Comprehensive genomic analyses of small cell lung cancer (SCLC) have revealed frequent mutually exclusive genomic amplification of MYC family members. Hence, it has been long suggested that they are functionally equivalent; however, more recently, their expression has been associated with specific neuroendocrine markers and distinct histopathology. Here, we explored a previously undescribed role of L-Myc and c-Myc as lineage-determining factors contributing to SCLC molecular subtypes and histology. Integrated transcriptomic and epigenomic analyses showed that L-Myc and c-Myc impart neuronal and non-neuroendocrine–associated transcriptional programs, respectively, both associated with distinct SCLC lineage. Genetic replacement of c-Myc with L-Myc in c-Myc–SCLC induced a neuronal state but was insufficient to induce ASCL1-SCLC. In contrast, c-Myc induced transition from ASCL1-SCLC to NEUROD1–SCLC characterized by distinct large-cell neuroendocrine carcinoma–like histopathology. Collectively, we characterize a role of historically defined general oncogenes, c-Myc and L-Myc, for regulating lineage plasticity across molecular and histological subtypes.

Tumor-initiating cells (TICs) are cancer cells endowed with self-renewal, multi-lineage differentiation, increased chemo-resistance, and in breast cancers the CD44+/CD24-/ALDH1+ phenotype. Triple negative breast cancers show lack of BRCA1 expression in addition to enhanced basal, epithelial-to-mesenchymal transition (EMT), and TIC phenotypes. BRCA1-IRIS (hereafter IRIS) is an oncogene produced by the alternative usage of the BRCA1 locus. IRIS is involved in induction of replication, transcription of selected oncogenes, and promoting breast cancer cells aggressiveness. Here, we demonstrate that IRIS overexpression (IRISOE) promotes TNBCs through suppressing BRCA1 expression, enhancing basal-biomarkers, EMT-inducers, and stemness-enforcers expression. IRISOE also activates the TIC phenotype in TNBC cells through elevating CD44 and ALDH1 expression/activity and preventing CD24 surface presentation by activating the internalization pathway EGFR→c-Src→cortactin. We show that the intrinsic sensitivity to an anti-CD24 cross-linking antibody-induced cell death in membranous CD24 expressing/luminal A cells could be acquired in cytoplasmic CD24 expressing IRISOE TNBC/TIC cells through IRIS silencing or inactivation. We show that fewer IRISOE TNBC/TICs cells form large tumors composed of TICs, resembling TNBCs early lesions in patients that contain metastatic precursors capable of disseminating and metastasizing at an early stage of the disease. IRIS-inhibitory peptide killed these IRISOE TNBC/TICs, in vivo and prevented their dissemination and metastasis. We propose IRIS inactivation could be pursued to prevent dissemination and metastasis from early TNBC tumor lesions in patients.

Resident mesenchymal stem cells (MSCs) promote cancer progression. However, pathways and mechanisms involved in recruiting MSCs into breast tumors remain largely undefined. Here we show that geminin-dependent acetylation releases HMGB1 from the chromatin to the cytoplasm and extracellular space. Extracellular acetylated HMGB1 (Ac-HMGB1) promotes geminin overexpressing (GemOE) cells survival by binding to RAGE and activating NF-κB signaling. Extracellular Ac-HMGB1 also triggers expression and activation of RAGE in the non-expressing MSCs. RAGE activation induces expression of CXCR4 in MSCs and directional migration towards SDF1 (aka CXCL12)-expressing GemOE cells in vitro and in vivo. These effects augmented by the necrotic and hypoxic environment in GemOE tumors, especially within their cores. Reciprocal interactions between newly recruited MSCs and GemOE tumor cells elevate tumor-initiating (TIC), basal and epithelial-to-mesenchymal transition (EMT) traits and enhance aggressiveness in vitro and in vivo in GemOE tumor cells. Indeed, faster, larger and more aggressive tumors develop when GemOE cells are co-injected with MSCs in orthotopic breast tumor model. Concurrently, inhibiting c-Abl (and thus geminin function), RAGE or CXCR4 prevented MSCs recruitment to GemOE cells in vitro and in vivo, and decreased the TIC, basal and EMT phenotypes in these tumor cells. Accordingly, we propose that GemOE tumor cells present within tumor cores represent metastatic precursors, and suppressing the GemOE→HMGB1/RAGE→SDF1/CXCR4 signaling circuit could be a valid target for therapies to inhibit GemOE tumors and their metastases.

The relationship between tumor initiation and tumor progression can follow a linear projection in which all tumor cells are equally endowed with the ability to progress into metastasis. Alternatively, not all tumor cells are equal genetically and/or epigenetically, and only few cells are induced to become metastatic tumor cells. The location of these cells within the tumor can also impact the fate of these cells. The most inner core of a tumor where an elevated pressure of adverse conditions forms, such as necrosis-induced inflammation and hypoxia-induced immunosuppressive environment, seems to be the most fertile ground to generate such tumor cells with metastatic potential. Here we will call this necrotic/hypoxic core the “aggressiveness niche” and will present data to support its involvement in generating these metastatic precursors. Within this niche, interaction of hypoxia-surviving cells with the inflammatory microenvironment influenced by newly recruited mesenchymal stromal cells (MSCs), tumor-associated macrophages (TAMs), and other types of cells and the establishment of bidirectional interactions between them elevate the aggressiveness of these tumor cells. Additionally, immune evasion properties induced in these cells most likely contribute in the formation and maintenance of such aggressiveness niche.

BackgroundRecently, we reported an association of a novel cancer testis (CT) antigen, sperm-associated antigen 9 (SPAG9) expression in breast cancer clinical samples, indicating its potential role in carcinogenesis. Around 15% breast cancers are designated as triple-negative for which treatment modalities are limited. Therefore, in the present study, we assessed the role of SPAG9 in triple-negative breast cancer cells.MethodsSPAG9 mRNA and protein expression was investigated in various breast cancer cells of different hormone receptor status and different subtypes by employing reverse transcriptase-polymerase chain reaction (RT-PCR), real time PCR, Western blotting, indirect immunofluorescence (IIF) and fluorescence activated cell sorting (FACS). Employing plasmid-based small interfering RNA (siRNA) approach, knockdown of SPAG9 was carried out in triple-negative breast cancer cells, MDA-MB-231, to assess its role on various malignant properties in vitro and in vivo.ResultsSPAG9 mRNA and protein expression was detected in all breast cancer cells. Further, IIF results showed that SPAG9 was predominantly localized in the cytoplasm of breast cancer cells. FACS analysis revealed distinct SPAG9 surface localization in breast cancer cells. Gene silencing of SPAG9 resulted in significant reduction in cellular proliferation, colony forming ability, migration, invasion and cellular motility of MDA-MB-231 cells. Further, ablation of SPAG9 expression resulted in reduction in the tumor growth of human breast cancer xenograft in nude mice in vivo.ConclusionsIn summary, our data indicated that down regulation of SPAG9 reduces growth and invasive potential of triple-negative breast cancer cells, suggesting that SPAG9 may be a potential target for therapeutic use.