Phosphoinositide 3-kinase (PI3K) signaling is hyperactivated in ~70% of breast cancers via mutations in oncogenes including PIK3CA or inactivation/depletion of phosphoinositide (PI)-phosphatases. Generation of PI(3,4,5)P3 by PI3K activates many downstream effectors, including AKT, that induce cellular proliferation in breast cancer. In this context PI(3,4,5)P3 is tightly regulated by PI-phosphatases, including the tumor suppressor PTEN and inositol polyphosphate 5-phosphatases such as PIPP/INPP5J. PTEN and PIPP dephosphorylate PI(3,4,5)P3 to form different lipid products, thereby individually regulating AKT activation. PI3K/AKT signaling is complex and the functional interplay between these PI-phosphatases in suppressing this pathway in vivo is unknown. Here, we utilize experimental models of breast cancer, both dependent and independent of PIK3CA mutation. Pipp ablation in Pten+/- mice increases mammary AKT signaling and cell proliferation, associated with increased hyperplasia and ductal thickening, characteristics linked with mammary epithelial cell transformation. In breast cancer cell lines, combined PIPP/PTEN knockdown increases AKT signaling and cell proliferation, independent of mutant PIK3CA, above any single PI-phosphatase knockdown. Notably, combined PIPP/PTEN loss is observed in a subset of human breast cancers, associated with reduced survival. Collectively, these findings support a model whereby loss of PIPP constitutes a co-operative step towards breast cancer progression in the context of PTEN deficiency.

Abstract Background: Rab25 is a member of RAS protein superfamily of small GTPases that belong to RAB11 subfamily located on 1q22 of chromosome. It is generally expressed by epithelial cells and its expression level varies by tissue types. From our previous research, Rab25 has been shown to be a significant player in moderating transformation of human mammary epithelial cells, as its loss promotes cellular transformation particularly in presence of RAS mutation. Rab25 is an ambiguous protein in cancer research as its role is cancer type dependent and remains unknown in melanoma. There is deep deletion of Rab25 in a section of melanomas (only∼3% profiled) in databases like the Cancer Genome Atlas and Cbioportal. Additionally, Rab25 shares a common consensus motif with the binding site of mutant H-RAS, Q61L. NRAS is mutated in 80% of RAS activated melanomas. Therefore, we are investigating the role of Rab25 loss in the development of NRAS mutant melanoma. Objectives: To study the status of Rab25 and its role in the transformation of melanocytes, we screened for Rab25 expression and protein level in different melanoma cell lines. We further did immunohistochemistry to examine the Rab25 expression in biopsy-proven melanoma sections. To uncover the function of Rab25, we overexpressed Rab25 in NRAS mutant melanoma cell lines and knocked down Rab25 in non-tumorigenic immortalized melanocytes. Further, we will use liposomal encapsulation technique to deliver a six-peptide sequence intracellularly to target NRAS. Results: There was significant loss of Rab25 in mRNA and protein level in melanoma cell lines. Rab25 is found in normal skin tissues but was significantly lost in melanoma sections and associated with reduced survival. NRAS mutant human melanoma cells transduced with Rab25 showed reduced proliferation, migration tendency, and loss of anchorage-independence growth as compared to control cell lines. Additionally, knockdown of Rab25 in non-cancerous immortalized human melanocytes showed resulted in enhanced proliferative behavior and gain of anchorage-independent growth in soft agar colony formation assay. Conclusion: These observations suggest that Rab25 loss results in gain of migratory potential, anchorage-independent growth, and enhanced proliferation potential which are requisite events in melanoma-genesis. Further studies on associated functional proteins of Rab GTPase family will be conducted to strengthen this data. Citation Format: Samikshya Kandel, Krishna Rao. Loss of Rab25 and associated mechanisms in cancerous transformation of melanocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 272.

The luminal-to-basal transition in mammary epithelial cells (MECs) is accompanied by changes in epithelial cell lineage plasticity; however, the underlying mechanism remains elusive. Here, we report that deficiency of Frmd3 inhibits mammary gland lineage development and induces stemness of MECs, subsequently leading to the occurrence of triple-negative breast cancer. Loss of Frmd3 in PyMT mice results in a luminal-to-basal transition phenotype. Single-cell RNA sequencing of MECs indicated that knockout of Frmd3 inhibits the Notch signaling pathway. Mechanistically, FERM domain-containing protein 3 (FRMD3) promotes the degradation of Disheveled-2 by disrupting its interaction with deubiquitinase USP9x. FRMD3 also interrupts the interaction of Disheveled-2 with CK1, FOXK1/2, and NICD and decreases Disheveled-2 phosphorylation and nuclear localization, thereby impairing Notch-dependent luminal epithelial lineage plasticity in MECs. A low level of FRMD3 predicts poor outcomes for breast cancer patients. Together, we demonstrated that FRMD3 is a tumor suppressor that functions as an endogenous activator of the Notch signaling pathway, facilitating the basal-to-luminal transformation in MECs.

The extracellular matrix (ECM) is a dynamic and complex microenvironment that modulates cell behavior and cell fate. Changes in ECM composition and architecture have been correlated with development, differentiation, and disease progression in various pathologies, including breast cancer [1]. Studies have shown that aligned fibers drive a pro-metastatic microenvironment, promoting the transformation of mammary epithelial cells into invasive ductal carcinoma via the epithelial-to-mesenchymal transition (EMT) [2]. The impact of ECM orientation on breast cancer metabolism, however, is largely unknown. Here, we employ two non-invasive imaging techniques, fluorescence-lifetime imaging microscopy (FLIM) and intensity-based multiphoton microscopy, to assess the metabolic states of cancer cells cultured on ECM-mimicking nanofibers in a random and aligned orientation. By tracking the changes in the intrinsic fluorescence of nicotinamide adenine dinucleotide and flavin adenine dinucleotide, as well as expression levels of metastatic markers, we reveal how ECM fiber orientation alters cancer metabolism and EMT progression. Our study indicates that aligned cellular microenvironments play a key role in promoting metastatic phenotypes of breast cancer as evidenced by a more glycolytic metabolic signature on nanofiber scaffolds of aligned orientation compared to scaffolds of random orientation. This finding is particularly relevant for subsets of breast cancer marked by high levels of collagen remodeling (e.g. pregnancy associated breast cancer), and may serve as a platform for predicting clinical outcomes within these subsets [3–6].

The loss of RAB25 expression-RAS superfamily of GTPase characteristic of numerous breast cancers-corresponds with H-RAS point mutations, particularly in triple-negative breast cancers (TNBC), a subtype associated with a poor prognosis. To address the poorly understood factors dictating the progression of TNBC tumors, we examine the cooperative effects that loss of RAB25 expression in human mammary epithelial cell (HMEC) lines with H-RAS mutations confers in tumorigenesis. HMECs were immortalized by transduction with LXSN CDK4 R24C, a mutant form of cyclin-dependent kinase, followed by transduction with hTERT, a catalytic subunit of the telomerase enzyme. We found that with the loss of RAB25 and overexpression of mutant H-RAS61L, immortal HMECs transformed toward anchorage-independent growth and acquired an increased ability to migrate. Furthermore, cells express low CD24, high CD44, and low claudin levels, indicating stem-like properties upon transformation. Besides, loss of RAB25 and overexpression of H-RAS61L resulted in increased expression of transcription factors Snail and Slug that drive these cells to lose E-cadherin and undergo epithelial-mesenchymal transition (EMT). This study confirms that loss of RAB25 and overexpression of mutant H-RAS can drive HMECs toward a mesenchymal stem-like state. Our findings reveal that RAB25 functions as a tumor suppressor gene, and loss of RAB25 could serve as a novel biomarker of the claudin-low type of TNBC.

Breast cancer arises from the malignant transformation of mammary epithelial cells under the influence of various carcinogenic factors, leading to a gradual increase in its prevalence. This disease has become the leading cause of mortality among female malignancies, posing a significant threat to the health of women. The timely identification of breast cancer remains challenging, often resulting in diagnosis at the advanced stages of the disease. Conventional therapeutic approaches, such as surgical excision, chemotherapy and radiotherapy, exhibit limited efficacy in controlling the progression and metastasis of the disease. Regulated cell death (RCD), a process essential for physiological tissue cell renewal, occurs within the body independently of external influences. In the context of cancer, research on RCD primarily focuses on cuproptosis, ferroptosis and pyroptosis. Mounting evidence suggests a marked association between these specific forms of RCD, and the onset and progression of breast cancer. For example, a cuproptosis vector can effectively bind copper ions to induce cuproptosis in breast cancer cells, thereby hindering their proliferation. Additionally, the expression of ferroptosis‑related genes can enhance the sensitivity of breast cancer cells to chemotherapy. Likewise, pyroptosis‑related proteins not only participate in pyroptosis, but also regulate the tumor microenvironment, ultimately leading to the death of breast cancer cells. The present review discusses the unique regulatory mechanisms of cuproptosis, ferroptosis and pyroptosis in breast cancer, and the mechanisms through which they are affected by conventional cancer drugs. Furthermore, it provides a comprehensive overview of the significance of these forms of RCD in modulating the efficacy of chemotherapy and highlights their shared characteristics. This knowledge may provide novel avenues for both clinical interventions and fundamental research in the context of breast cancer.

Cholesterol (CHOL) is a multifaceted lipid molecule. It is an essential structural component of cell membranes, where it cooperates in regulating the intracellular trafficking and signaling pathways. Additionally, it serves as a precursor for vital biomolecules, including steroid hormones, isoprenoids, vitamin D, and bile acids. Although CHOL is normally uptaken from the bloodstream, cells can synthesize it de novo in response to an increased requirement due to physiological tissue remodeling or abnormal proliferation, such as in cancer. Cumulating evidence indicated that increased CHOL biosynthesis is a common feature of breast cancer and is associated with the neoplastic transformation of normal mammary epithelial cells. After an overview of the multiple biological activities of CHOL and its derivatives, this review will address the impact of de novo CHOL production on the promotion of breast cancer with a focus on mammary stem cells. The review will also discuss the effect of de novo CHOL production on in situ and invasive carcinoma and its impact on the response to adjuvant treatment. Finally, the review will discuss the present and future therapeutic strategies to normalize CHOL biosynthesis.

Adipocyte-derived kynurenine stimulates malignant transformation of mammary epithelial cells through the aryl hydrocarbon receptor

Abstract Background: RAB25 is a protein that belongs to the RAB11 subfamily located in the lq22 locus of chromosome and is ubiquitously expressed in cells of the gastrointestinal mucosa, ileum, and kidney. Its role in tumorigenesis is variable but is unclear in the case of melanoma progression. Our previous studies showed that loss of RAB25 has a special role in mediating the tumorigenic transformation of human mammary epithelial cells. Its loss particularly cooperated with mutant Ras isoforms to promote transformation in vitro. We are now examining the role of RAB25 in melanomas as melanomas exhibit a high rate of N-Ras mutations. We hypothesize that the presence of NRAS types of mutation and expression of RAB25 are mutually exclusive as RAB25 functions are a tumor suppressor to inhibit this mutation. Methods: Different NRAS mutant melanoma cell lines (SKMEL-2, SKMEL-28, WM1366 and WM266-4) were examined forRab25 expression through qRT-PCR and Western Blotting. The positive control was MCF cell lines and MDA-MB-231 was considered as negative control. Immunohistochemistry was done with biopsy proven melanoma tissue samples and normal adjacent tissue to observe the trend of Rab25 expression. Keratinocytes and fibroblasts in the tissue samples were considered as internal controls.Results: We found a significant decrease in Rab25 expression in NRAS mutant melanoma cell lines compared to Raf mutants and primary melanocytes. The reduction was prominent at both the RNA and protein level. RAB25 expression was also reduced in melanoma tumors as compared to surrounding tissue. Conclusion: RAB25 loss may indeed cooperate with NRAS mutation to promote transformation in melanomas. Further studies including overexpression experiment in the cell line model followed by migration assay, invasion assay, and in vivo nude mouse experiments will provide further confirmation of our hypothesis. Citation Format: Samikshya Kandel, Krishna Rao. Role of Rab25 in Melanoma and its potential targeting [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 2591.

Abstract Background: Low molecular weight cyclin E (LMW-E) are oncogenic forms of cyclin E that are post translationally generated from the full-length cyclin E1 (FL-cycE). LMW-E is detected in breast cancer cells and tumor tissues, but not in normal mammary epithelial cells or adjacent normal tissues. Unlike FL-cycE, LMW-E drives mammary epithelial cell transformation in human cells and spontaneous mammary tumor formation in transgenic mouse models, but the oncogenic mechanisms of LMW-E and its unique function(s) independent of FL-cycE are not fully understood. It is currently assumed that LMW-E drives the tumorigenic process by promoting G1/S cell cycle transition and accelerating mitotic exit. Biochemical features such as longer protein half-life, higher affinity to its kinase partner CDK2, and resistance to endogenous CDK inhibitors such as p21 and p27 all promote the tumorigenic ability of LMW-E. Clinical studies in breast cancer reveal that overexpression of LMW-E predicts recurrence and poor survival in breast cancer patients independent of molecular subtype, Ki67 status, nodal status, or tumor grade, suggesting LMW-E may drive breast cancer development independent of its role in cell proliferation. In the current study, we tested the hypothesis that LMW-E promotes genomic instability by deregulating DNA replication and damage repair. Results: We generated immortalized pre-cancerous human mammary epithelial cells (hMECs) to express doxycycline inducible LMW-E or FL-cycE in CCNE1 knock-out background. We found that FL-cycE overexpression led to DNA replication stress and DNA damage accumulation, resulting in reduced cell viability. LMW-E overexpression, on the other hand, promoted cell survival under replication stress, resulting in persistent genomic instability. RNA-sequencing results showed LMW-E but not FL-cycE overexpression enhanced DNA replication and damage repair pathways. Molecularly, LMW-E interacted with and facilitated pre-replication complex assembly. LMW-E also mediated DNA repair by upregulating RAD51 and C17orf53, showing a dominant repairing effect over DNA damage induced by FL-cycE. Moreover, targeting the replication stress response pathway ATR-CHK1-RAD51 with small molecule inhibitors significantly decreased viability of LMW-E overexpressing hMECs and breast cancer cells. Lastly, we showed that positive LMW-E status was associated with genomic instability in tumors from a cohort of 725 patients diagnosed with early-stage breast cancer, further supporting our hypothesis that LMW-E promotes genomic instability to fuel breast cancer development. Conclusions: Collectively, our findings delineated a novel role for LMW-E in breast tumorigenesis mediated by replication stress tolerance and genomic instability, providing novel therapeutic strategies for LMW-E overexpressing breast cancers. Citation Format: Mi Li, Spiridon Tsavachidis, Fuchenchu Wang, Tuyen Bui, Tuyen D. Nguyen, Linjie Luo, Asha S. Multani, Melissa L. Bondy, Kelly K. Hunt, Khandan Keyomarsi. Low molecular weight cyclin E deregulates DNA replication and damage repair to promote genomic instability in breast 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 308.

BackgroundIFN-γ has been traditionally recognized as an inflammatory cytokine that involves in inflammation and autoimmune diseases. Previously we have shown that sustained IFN-γ induced malignant transformation of bovine mammary epithelial cells (BMECs) via arginine depletion. However, the molecular mechanism underlying this is still unknown.MethodsIn this study, the amino acids contents in BMECs were quantified by a targeted metabolomics method. The acquisition of differentially expressed genes was mined from RNA-seq dataset and analyzed bioinformatically. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), western blotting, and immunohistochemistry (IHC) assay were performed to detect gene mRNA and protein expression levels. CCK-8 and would healing assays were used to detect cell proliferation and migration abilities, respectively. Cell cycle phase alternations were analyzed by flow cytometry.ResultsThe targeted metabolomics analysis specifically discovered IFN-γ induced arginine depletion through accelerating arginine catabolism and inhibiting arginine anabolism in BMECs. Transcriptome analysis identified leucine aminopeptidase 3 (LAP3), which was regulated by p38 and ERK MAPKs, to downregulate arginine level through interfering with argininosuccinate synthetase (ASS1) as IFN-γ stimulated. Moreover, LAP3 also contributed to IFN-γ-induced malignant transformation of BMECs by upregulation of HDAC2 (histone deacetylase 2) expression and promotion of cell cycle proteins cyclin A1 and D1 expressions. Arginine supplementation did not affect LAP3 and HDAC2 expressions, but slowed down cell cycle process of malignant BMECs. In clinical samples of patients with breast cancer, LAP3 was confirmed to be upregulated, while ASS1 was downregulated compared with healthy control.ConclusionsThese results demonstrated that LAP3 mediated IFN-γ-induced arginine depletion to malignant transformation of BMECs. Our findings provide a potential therapeutic target for breast cancer both in humans and dairy cows.

p21-Activated kinase-1 (Pak1) is frequently overexpressed and/or amplified in human breast cancer and is necessary for transformation of mammary epithelial cells. Here, we show that Pak1 interacts with and phosphorylates the Calcium/Calmodulin-dependent Protein Kinase II (CaMKII), and that pharmacological inhibition or depletion of Pak1 leads to diminished activity of CaMKII. We found a strong correlation between Pak1 and CaMKII expression in human breast cancer samples, and combined inhibition of Pak1 and CaMKII with small-molecule inhibitors was synergistic and induced apoptosis more potently in Her2 positive and triple negative breast cancer (TNBC) cells. Co-adminstration of Pak and CaMKII small-molecule inhibitors resulted in a dramatic reduction of proliferation and an increase in apoptosis in a 3D cell culture setting, as well as an impairment in migration and invasion of TNBC cells. Finally, mice bearing xenografts of TNBC cells showed a significant delay in tumor growth when treated with small-molecule inhibitors of Pak and CaMKII. These data delineate a signaling pathway from Pak1 to CaMKII that is required for efficient proliferation, migration and invasion of mammary epithelial cells, and suggest new therapeutic strategies in breast cancer.

Abstract Despite an enhanced interest, the origin of cancer stem cells (CSCs) remains equivocal. A strong body of evidence suggests that, in some adult carcinomas, CSC may originate from normal mature differentiated cells through a dedifferentiation process and the acquisition of stem-like properties. At the crux of this concept is the epithelial-mesenchymal transition (EMT), an embryonic transdifferentiation process that can be abnormally reactivated over the course of tumor development as a result of oncogenic or microenvironmental cues. High expression of EMT genes and stemness features are hallmarks of claudin-low (CL) breast cancers, a rare subtype of breast malignancies, generally considered as the most primitive breast cancers. We have recently reported that this molecular subtype of breast cancers exhibits a significant diversity, comprising three main subgroups (CL1, CL2 and CL3), that emerge from unique evolutionary processes. Genetic and epigenetic analyses support the hypothesis that CL1 tumors generate from the transformation of a normal mammary stem cells, whereas CL2 and CL3 arise from the reactivation of an EMT process over the course of tumor progression. Interestingly, CL tumors share the frequent activation of the RAS-MAPK pathway, suggesting a potent role for the RAS pathway in the development of CL tumors. Based on these findings, we wondered whether RAS pathway activation could induce the reactivation of EMT-TFs allowing the emergence of CSCs displaying CL2 and CL3 characteristics. By expressing an oncogenic form of RAS in differentiated epithelial cells (HME), we observed a phenotypic switch towards a CD24-/low/CD44+ phenotype, associated with CSC properties, and suggesting an acquisition of cellular plasticity after RAS activation. By using CRISPR/Cas9 knock-down experiments we have shown i) the emergence of CD24-/low/CD44+ cells, ii) the acquisition of transformation and stemness features, and iii) that the cellular plasticity acquired after RAS activation in differentiated HME strongly rely on ZEB1. Single-cell RNA sequencing analyses after RAS activation in HME-RAS cells suggested two different outcomes: senescence or EMT. Treatment with a senolytic drug resulted in reduction of RAS-induced senescent cells associated with a decrease in the emergence of CD24-/low/CD44+ cells and ZEB1 expression. Finally, a co-culture reporter system and a depletive approach with cytokine-neutralizing antibodies highlighted a paracrine effect of IL-6 and IL-1α, produced by senescent RAS-expressing cells on their neighboring differentiated cells, leading to the acquisition of ZEB1-dependent cellular plasticity. Hence, we unveiled a new paracrine process, in which cytokines secreted by cells undergoing oncogene-induced senescence drive ZEB1 expression, thus promoting cellular plasticity correlated with the emergence of CSCs. Citation Format: ALAIN PUISIEUX, Hadrien De Blander, Laurie Tonon, Frédérique Fauvet, Roxane Pommier, Christelle Lamblot, Rahma Benhassoun, Benjamin Gibert, Maria Ouzounova, Anne-Pierre Morel. RAS-induced transformation of mammary epithelial cells relies upon a ZEB1-dependent cellular reprogramming through a paracrine process [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 2667.

Abstract Approximately 20% to a quarter of breast cancer cases are estrogen receptor (ER)-negative breast cancer (ENBC), including the most aggressive subtype, triple-negative breast cancer (TNBC), which spreads early and shows poor prognosis. ENBCs do not respond to selective ER modulators such as tamoxifen. Therefore, there is an urgent need for non-ER-based therapies for ENBC. Recently, STAT3 was found to play an important role in maintaining cancer stem cells in vitro and in mouse models of human cancers. In addition, during mammary carcinogenesis, STAT3 is highly activated in mammary epithelial cells involving in mammary epithelial cell transformation, tumor initiation, progression, and metastasis. STAT3 has been traditionally considered as non-targetable or undruggable, and there is currently a lack of FDA-approved STAT3 inhibitors for clinical use. We recently developed a putative STAT3 inhibitor, HJC0152, and a series of HJC0152-based analogues as orally bioavailable STAT3 inhibitors. These small molecules have been evaluated and a number of them showed low micro-molar potency in inhibiting the proliferation of a panel of breast cancer cell lines. Compound JMX0804 showed improved anti-proliferative effects against the highly aggressive TNBC cell line MDA-MB-231 and other ER+ and ER- breast cancer cell lines in comparison to HJC0152. JMX0804 inhibited STAT3 phosphorylation at the Tyr705 residue and induced apoptosis in MDA-MB-231 cells. In vivo, mice treated with JMX0804 at a 10 mg/kg dose via i.p. showed reduced growth of xenograft tumors arisen from MDA-MB-231 cells, with minimal impact on general health and body weight. Our current research supports the notion that JMX0804 suppresses breast cancer growth by inhibiting breast cancer cell proliferation and inducing apoptosis via mediating STAT3 signaling. JMX0804 is currently undergoing further characterization for breast cancer metastasis and mechanisms of action as well as optimization to improve drug properties including potency and safety profile. This work is supported by NIH/NCI grant R01 CA231150. Citation Format: Hyejin Kim, Doerte R. Fricke, Jimin Xu, Haiying Chen, Jia Zhou, Qiang Shen. Treating estrogen receptor (ER)-negative and triple-negative breast cancer by targeting STAT3 signaling with putative STAT3 inhibitors [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 1238.

Histone methyltransferase NSD3 is frequently dysregulated in human cancers, yet the epigenetic role of NSD3 during cancer development remains elusive. Here we report that NSD3-induced methylation of H3K36 is crucial for breast tumor initiation and metastasis. In patients with breast cancer, elevated expression of NSD3 was associated with recurrence, distant metastasis, and poor survival. In vivo, NSD3 promoted malignant transformation of mammary epithelial cells, a function comparable to that of HRAS. Furthermore, NSD3 expanded breast cancer-initiating cells and promoted epithelial-mesenchymal transition to trigger tumor invasion and metastasis. Mechanistically, the long isoform (full-length transcript) of NSD3, but not its shorter isoform lacking a catalytic domain, cooperated with EZH2 and RNA polymerase II to stimulate H3K36me2/3-dependent transactivation of genes associated with NOTCH receptor cleavage, leading to nuclear accumulation of NICD and NICD-mediated transcriptional repression of E-cadherin. Furthermore, mice harboring primary and metastatic breast tumors with overexpressed NSD3 showed sensitivity to NOTCH inhibition. Together, our findings uncover the critical epigenetic role of NSD3 in the modulation of NOTCH-dependent breast tumor progression, providing a rationale for targeting the NSD3-NOTCH signaling regulatory axis in aggressive breast cancer. SIGNIFICANCE: This study demonstrates the functional significance of histone methyltransferase NSD3 in epigenetic regulation of breast cancer stemness, EMT, and metastasis, suggesting NSD3 as an actionable therapeutic target in metastatic breast cancer.

Gene transcription is regulated by distant regulatory elements via combinatorial binding of transcription factors. It is increasingly recognized that alterations in chromatin state and transcription factor binding in these distant regulatory elements may have key roles in cancer development. Here we focused on the first stages of oncogene-induced carcinogenic transformation, and characterized the regulatory network underlying transcriptional changes associated with this process. Using Hi-C data, we observe spatial coupling between differentially expressed genes and their differentially accessible regulatory elements and reveal two candidate transcription factors, p53 and CTCF, as determinants of transcriptional alterations at the early stages of oncogenic HRas-induced transformation in human mammary epithelial cells. Strikingly, the malignant transcriptional reprograming is promoted by redistribution of chromatin binding of these factors without major variation in their expression level. Our results demonstrate that alterations in the regulatory landscape have a major role in driving oncogene-induced transcriptional reprogramming.

Abstract The physical microenvironment of tumor cells plays an important role in cancer initiation and progression. Here, we present evidence that confinement - a new physical parameter that is apart from matrix stiffness - can also induce malignant transformation in mammary epithelial cells. We discovered that MCF10A cells, a benign mammary cell line that forms growth-arrested polarized acini in Matrigel, transforms into cancer-like cells within the same Matrigel material following confinement in alginate shell hydrogel microcapsules. The confined cells exhibited a range of tumor-like behaviors, including uncontrolled cellular proliferation and invasion. Additionally, 4-6 weeks after transplantation into the mammary fad pads of immunocompromised mice, the confined cells formed large palpable masses that exhibited histological features similar to that of carcinomas. Taken together, our findings suggest that physical confinement represents a previously unrecognized mechanism for malignancy induction in mammary epithelial cells and also provide a new, microcapsule-based, high throughput model system for testing new breast cancer therapeutics. Citation Format: Yen-Chun Lu. Physical confinement induces malignant transformation in mammary epithelial cells [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 3963.

e13578 Background: General/overall obesity and visceral/central obesity are associated with increased breast cancer risk and poorer cancer outcomes; however, the precise etiology of these observations is not fully known. Our previous research suggests that adipose tissue-derived fibroblast growth factor 2 (FGF2) promotes malignant transformation of mammary epithelial cells through the activation of its primary receptor, fibroblast growth factor receptor 1 (FGFR1). Both FGF2 and leptin have been shown to be elevated in the serum in obesity (body mass index (BMI) > 25kg/m2). It is well established that elevated sera leptin promotes breast cancer through multiple mechanisms. Therefore, we hypothesized that tumors that express elevated FGFR1 and leptin receptor (LepR) may have a poorer prognosis in obese patients. Methods: Databases that house gene-sequenced breast cancer tumors and normal breast tissue were analyzed to discern relationships between gene copy number and mRNA levels of leptin, leptin receptor, FGFR1 and janus kinase 2 (Jak2), as Jak2 was shown to be a common downstream mediator of both FGFR1 and leptin receptor activation by Cytoscape network analysis. Furthermore, we tested the efficacy of Jak2 inhibitors for preventing mammary epithelial cell malignant transformation using our well-established models of adipose tissue-stimulated growth in soft agar. The anchorage-independent growth in soft agar is a surrogate marker for malignant transformation. Results: A much stronger significant relationship exists between FGFR1 mRNA and leptin mRNA in primary breast cancer (p = 3.17*10−9) compared with normal breast epithelium (p = 0.0013). In primary breast tumors, gene copy number of FGFR1 positively correlates with leptin copy number (p = 8.956*10−8, r = 0.19). Moreover, two separate Jak2 inhibitors attenuated both leptin+FGF2-stimulated and mouse adipose tissue-stimulated MCF-10A cell transformation. Conclusions: Taken together, these results suggest that elevated sera FGF2 and leptin in obese patients may promote breast tumorigenesis in tumors that express elevated FGFR1 and LepR and that Jak2 inhibitors may be a novel therapeutic option for adiposity-driven breast cancers. Future work will examine the relationship between FGFR1/leptin receptor expression and survival and how these relationships are influenced by BMI status.

Oncogenic transformation of mammary epithelial cells (MECs) is a critical step in epithelial-to-mesenchymal transition (EMT), but evidence also shows that MECs undergo EMT with increasing matrix stiffness; the interplay of genetic and environmental effects on EMT is not clear. To understand their combinatorial effects on EMT, premalignant MCF10A and isogenic Ras-transformed MCF10AT are cultured on polyacrylamide gels ranging from normal mammary stiffness, ≈150 Pa, to tumor stiffness, ≈5700 Pa. Though cells spread on stiff hydrogels independent of transformation, only 10AT cells exhibit heterogeneous spreading behavior on soft hydrogels. Within this mixed population, spread cells exhibit an elongated, mesenchymal-like morphology, disrupted localization of the basement membrane, and nuclear localization of the EMT transcription factor TWIST1. MCF10AT spreading is not driven by typical mechanosensitive pathways including YAP and TGF-β or by myosin contraction. Rather, ERK activation induces spreading of MCF10AT cells on soft hydrogels and requires dynamic microtubules. These findings indicate the importance of oncogenic signals, and their hierarchy with substrate mechanics, in regulating MEC EMT.

Physical confinement induces malignant transformation in mammary epithelial cells