NCT03786094). Clinical proof-of-concept in combination with eribulin was demonstrated in a PhI single arm dose-escalation trial in patients with metastatic HER2-negative BC (NCT01837095). The objective response rate for a dose of 5.5mg/kg balixafortide and 1.4mg/m2 eribulin) was 38% (median duration 4.4 months), and the clinical benefit rate was 63% (median duration 8.1 months). Tolerability and safety of eribulin in combination with balixafortide was comparable to that of eribulin monotherapy. Balixafortide was combined with paclitaxel in a humanized BC patient-derived xenograft (PDX) model to explore the efficacy in combination with another tubulin-binding drugs. Human immunophenotyping confirmed successful human CD34+ cell engraftment and expansion of human immune cells prior implantation of an invasive ductal carcinoma (ypT1a ypN1a R0 G3 L1 V1), ER/PR/Her-2 negative tumor. 10mg/kg paclitaxel was given 2x weekly iv. Dosing of balixafortide was 20mg/kg bid sc for 5 days with 2 days break. Total treatment duration was 28 days. Partial remission was achieved in the combination arm balixafortide+paclitaxel (T/C 13%, 87% inhibition of tumor volume vs vehicle control) which was stat. significantly better than paclitaxel monotherapy (T/C 42%). There was no decrease in tolerability in balixafortide combination vs paclitaxel alone. This data suggests combination with balixafortide is more efficacious and equally tolerated to paclitaxel single agent treatment. Citation Format: Daniel Obrecht, Johann Zimmermann. Efficacy of Balixafortide (POL6326) and Paclitaxel alone and in combination in humanized breast cancer PDX [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 414.

Inhibitory Effects of Coumestrol in Triple‐Negative Inflammatory Breast Cancer

Breast cancer is the second leading cause of death in females in the United States, being Inflammatory Breast Cancer (IBC) the most aggressive subtype. Usually, this cancer is diagnosed in an advanced stage with metastasis. Experts in IBC pathogenesis think that IBC rapid metastasis is induces by tumor emboli formation, a hallmark of IBC. The poor prognosis for patients with IBC emphasizes the need to better understand the molecular signature of this disease to develop effective targeted therapeutics. To this date, there is no effective targeted therapeutics, especially for those patients that account for approximately 20-40% of IBC cases with triple-negative breast cancer (TNBC) classification. Importantly, several studies have shown that estrogen can exert non-genomic effects in IBC and non-IBC TNBC, mediated by the expression of an alternate isoform of estrogen receptor alpha-66, estrogen receptor alpha-36, and GPR30. In this context, estrogen can activate non-genomic signaling pathways involved in the acquisition of oncogenic phenotypes such as increased motility and invasion in IBC cells. Phytoestrogens, like coumestrol (Cou), are natural compounds found in plants, specifically soybeans or clover sprouts. Also, it is a polyphenolic compound that is structurally similar to estradiol (E2) and several studies have shown that coumestrol induces a cytotoxic effect in TNBC. We hypothesize (1) that estrogen non-genomic signaling has an active role in the aggressive metastatic phenotype of IBC, and (2) that Cou has anticancer activity by inhibiting estrogen non-genomic signaling in IBC. To test this hypothesis, IBC cells were treated with E2 or Cou, and activation of downstream kinases was analyzed by Western blot and using a Human Phospho-Kinase Array. A dose-response curve in 2D and 3D models were generated to determine the half-maximal inhibitory concentration (IC50) of Cou. Finally, treatment using the IC50 of Cou caused a decrease in cell viability in the TNBC cell line. Also, in comparison to E2, Cou decrease migration, proliferation, and tumor emboli formation in IBC cell line. In summary, IBC cells are responsive to E2 treatment making this signaling pathway attractive for the development of innovative therapeutic strategies. Importantly, these Cou preliminary studies show a potential anticancer activity in IBC cell lines.

Abstract PS12-13: Balixafortide (a CXCR4 antagonist) plus eribulin in HER2 negative metastatic breast cancer: Final analysis from the Phase 1 single arm trial

Abstract Background: Balixafortide (B) is a potent, selective antagonist of the chemokine receptor CXCR4. High CXCR4 levels correlate with aggressive metastatic phenotypes and poor prognosis in metastatic breast cancer (MBC). Efficacy and safety data were published recently from the Phase 1 trial investigating B + eribulin (E) in patients with HER2 negative MBC1. We report the final safety and efficacy analyses from this trial, including an assessment of dose-response and adverse events of particular interest (AEPIs) (e.g. neutropenia, peripheral neuropathy). Methods: In this single-arm, dose escalation trial, patients (pts) received E + increasing doses of B using a 3+3 design in 3 parts: Part I cohorts received low B doses (0.5−1mg/kg) + increasing E doses (1.1−1.4mg/m2); Part II dose-escalation cohort for B (1−5.5mg/kg) + 1.4mg/m2 E; Expanded Cohort (EC) to confirm safety and efficacy of B 5.5mg/kg + 1.4mg/m2 E. Most cohorts received E on days 2 and 9, and B on days 1−3 and 8−10 of 21-day cycles. Results: At entry, all 56 women (age range 33−82 years) were HER2 negative, CXCR4 positive. Most pts were Caucasian and heavily pretreated in the metastatic setting (line of chemotherapy on study: 29% 2nd line, 50% 3rd line, 21% 4th line). 75% were hormone receptor positive and 23% had triple negative breast cancer. A linear dose-exposure was observed over the entire dose range tested for B. Cmax and AUC for E were within published ranges. Safety findings (including AEPIs) remained similar to those reported previously1. No dose-limiting toxicities were confirmed; therefore, the maximum tolerated dose of B was not reached. The highest B dose evaluated was 5.5mg/kg; pharmacokinetic evaluation showed that further protocolled dose increments of B would not have provided a sufficient increase in plasma levels. In addition, the objective response rate in Part II was 3-fold greater than published for eribulin alone which suggested that the anti-tumor activity of B was worthy of further exploration at 5.5mg/kg in the EC. Efficacy data for the trial are shown in the table. These data suggest a potential dose-response relationship for B across all efficacy endpoints, with efficacy being numerically greatest in the EC. While PFS and OS should be interpreted with caution in single arm trials, these data suggest potential benefit for this combination. Further analyses will be presented. Responses were observed regardless of line of chemotherapy on study or extent of CXCR4 expression and were numerically higher in hormone receptor positive patients. Conclusions: A consistent dose response effect for B + E was suggested across all efficacy endpoints for heavily pretreated pts with HER2 negative MBC. When these results are compared with published data for E monotherapy in similar populations, the EC consistently shows numerically greater benefit for all efficacy endpoints2, 3. The safety and tolerability of B + E appear comparable to published data on E or B alone, particularly for neutropenia and peripheral neuropathy1. These results suggest that B + E could potentially provide a new treatment option in heavily pretreated patients with HER2 negative MBC. A Phase 3 trial exploring efficacy and safety of B 5.5mg/kg + E is ongoing. 1. Pernas S et al. Lancet Oncol. 2018; 19: 812−242. Cortes J et al. Lancet. 2011; 377: 914−9233. Kaufman PA et al. J Clin Oncol. 2015; 33: 594−601 Part II(N=21)Expanded Cohort(N=24)Overall Efficacy Population(N=54)Objective Response Rate (95% CI)33% (15−57)38% (19−59)30% (18−44)median duration in months (IQR)2.8 (1.4−3.3)4.4 (3.1−5.3)3.2 (2.2−4.5)Clinical Benefit Rate (95% CI)43% (22−66)63% (41−81)44% (31−59)median duration in months (IQR)5.4 (4.2−6.7)8.1 (6.3−10.8)6.9 (5.4−10.3)median PFS in months (95% CI)4.2 (3−5.4)6.2 (2.9−8.1)4.6 (3.2–5.7)median OS in months (95% CI)10.4 (7.7−18.4)18 (12.2–27.2)16.8 (10.6–18.4)Landmark OS estimate12 months (95% CI)40% (19−60)75% (53−88)60% (45−72)18 months (95% CI)30% (12−50)50% (29−68)42% (29−55)24 months (95% CI)20% (6−39)33% (16−52)25% (14−37)CI: confidence interval; IQR: interquartile range; OS: overall survival; PFS: progression free survival Citation Format: Peter A. Kaufman, Sonia Pernas, Miguel Martin, Marta Gil-Martin, Patricia Gomez Pardo, Sara Lopez-Tarruella, Luis Manso, Eva Ciruelos, Jose Alejandro Perez-Fidalgo, Cristina Hernando, Foluso O Ademuyiwa, Katherine Weilbaecher, Ingrid A Mayer, Timothy J. Pluard, Maria Martinez Garcia, Francois Ringeisen, Daniela Schmitter, Javier Cortes. Balixafortide (a CXCR4 antagonist) plus eribulin in HER2 negative metastatic breast cancer: Final analysis from the Phase 1 single arm trial [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS12-13.

Abstract 6551: Novel leads from Prosopis juliflora (Sw.) arrest the growth of B16F10 melanoma cancer cells by inducing apoptosis and down regulating the cell proliferating signalling pathways

Abstract The evolving drug resistance and cancer heterogeneity is limiting the efficacy of current chemotherapy drugs. In the present state of the art, natural product and drug discovery has remained a significant hope for the identification of novel leads against series anti-cancer drug targets. The major issue is to convert the traditional knowledge into clinical reality by applying omics approaches. As a part of our continues efforts in exploring the efficacy of Prosopis juliflora as possible resource for the management of cancer, we have carried out the efficacy of P. juliflora against melanoma cancer using B16F10 as a melanoma model. The rational in selecting the melanoma lies in its aggressive metastatic phenotype and resistance to present chemotherapy drugs. Prosopis juliflora leaves methanol extract (PJLME) was assessed for its anticancer activity against B16F10 skin melanoma cancer cell line. Series of in vitro and in vivo experiments demonstrated that PJLME is cytotoxic (IC5017ug/ml), inhibited the migration ability of cells, inhibited the colony formation, arrested cell cycle at G0-G1 phase, induced apoptosis by up regulating the pro-apoptic markers like Bax & Bad, along with successful cleavage of Caspase-9 and PARP-1 and down regulated the anti-apoptotic marker Bcl2. The PJLME also suppressed the expression of Akt, mTOR and ERK signalling pathways. The in vivo xenograft studies in BALB/c mice model demonstrated impressive activity against the melanoma cancer. As a part of understanding the possible mechanism of action, our studies indicate that, PJLME induces cell death via apoptosis mechanism and by down regulating the cell proliferation signalling pathways. The HPLC-MS analysis of PJLME indicates the diversity of compounds, few of them are known for their anticancer effects. Our studies unraveled the significance of PJLME as a novel natural source for the management of melanoma cancer. Citation Format: Raju Nivarti Gacche. Novel leads from Prosopis juliflora (Sw.) arrest the growth of B16F10 melanoma cancer cells by inducing apoptosis and down regulating the cell proliferating signalling pathways [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 6551.

Prognostic Molecular Signatures for Metastatic Potential in Clinically Low-Risk Stage I and II Clear Cell Renal Cell Carcinomas.

Introduction: For patients with localized node-negative (Stage I and II) clear cell renal cell carcinomas (ccRCC), current clinicopathological staging has limited predictive capability because of their low risk. Analyzing molecular signatures at the time of nephrectomy can aid in understanding future metastatic potential.Objective: Develop a molecular signature that can stratify patients who have clinically low risk ccRCC, but have high risk genetic changes driving an aggressive metastatic phenotype.Patients, Materials, and Methods: Presented is the differential expression of mRNA and miRNA in 44 Stage I and Stage II patients, 21 who developed metastasis within 5 years of nephrectomy, compared to 23 patients who remained disease free for more than 5 years. Extracted RNA from nephrectomy specimens preserved in FFPE blocks was sequenced using RNAseq. MiRNA expression was performed using the TaqMan OpenArray qPCR protocol.Results: One hundred thirty one genes and 2 miRNA were differentially expressed between the two groups. Canonical correlation (CC) analysis was applied and four CCs (CC32, CC20, CC9, and CC7) have an AUC > 0.65 in our dataset with similar predictive power in the TCGA-KIRC dataset. Gene set enrichment showed CC9 as kidney development/adhesion, CC20 as oxidative phosphorylation pathway, CC32 as RNA binding/spindle and CC7 as immune response. In a multivariate Cox model, the four CCs were able to identify high/low risk groups for metastases in the TCGA-KIRC (p < 0.05) with odds ratios of CC32 = 5.7, CC20 = 4.4, CC9 = 3.6, and CC7 = 2.7.Conclusion: These results identify molecular signatures for more aggressive tumors in clinically low risk ccRCC patients who have a higher potential of metastasis than would be expected.

ZEB1/miR-200c/AGR2: A New Regulatory Loop Modulating the Epithelial-Mesenchymal Transition in Lung Adenocarcinomas.

Epithelial-mesenchymal transition (EMT) is a process involved not only in morphogenesis and embryonic development, but also in cancer progression, whereby tumor cells obtain a more aggressive metastatic phenotype. Anterior gradient protein 2 (AGR2) maintains the epithelial phenotype and blocks the induction of EMT, thus playing an undeniable role in tumor progression. However, the mechanism through which AGR2 expression is regulated, not only during EMT, but also in the early stages of cancer development, remains to be elucidated. In the present study, we show an inverse correlation of AGR2 with ZEB1 (zinc finger enhancer binding protein, δEF1) that was verified by analysis of several independent clinical data sets of lung adenocarcinomas. We also identified the ZEB1 binding site within the AGR2 promoter region and confirmed AGR2 as a novel molecular target of ZEB1. The overexpression of ZEB1 decreased the promoter activity of the AGR2 gene, which resulted in reduced AGR2 protein level and the acquisition of a more invasive phenotype of these lung cancer cells. Conversely, silencing of ZEB1 led not only to increased levels of AGR2 protein, but also attenuated the invasiveness of tumor cells. The AGR2 knockout, vice versa, increased ZEB1 expression, indicating that the ZEB1/AGR2 regulatory axis may function in a double negative feedback loop. In conclusion, we revealed for the first time that ZEB1 regulates AGR2 at the transcriptional level, while AGR2 presence contributes to ZEB1 mRNA degradation. Thus, our data identify a new regulatory mechanism between AGR2 and ZEB1, two rivals in the EMT process, tightly associated with the development of metastasis.

Abstract A096: Oncogenic TRIM37 is a genetic determinant of racial disparity in TNBC patients

Abstract Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype that disproportionately affects younger African American (AA) women. A 5-year survival rate for AA TNBC patients is only 14% compared to 36% of Caucasian patients, which is attributed to early relapse, and metastasis to lung, liver and brain. Although disparities in treatment, co-morbid disease, and access to health care contribute to poor prognosis in AA women, a race-specific genetic component to TNBC disparity cannot be ruled out. To this end, our comprehensive preliminary studies have discovered genetic variations in tripartite motif-containing protein 37 (TRIM37) protein that strongly correlates with TNBC disparity and aggressive metastatic phenotype. Originally, we discovered and characterized TRIM37 as a new breast cancer oncogene that catalyzes histone H2A ubiquitination, a repressive epigenetic modification (Bhatnagar et. al., Nature 2014). Our recent follow-up studies combined with analysis of ethnically diverse genotype-tissue expression, quantitative trait loci, and 1000 genome datasets have discovered single nucleotide polymorphism in TRIM37 that segregate racially. Critically, we have identified TRIM37 polymorphic variants that are predominant in AA women and strongly associate with high TRIM37 in breast tissue. Furthermore, our genetic and biochemical studies establish a strong correlation between TRIM37 expression, metastasis and poor overall-survival using murine TNBC models and TNBC patient tissue analysis. Our comparative mechanistic investigations provide compelling evidence that TRIM37 accelerates TNBC metastasis by enhancing resistance to chemotherapy and inhibiting anoikis pathway, preferentially in AA TNBC relative to non-African American (non-AA) TNBC cells. Therefore, we hypothesize that TRIM37 is a genetic determinant of a high predisposition to metastatic TNBC in AA women, and a strategic target for clinical intervention. Clinical data indicate that AA women have a higher rate of metastatic recurrences compared to non-AA patients. An aggressive clinical course and our preliminary findings strongly suggest that TRIM37 preferentially influences clinical outcome of TNBC in AA women and represents a novel therapeutic target. In this proposal, we will test a highly innovative TRIM37 targeting approach that makes use of cutting-edge antibody-nanoparticle conjugates and antisense oligonucleotide strategy. The overall objectives of this proposal are to demonstrate that TRIM37 is a genetic variant associated with a high risk of metastatic TNBC in AA women and generate proof-of-concept data to treat metastatic TNBC by inhibiting TRIM37 in vivo. Our high precision-targeted approach offers a promising path for selective and effective TNBC treatment. If successful, the described strategy also fits into a broader goal of effective and superior TNBC treatments, and therefore, the potential to generate and replicate additional therapies using other TNBC regulators is very high. Citation Format: Rachisan Gabriel Djiake Tihagam. Oncogenic TRIM37 is a genetic determinant of racial disparity in TNBC patients [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr A096.