In the last decades, progresses in medical oncology have ameliorated the treatment of patients and their outcome. However, further improvements are still necessary, in particular for certain types of tumors such as pancreatic, gastric, and lung cancer as well as acute myeloid leukemia where early detection and monitoring of the disease are crucial for final patient outcome. Liquid biopsy represents a great advance in the field because it is less invasive, less time-consuming, and safer compared to classical biopsies and it can be useful to monitor the evolution of the disease as well as the response of patients to therapy. Liquid biopsy allows the detection of circulating tumor cells, nucleic acids, and exosomes not only in blood but also in different biological fluids: urine, saliva, pleural effusions, cerebrospinal fluid, and stool. Among the potential biomarkers detectable in liquid biopsies, microRNAs (miRNAs) are gaining more and more attention, since they are easily detectable, quite stable in biological fluids, and show high sensitivity. Many data demonstrate that miRNAs alone or in combination with other biomarkers could improve the diagnostic and prognostic power for many different tumors. Despite this, standardization of methods, sample preparation, and analysis remain challenging and a huge effort should be made to address these issues before miRNA biomarkers can enter the clinic. This review summarizes the main findings in the field of circulating miRNAs in both solid and hematological tumors.

Melanoma is a highly aggressive tumor and almost always fatal when metastatic. Herein, we discuss recent findings on the mechanisms of resistance of human cutaneous melanoma. To achieve a precision medicine approach, the heterogeneity and plasticity of tumor cells are two crucial aspects to be investigated in depth. In fact, to understand the mechanisms that cells use to acquire a resistant phenotype after chemotherapy or how resistant cells inside the tumor are selected, it is the most important issue for a successful therapy. Since new therapeutic strategies are trying to go in this direction, we discuss here the state of the art of the research and the clinical impact of these strategies. We also discuss and suggest future research directions to develop approaches able to define the best concentration and time of exposure of the drug or the cocktails of drugs for each specific patient based on his/her biological features.

The type 2 DNA topoisomerases (Top2) are conserved enzymes and biomarkers for cell proliferation. The catalytic activities of the human isoform Top2α are essential for the regulation of DNA topology during DNA replication, transcription, and chromosome segregation. Top2α is a prominent target for anti-cancer drugs and is highly regulated by post-translational modifications (PTM). Despite an increasing number of proteomic studies, the extent of PTM in cancer cells and its importance in drug response remains largely uncharacterized. In this review, we highlight the different modifications affecting the human Top2α in healthy and cancer cells, taking advantage of the structure-function information accumulated in the past decades. We also overview the regulation of Top2α by PTM, the level of PTM in cancer cells, and the resistance to therapeutic compounds targeting the Top2 enzyme. Altogether, this review underlines the importance of future studies addressing more systematically the interplay between PTM and Top2 drug resistance.

DNA topoisomerase IIα (170 kDa, TOP2α/170) induces transient DNA double-strand breaks in proliferating cells to resolve DNA topological entanglements during chromosome condensation, replication, and segregation. Therefore, TOP2α/170 is a prominent target for anticancer drugs whose clinical efficacy is often compromised due to chemoresistance. Although many resistance mechanisms have been defined, acquired resistance of human cancer cell lines to TOP2α interfacial inhibitors/poisons is frequently associated with a reduction of Top2α/170 expression levels. Recent studies by our laboratory, in conjunction with earlier findings by other investigators, support the hypothesis that a major mechanism of acquired resistance to TOP2α-targeted drugs is due to alternative RNA processing/splicing. Specifically, several TOP2α mRNA splice variants have been reported which retain introns and are translated into truncated TOP2α isoforms lacking nuclear localization sequences and subsequent dysregulated nuclear-cytoplasmic disposition. In addition, intron retention can lead to truncated isoforms that lack both nuclear localization sequences and the active site tyrosine (Tyr805) necessary for forming enzyme-DNA covalent complexes and inducing DNA damage in the presence of TOP2α-targeted drugs. Ultimately, these truncated TOP2α isoforms result in decreased drug activity against TOP2α in the nucleus and manifest drug resistance. Therefore, the complete characterization of the mechanism(s) regulating the alternative RNA processing of TOP2α pre-mRNA may result in new strategies to circumvent acquired drug resistance. Additionally, novel TOP2α splice variants and truncated TOP2α isoforms may be useful as biomarkers for drug resistance, prognosis, and/or direct future TOP2α-targeted therapies.

Mutated or rearranged driver kinases in non-small cell lung cancer (NSCLC) cells are clinically amenable to treatment with tyrosine kinase inhibitors (TKIs) resulting in prolonged survival and significant benefit compared to cytotoxic chemotherapy. The most frequent genomic alterations are observed for epidermal growth factor receptor and anaplastic lymphoma kinase, which can be blocked by a range of specific TKIs in sequence. In clinics, resistance to TKIs emerges after approximately one year and comprises secondary mutations of the kinases (on-target) or alternative pathways circumventing the original kinase (off-target) alterations. A special feature of NSCLC is the occurrence of histological transformation to small cell lung cancer (SCLC) in up to 14% of cases, which, in general, is accompanied by resistance to the original TKIs. SCLC transformed tumors may be treated with the classical platinum/etoposide regimen but thus far there are no definitive guidelines. Four transformed pleural SCLC lines in our lab indicate the presence of a gradual NSCLC-SCLC shift with overlapping drug sensitivities. In conclusion, the treatment of NSCLC-SCLC transformed cancer cells would need a better chemosensitivity assessment using functional genomics to guide further therapy.

Approximately 20% of invasive breast cancers have upregulation/gene amplification of the oncogene human epidermal growth factor receptor-2 (HER2/ErbB2). Of these, some also express steroid receptors (the so-called Luminal B subtype), whereas others do not (the HER2 subtype). HER2 abnormal breast cancers are associated with a worse prognosis, chemotherapy resistance, and sensitivity to selected anti-HER2 targeted therapeutics. Transcriptional data from over 3000 invasive breast cancers suggest that this approach is overly simplistic; rather, the upregulation of HER2 expression resulting from gene amplification is a driver event that causes major transcriptional changes involving numerous genes and pathways in breast cancer cells. Most notably, this includes a shift from estrogenic dependence to regulatory controls driven by other nuclear receptors, particularly the androgen receptor. We discuss members of the HER receptor tyrosine kinase family, heterodimer formation, and downstream signaling, with a focus on HER2 associated pathology in breast carcinogenesis. The development and application of anti-HER2 drugs, including selected clinical trials, are discussed. In light of the many excellent reviews in the clinical literature, our emphasis is on recently developed and successful strategies to overcome targeted therapy resistance. These include combining anti-HER2 agents with programmed cell death-1 ligand or cyclin-dependent kinase 4/6 inhibitors, targeting crosstalk between HER2 and other nuclear receptors, lipid/cholesterol synthesis to inhibit receptor tyrosine kinase activation, and metformin, a broadly inhibitory drug. We seek to facilitate a better understanding of new approaches to overcome anti-HER2 drug resistance and encourage exploration of two other therapeutic interventions that may be clinically useful for HER+ invasive breast cancer patients.

Aim: Improved treatment strategies are desperately needed for eradicating cancer stem cells (CSCs), which drive malignancy and recurrence in glioblastoma multiforme. Hypoxic regions within the tumor microenvironment help maintain and promote the proliferation of CSCs. Here, we explored the effects of silencing hypoxia inducible factor-2α (HIF-2α) because of its specificity for CSCs within the hypoxic environment.

Methods: Cancer stem cell neurospheres were formed by enriching from both the glioblastoma cell line U87 and from brain tumor stem cells isolated directly from human brain tumors. Silencing of human HIF-2α was performed using both commercial and in-house transfection of a validated short interfering RNA, with all results compared to an established non-silencing control short interfering RNA. Silencing of HIF-2α was established by Western blotting, and phenotypic effects were assayed by cell migration assays, cell viability measurements, and immunofluorescence staining of differentiation markers.

Results: Transfection with either our previously reported pH-sensitive, cationic amphiphilic macromolecule-based delivery system or Lipofectamine was similarly effective in silencing HIF-2α. The chemotherapeutic resistance and neurosphere formation were reduced when HIF-2α was silenced. Migratory capacities in the presence of macrophage conditioned media were modulated. HIF-2α silencing was complementary to temozolomide treatment in producing phenotypic rather than cytotoxic effects.

Conclusion: HIF-2α silencing under hypoxia inhibited CSC phenotypes while promoting differentiated cell phenotypes and is complementary to existing DNA alkylating treatments in inhibiting glioma CSC activity.

Aim: Several cationic radiotracers originally developed as myocardial perfusion agents have shown potential for both early detection of cancer and non-invasive monitoring of multiple drug resistance (MDR) by single photon emission computed tomography. We have introduced two cationic complexes, ^99mTc-DMEOP [di-methoxy-tris-pyrazolyl-^99mTc-(CO)₃] and ^99mTc-TMEOP [tri-methoxy-tris-pyrazolyl-^99mTc-(CO)₃], which showed excellent preclinical results as cardiac imaging probes, namely a persistent heart uptake with rapid blood and liver clearance. This study aimed at the evaluation of their usefulness for tumoral detection and functional assessment of MDR.

Methods: The uptake and efflux kinetics of ^99mTc-DMEOP and ^99mTc-TMEOP were evaluated in human prostate, lung, and breast cancer cell lines, including drug-resistant cell lines that are known to overexpress the MDR P-glycoprotein (Pgp). The effects of MDR modulators were also studied. In vivo studies were performed in xenografted tumor models, and the MDR phenotype of the tumors was confirmed by Western blot.

Results: The uptake kinetics of both complexes in human cancer cell lines is comparable with the one found for ^99mTc-Sestamibi, increasing over time. The uptake of ^99mTc-TMEOP is greatly reduced in cells overexpressing Pgp and increased in the presence of a Pgp modulator. In nude mice, the tumor uptake of ^99mTc-TMEOP was higher in the MCF-7 xenografts compared with the MCF7 Pgp tumors.

Conclusion: The uptake kinetics of both complexes in human cancer cell lines is comparable with the one found for ^99mTc-Sestamibi, increasing over time. The uptake of ^99mTc-TMEOP is greatly reduced in cells overexpressing Pgp, and increased in the presence of a Pgp modulator. In nude mice, the tumor uptake of ^99mTc-TMEOP was higher in the MCF-7 xenografts compared with the MCF7 Pgp tumors.

The genetic and epigenetic aberrations that underlie immune resistance lead to tumors that are refractory to clinically established and experimental immunotherapies, including monoclonal antibodies and T cell-based therapies. From various forms of cytotoxic T cells to small molecule inhibitors that revamp the tumor microenvironment, these therapies have demonstrated notable responses in cancer models and a resistant subset of cancer patients, used both alone and in combination. However, even current approaches, such as those targeting checkpoint molecules, tumor ligands, and involving gene-related therapies, present a challenge in non-responding patients. In this perspective, we discuss the most common mechanisms of immune resistance, including tumor heterogeneity, tumor ligand and major histocompatibility complex modulation, anti-apoptotic pathways, checkpoint inhibitory ligands, immunosuppressive cells and factors in the tumor microenvironment, and activation-induced cell death. In addition, we discuss the strategies designed to circumvent these resistance pathways to showcase the potential of emerging technologies in battling the rise of resistance.