Diagnostic potential of exosomal extracellular vesicles in oncology.

Liquid biopsy can detect circulating cancer cells or tumor cell-derived DNA at various stages of cancer. The fluid from these biopsies contains extracellular vesicles (EVs), such as apoptotic bodies, microvesicles, exomeres, and exosomes. Exosomes contain proteins and nucleic acids (DNA/RNA) that can modify the microenvironment and promote cancer progression, playing significant roles in cancer pathology. Clinically, the proteins and nucleic acids within the exosomes from liquid biopsies can be biomarkers for the detection and prognosis of cancer. We review EVs protein and miRNA biomarkers identified for select cancers, specifically melanoma, glioma, breast, pancreatic, hepatic, cervical, prostate colon, and some hematological malignancies. Overall, this review demonstrates that EV biomolecules have great potential to expand the diagnostic and prognostic biomarkers used in Oncology; ultimately, EVs could lead to earlier detection and novel therapeutic targets. Clinical implicationsEVs represent a new paradigm in cancer diagnostics and therapeutics. The potential use of exosomal contents as biomarkers for diagnostic and prognostic indicators may facilitate cancer management. Non-invasive liquid biopsy is helpful, especially when the tumor is difficult to reach, such as in pancreatic adenocarcinoma. Moreover, another advantage of using minimally invasive liquid biopsy is that monitoring becomes more manageable. Identifying tumor-derived exosomal proteins and microRNAs would allow a more personalized approach to detecting cancer and improving treatment.

Evaluation of the Role of Human DNAJAs in the Response to Cytotoxic Chemotherapeutic Agents in a Yeast Model System.

Heat-shock proteins (HSPs) play a crucial role in maintaining protein stability for cell survival during stress-induced insults. Overexpression of HSPs in cancer cells results in antiapoptotic activity contributing to cancer cell survival and restricting the efficacy of cytotoxic chemotherapy, which continues to play an important role in the treatment of many cancers, including triple-negative breast cancer (TNBC). First-line therapy for TNBC includes anthracycline antibiotics, which are associated with serious dose-dependent side effects and the development of resistance. We previously identified YDJ1, which encodes a heat-shock protein 40 (HSP40), as an important factor in the cellular response to anthracyclines in yeast, with mutants displaying over 100-fold increased sensitivity to doxorubicin. In humans, the DNAJA HSP40s are homologues of YDJ1. To determine the role of DNAJAs in the cellular response to cytotoxic drugs, we investigated their ability to rescue ydj1Δ mutants from exposure to chemotherapeutic agents. Our results indicate that DNAJA1 and DNAJA2 provide effective protection, while DNAJA3 and DNAJA4 did not. The level of complementation was also dependent on the agent used, with DNAJA1 and DNAJA2 rescuing the ydj1Δ strain from doxorubicin, cisplatin, and heat shock. DNAJA3 and DNAJA4 did not rescue the ydj1Δ strain and interfered with the cellular response to stress when expressed in wild type background. DNAJA1 and DNAJA2 protect the cell from proteotoxic damage caused by reactive oxygen species (ROS) and are not required for repair of DNA double-strand breaks. These data indicate that the DNAJAs play a role in the protection of cells from ROS-induced cytotoxic stress.

Synergistic Effect of Endogenous and Exogenous Aldehydes on Doxorubicin Toxicity in Yeast.

Anthracyclines are frequently used to treat many cancers including triple negative breast cancer, which is commonly observed in African-American women (AA), and tend to be more aggressive, carry worse prognoses, and are harder to manage because they lack molecular targets. Although effective, anthracyclines use can be limited by serious side effects and eventually the development of drug resistance. In S. cerevisiae, mutants of HOM6 display hypersensitivity to doxorubicin. HOM6 is required for synthesis of threonine and interruption of the pathway leads to accumulation of the threonine intermediate L-aspartate-semialdehyde. This intermediate may synergize with doxorubicin to kill the cell. In fact, deleting HOM3 in the first step, preventing the pathway to reach the HOM6 step, rescues the sensitivity of the hom6 strain to doxorubicin. Using several S. cerevisiae strains (wild type, hom6, hom3, hom3hom6, ydj1, siz1, and msh2), we determined their sensitivity to aldehydes and to their combination with doxorubicin, cisplatin, and etoposide. Combination of formaldehyde and doxorubicin was most effective at reducing cell survival by 31-fold-39-fold (in wild type cells) relative to doxorubicin and formaldehyde alone. This effect was dose dependent on doxorubicin. Cotreatment with formaldehyde and doxorubicin also showed increased toxicity in anthracycline-resistant strains siz1 and msh2. The hom6 mutant also showed sensitivity to menadione with a 2.5-fold reduction in cell survival. The potential use of a combination of aldehydes and cytotoxic drugs could potentially lead to applications intended to enhance anthracycline-based therapy.

The Role of HSP40 Conserved Motifs in the Response to Cytotoxic Stress.

Doxorubicin, a highly effective therapeutic agent against several types of cancer, is associated with serious side-effects, particularly cardiotoxicity. In addition, drug resistance leads to unsuccessful outcomes in many patients. There are no current biomarkers to indicate doxorubicin treatment response in patients. To understand the mechanisms of toxicity of doxorubicin, a whole-genome sensitivity screen was performed in the yeast S. cerevisiae. A deletion mutant of the yeast DNAJ (YDJ1), a J-domain heat-shock protein 40 (HSP40) was among the most sensitive strains. HSP40 is a co-chaperone to HSP70 and together refold denatured proteins into native conformation. The HSP40 YDJ1 is comprised of several highly-conserved domains and motifs that are essential in the heat-shock response. The cysteine-rich region has been implicated in protein-protein interaction with client proteins, farnesylation of YDJ1 facilitates attachment of YDJ1 to the ER and perinuclear membranes, and the histidine-proline-aspartic acid (HPD) tripeptide motif present in the J-domain, is responsible for the regulation of the ATPase activity of HSP70s. We have investigated the role of these motifs in the protection cytotoxic stress. We find that mutations in the HPD motif and cysteine-rich region of YDJ1 sensitize cells to doxorubicin and cisplatin, while a mutation in farnesylation results in a slightly protective effect. The sensitivity of the HPD and cysteine mutants is specific to oxidative stress and not to DNA double-strand breaks.

THE ROLE OF PROTEIN CHAPERONES IN THE SURVIVAL FROM ANTHRACYCLINE-INDUCED OXIDATIVE STRESS IN SACCHAROMYCES CEREVISIAE.

Several S. cerevisiae deletion strains involving heat-shock response factors were among the most sensitive mutants identified in a previous genetic screen for doxorubicin hypersensitivity. These strains included ydj1Δ, ssz1Δ and zuo1Δ mutants. In addition, new1Δ, whose function was unknown, also displayed significant sensitivity to anthracyclines. We further investigated the basis for the sensitivity of these mutants. We determined that heat-shock could partially rescue the sensitivity of the strains to doxorubicin, including the homologous recombination mutant rad52Δ, which is sensitive to doxorubicin-mediated DNA double strand breaks (DSBs). However, none of the heat-shock response mutants were sensitive to DSBs, but were highly sensitive to reactive oxygen species (ROS) generated by quinone-ring-containing agents, such as anthracyclines and menadione. A fluorescent-based assay indicates that doxorubicin causes protein aggregation. Interestingly, the disaggregase mutant hsp104Δ is not sensitive to anthracyclines or menadione suggesting that Hsp104p does not play a role in disaggregating doxorubicin-induced protein aggregates. However New1p, which has been recently shown to be a novel disaggregase, is essential for cell viability after exposure to anthracyclines and menadione and it is not involved in thermotolerance. Our data suggest that in S. cerevisiae, doxorubicin produces protein aggregation through ROS and requires Ydj1p and New1p for resolution.

Inactivation of chromatin remodeling factors sensitizes cells to selective cytotoxic stress.

The SWI/SNF chromatin-remodeling complex plays an essential role in several cellular processes including cell proliferation, differentiation, and DNA repair. Loss of normal function of the SWI/SNF complex because of mutations in its subunits correlates with tumorigenesis in humans. For many of these cancers, cytotoxic chemotherapy is the primary, and sometimes the only, therapeutic alternative. Among the antineoplastic agents, anthracyclines are a common treatment option. Although effective, resistance to these agents usually develops and serious dose-related toxicity, namely, chronic cardiotoxicity, limits its use. Previous work from our laboratory showed that a deletion of the SWI/SNF factor SNF2 resulted in hypersensitivity to doxorubicin. We further investigated the contribution of other chromatin remodeling complex components in the response to cytotoxic chemotherapy. Our results indicate that, of the eight SWI/SNF strains tested, snf2, taf14, and swi3 were the most sensitive and displayed distinct sensitivity to different cytotoxic agents, while snf5 displayed resistance. Our experimental results indicate that the SWI/SNF complex plays a critical role in protecting cells from exposure to cytotoxic chemotherapy and other cytotoxic agents. Our findings may prove useful in the development of a strategy aimed at targeting these genes to provide an alternative by hypersensitizing cancer cells to chemotherapeutic agents.

Defects in base excision repair sensitize cells to manganese in S. cerevisiae.

Manganese (Mn) is essential for normal physiologic functioning; therefore, deficiencies and excess intake of manganese can result in disease. In humans, prolonged exposure to manganese causes neurotoxicity characterized by Parkinson-like symptoms. Mn(2+) has been shown to mediate DNA damage possibly through the generation of reactive oxygen species. In a recent publication, we showed that Mn induced oxidative DNA damage and caused lesions in thymines. This study further investigates the mechanisms by which cells process Mn(2+)-mediated DNA damage using the yeast S. cerevisiae. The strains most sensitive to Mn(2+) were those defective in base excision repair, glutathione synthesis, and superoxide dismutase mutants. Mn(2+) caused a dose-dependent increase in the accumulation of mutations using the CAN1 and lys2-10A mutator assays. The spectrum of CAN1 mutants indicates that exposure to Mn results in accumulation of base substitutions and frameshift mutations. The sensitivity of cells to Mn(2+) as well as its mutagenic effect was reduced by N-acetylcysteine, glutathione, and Mg(2+). These data suggest that Mn(2+) causes oxidative DNA damage that requires base excision repair for processing and that Mn interferes with polymerase fidelity. The status of base excision repair may provide a biomarker for the sensitivity of individuals to manganese.