Evidence has been accumulating to indicate that extracellular vesicles (EVs), including exosomes, released by cancer cells can foster tumour progression. The molecular chaperones – CDC37, HSP90α and HSP90β play key roles in cancer progression including epithelial‐mesenchymal transition (EMT), although their contribution to EVs‐mediated cell–cell communication in tumour microenvironment has not been thoroughly examined. Here we show that triple depletion of the chaperone trio attenuates numerous cancer malignancy events exerted through EV release. Metastatic oral cancer‐derived EVs (MEV) were enriched with HSP90α HSP90β and cancer‐initiating cell marker CD326/EpCAM. Depletion of these chaperones individually induced compensatory increases in the other chaperones, whereas triple siRNA targeting of these molecules markedly diminished the levels of the chaperone trio and attenuated EMT. MEV were potent agents in initiating EMT in normal epithelial cells, a process that was attenuated by the triple chaperone depletion. The migration, invasion, and in vitro tumour initiation of oral cancer cells were significantly promoted by MEV, while triple depletion of CDC37/HSP90α/β reversed these MEV‐driven malignancy events. In metastatic oral cancer patient‐derived tumours, HSP90β was significantly accumulated in infiltrating tumour‐associated macrophages (TAM) as compared to lower grade oral cancer cases. HSP90‐enriched MEV‐induced TAM polarization to an M2 phenotype, a transition known to support cancer progression, whereas the triple chaperone depletion attenuated this effect. Mechanistically, the triple chaperone depletion in metastatic oral cancer cells effectively reduced MEV transmission into macrophages. Hence, siRNA‐mediated knockdown of the chaperone trio (CDC37/HSP90α/HSP90β) could potentially be a novel therapeutic strategy to attenuate several EV‐driven malignancy events in the tumour microenvironment. Abbreviations: CDC37: cell division control 37; EMT: epithelial‐mesenchymal transmission; EV: extracellular vesicles; HNSCC: head and neck squamous cell carcinoma; HSP90: heat shock protein 90; TAM: tumour‐associated macrophage

Tumor cells exhibit therapeutic stress resistance-associated secretory phenotype involving extracellular vesicles (EVs) such as oncosomes and heat shock proteins (HSPs). Such a secretory phenotype occurs in response to cell stress and cancer therapeutics. HSPs are stress-responsive molecular chaperones promoting proper protein folding, while also being released from cells with EVs as well as a soluble form known as alarmins. We have here investigated the secretory phenotype of castration-resistant prostate cancer (CRPC) cells using proteome analysis. We have also examined the roles of the key co-chaperone CDC37 in the release of EV proteins including CD9 and epithelial-to-mesenchymal transition (EMT), a key event in tumor progression. EVs derived from CRPC cells promoted EMT in normal prostate epithelial cells. Some HSP family members and their potential receptor CD91/LRP1 were enriched at high levels in CRPC cell-derived EVs among over 700 other protein types found by mass spectrometry. The small EVs (30–200 nm in size) were released even in a non-heated condition from the prostate cancer cells, whereas the EMT-coupled release of EVs (200–500 nm) and damaged membrane vesicles with associated HSP90α was increased after heat shock stress (HSS). GAPDH and lactate dehydrogenase, a marker of membrane leakage/damage, were also found in conditioned media upon HSS. During this stress response, the intracellular chaperone CDC37 was transcriptionally induced by heat shock factor 1 (HSF1), which activated the CDC37 core promoter, containing an interspecies conserved heat shock element. In contrast, knockdown of CDC37 decreased EMT-coupled release of CD9-containing vesicles. Triple siRNA targeting CDC37, HSP90α, and HSP90β was required for efficient reduction of this chaperone trio and to reduce tumorigenicity of the CRPC cells in vivo. Taken together, we define “stressome” as cellular stress-induced all secretion products, including EVs (200–500 nm), membrane-damaged vesicles and remnants, and extracellular HSP90 and GAPDH. Our data also indicated that CDC37 is crucial for the release of vesicular proteins and tumor progression in prostate cancer.

Matrix metalloproteinase 3 (MMP3) plays multiple roles in extracellular proteolysis as well as intracellular transcription, prompting a new definition of moonlighting metalloproteinase (MMP), according to a definition of protein moonlighting (or gene sharing), a phenomenon by which a protein can perform more than one function. Indeed, connective tissue growth factor (CTGF, aka cellular communication network factor 2 (CCN2)) is transcriptionally induced as well as cleaved by MMP3. Moreover, several members of the MMP family have been found within tumor-derived extracellular vesicles (EVs). We here investigated the roles of MMP3-rich EVs in tumor progression, molecular transmission, and gene regulation. EVs derived from a rapidly metastatic cancer cell line (LuM1) were enriched in MMP3 and a C-terminal half fragment of CCN2/CTGF. MMP3-rich, LuM1-derived EVs were disseminated to multiple organs through body fluid and were pro-tumorigenic in an allograft mouse model, which prompted us to define LuM1-EVs as oncosomes in the present study. Oncosome-derived MMP3 was transferred into recipient cell nuclei and thereby trans-activated the CCN2/CTGF promoter, and induced CCN2/CTGF production in vitro. TRENDIC and other cis-elements in the CCN2/CTGF promoter were essential for the oncosomal responsivity. The CRISPR/Cas9-mediated knockout of MMP3 showed significant anti-tumor effects such as the inhibition of migration and invasion of tumor cells, and a reduction in CCN2/CTGF promoter activity and fragmentations in vitro. A high expression level of MMP3 or CCN2/CTGF mRNA was prognostic and unfavorable in particular types of cancers including head and neck, lung, pancreatic, cervical, stomach, and urothelial cancers. These data newly demonstrate that oncogenic EVs-derived MMP is a transmissive trans-activator for the cellular communication network gene and promotes tumorigenesis at distant sites.

Tumor growth, progression, and therapy resistance are crucial factors in the prognosis of cancer. The properties of three-dimensional (3D) tumor-like organoids (tumoroids) more closely resemble in vivo tumors compared to two-dimensionally cultured cells and are therefore effectively used for assays and drug screening. We here established a repurposed drug for novel anticancer research and therapeutics using a 3D tumoroid-based screening system. We screened six pharmacologically active compounds by using an original tumoroid-based multiplex phenotypic screening system with a matrix metalloproteinase 9 (MMP9) promoter-driven fluorescence reporter for the evaluation of both tumoroid formation and progression. The antiparkinson drug benztropine was the most effective compound uncovered by the screen. Benztropine significantly inhibited in vitro tumoroid formation, cancer cell survival, and MMP9 promoter activity. Benztropine also reduced the activity of oncogenic signaling transducers and trans-activators for MMP9, including STAT3, NF-κB, and β-catenin, and the properties of cancer stem cells/cancer-initiating cells. Benztropine and GBR-12935 directly targeted the dopamine transporter DAT/SLC6A3, whose genetic alterations such as amplification were correlated with poor prognosis for cancer patients. Benztropine also inhibited the tumor growth, circulating tumor cell (CTC) number, and rate of metastasis in a tumor allograft model in mice. In conclusion, we propose the repurposing of benztropine for anticancer research and therapeutics that can suppress tumor progression, CTC, and metastasis of aggressive cancers by reducing key pro-tumorigenic factors.

Replicative DNA polymerases are blocked by nearly all types of DNA damage. The resulting DNA replication stress threatens genome stability. DNA replication stress is also caused by depletion of nucleotide pools, DNA polymerase inhibitors, and DNA sequences or structures that are difficult to replicate. Replication stress triggers complex cellular responses that include cell cycle arrest, replication fork collapse to one-ended DNA double-strand breaks, induction of DNA repair, and programmed cell death after excessive damage. Replication stress caused by specific structures (e.g., G-rich sequences that form G-quadruplexes) is localized but occurs during the S phase of every cell division. This review focuses on cellular responses to widespread stress such as that caused by random DNA damage, DNA polymerase inhibition/nucleotide pool depletion, and R-loops. Another form of global replication stress is seen in cancer cells and is termed oncogenic stress, reflecting dysregulated replication origin firing and/or replication fork progression. Replication stress responses are often dysregulated in cancer cells, and this too contributes to ongoing genome instability that can drive cancer progression. Nucleases play critical roles in replication stress responses, including MUS81, EEPD1, Metnase, CtIP, MRE11, EXO1, DNA2-BLM, SLX1-SLX4, XPF-ERCC1-SLX4, Artemis, XPG, FEN1, and TATDN2. Several of these nucleases cleave branched DNA structures at stressed replication forks to promote repair and restart of these forks. We recently defined roles for EEPD1 in restarting stressed replication forks after oxidative DNA damage, and for TATDN2 in mitigating replication stress caused by R-loop accumulation in BRCA1-defective cells. We also discuss how insights into biological responses to genome-wide replication stress can inform novel cancer treatment strategies that exploit synthetic lethal relationships among replication stress response factors.

Cell division control 37 (CDC37) increases the stability of heat shock protein 90 (HSP90) client proteins and is thus essential for numerous intracellular oncogenic signaling pathways, playing a key role in prostate oncogenesis. Notably, elevated expression of CDC37 was found in prostate cancer cells, although the regulatory mechanisms through which CDC37 expression becomes increased are unknown. Here we show both positive and negative regulation of CDC37 gene transcription by two members of the SREZBP-CTfin51-AW1-Number 18 cDNA (SCAN) transcription factor family—MZF1 and SCAND1, respectively. Consensus DNA-binding motifs for myeloid zinc finger 1 (MZF1/ZSCAN6) were abundant in the CDC37 promoter region. MZF1 became bound to these regulatory sites and trans-activated the CDC37 gene whereas MZF1 depletion decreased CDC37 transcription and reduced the tumorigenesis of prostate cancer cells. On the other hand, SCAND1, a zinc fingerless SCAN box protein that potentially inhibits MZF1, accumulated at MZF1-binding sites in the CDC37 gene, negatively regulated the CDC37 gene and inhibited tumorigenesis. SCAND1 was abundantly expressed in normal prostate cells but was reduced in prostate cancer cells, suggesting a potential tumor suppressor role of SCAND1 in prostate cancer. These findings indicate that CDC37, a crucial protein in prostate cancer progression, is regulated reciprocally by MZF1 and SCAND1.