Protein stability is largely regulated by post-translational modifications, such as ubiquitination, which is mediated by ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3 with substrate specificity. Membrane-associated RING-CH (MARCH) proteins represent one novel family of transmembrane E3 ligases which target glycoproteins for lysosomal destruction. While most of the MARCH family members are known to degrade membrane proteins in immune cells, their tumor-intrinsic role is largely unknown. In this study, we found that the expression of one MARCH family member, MARCH8, is specifically downregulated in breast cancer tissues and positively correlated with breast cancer survival rate according to bioinformatic analysis of The Cancer Genomic Atlas (TCGA) dataset. MARCH8 protein expression was also lower in a variety of human breast cancer cell lines in comparison to immortalized human mammary epithelial MCF-12A cells. Restoration of MARCH8 expression induced apoptosis in human breast cancer cell lines MDA-MB-231 and BT549. Stable expression of MARCH8 inhibited tumorigenesis and lung metastases of MDA-MB-231 cells in mice. Moreover, we discovered that the breast cancer stem-cell marker and metastasis driver CD44, a membrane protein, interacts with MARCH8 and is one of the glycoprotein targets subject to MARCH8-dependent lysosomal degradation. Unexpectedly, we identified a nonmembrane protein, signal transducer and transcription activator 3 (STAT3), as another essential ubiquitination target of MARCH8, whose degradation through the proteasome pathway is responsible for the proapoptotic changes mediated by MARCH8. These findings highlight a novel tumor-suppressing function of MARCH8 in targeting both membrane and nonmembrane protein targets required for the survival and metastasis of breast cancer cells. Full article

Background: IL13Rα2 is reportedly a promising therapeutic target in different cancers. Still, no specific antagonists have reached the clinics yet. We investigated the use of a IL-13/IL13Rα2 binding motif, called D1, as a new target for the development of therapeutic monoclonal antibodies (mAbs) for colorectal cancer (CRC) metastasis. Methods: IL13Rα2 D1 peptides were prepared and used for immunization and antibody development. Antibodies were tested for inhibition of cellular invasion through Matrigel using CRC cell lines. Effects of the mAbs on cell signaling, receptor internalization and degradation were determined by western blot and flow cytometry. Swiss nude mice were used for survival analysis after treatment with IL13Rα2-specific mAbs and metastasis development. Results: IL13Rα2 D1 peptides were used to generate highly selective mAbs that blocked IL13/IL13Rα2-mediated SRC activation and cell invasion in colorectal cancer cells. Antibodies also provoked a significant reduction in cell adhesion and proliferation of metastatic cancer cells. Treatment with mAbs impaired the FAK, SRC and PI3K/AKT pathway activation. Blocking effectivity was shown to correlate with the cellular IL13Rα2 expression level. Despite mAb 5.5.4 partially blocked IL-13 mediated receptor internalization from the cancer cell surface it still promotes receptor degradation. Compared with other IL13Rα2-specific antibodies, 5.5.4 exhibited a superior efficacy to inhibit metastatic growth in vivo, providing a complete mouse survival in different conditions, including established metastasis. Conclusions: Monoclonal antibody 5.5.4 showed a highly selective blocking capacity for the interaction between IL-13 and IL13Rα2 and caused a complete inhibition of liver metastasis in IL13Rα2-positive colorectal cancer cells. This capacity might be potentially applicable to other IL13Rα2-expressing tumors. Full article

In rodents, we identified a physiologic process within the normal liver that creates a pre-metastatic niche. This physiology is weaning-induced liver involution, characterized by hepatocyte cell death, immune influx, and extracellular matrix remodeling. Here, using weaning-induced liver involution as a model of a physiologically regulated pro-metastatic niche, we investigate how liver involution supports breast cancer metastasis. Liver metastases were induced in BALB/c immune competent hosts by portal vein injection of D2OR (low metastatic) or D2A1 (high metastatic) mouse mammary tumor cells. Tumor incidence and multiplicity increased in involution hosts with no evidence of a proliferation advantage. D2OR tumor cell extravasation, seeding, and early survival were not enhanced in the involuting group compared to the nulliparous group. Rather, the involution metastatic advantage was observed at 14 days post tumor cell injection. This metastatic advantage associated with induction of immune tolerance in the involution host liver, reproductive state dependent intra-tumoral immune composition, and CD8-dependent suppression of metastases in nulliparous hosts. Our findings suggest that the normal postpartum liver is in an immune suppressed state, which can provide a pro-metastatic advantage to circulating breast cancer cells. Potential relevance to women is suggested as a postpartum diagnosis of breast cancer is an independent predictor of liver metastasis. Full article

The EphA2 tyrosine kinase receptor is highly expressed in several types of solid tumors. In our recent studies, we targeted EphA2 in pancreatic cancer with agonistic agents and demonstrated that suppression of EphA2 significantly reduced cancer-cell migration in cell-based assays. In the present study, we focused on targeting EphA2 in prostate cancer. While not all prostate cancers express EphA2, we showed that enzalutamide induced EphA2 expression in prostate cancer cells and in a patient-derived xenograft (PDX) animal model, which provides further impetus to target EphA2 in prostate cancer. Western blot studies showed that agonistic dimeric synthetic (135H12) and natural (ephrinA1-Fc) ligands effectively degraded EphA2 receptor in the prostate cancer cell line PC-3. The agents also delayed cell migration of prostate cancer (PC-3) cells, while an in vivo PC-3 orthotopic metastatic nude-mouse model also revealed that administration of ephrinA1-Fc or 135H12 strongly reduced metastases. The present study further validates EphA2 as an important target in metastatic prostate cancer treatment. Our results should incentivize further efforts aimed at developing potent and effective EphA2 synthetic agonistic agents for the treatment of EphA2-driven aggressive metastatic tumors including prostate, pancreatic, and breast cancer. Full article

This retrospective multi-center analysis aimed to assess the clinical response and stabilizing effects of palliative radiotherapy (RT) for spinal bone metastases (SBM) in head and neck cancer (HNC), and to establish potential predictive factors for stability and overall survival (OS). Patients included in this analysis were treated at the University Hospitals of Mainz, Freiburg, and Heidelberg between 2001 and 2019. Clinical information was taken from the medical records. The stability of affected vertebral bodies was assessed according to the validated spine instability neoplastic score (SINS) based on CT-imaging before RT, as well as 3 and 6 months after RT. OS was quantified as the time between the start of palliative RT and death from any cause or last follow-up. Potential predictive factors for stability and OS were analyzed using generalized estimating equations and Cox regression for time-varying covariates to take into account multiple observations per patient. The mean follow-up time of 66 included patients after the first palliative RT was 8.1 months (range 0.3–85.0 months). The majority of patients (70%; n = 46) had squamous cell carcinomas (SCC) originating from the pharynx, larynx and oral cavity, while most of the remaining patients (26%; n = 17) suffered from salivary glands tumors. A total of 95 target volumes including 178 SBM were evaluated that received a total of 81 irradiation series. In patients with more than one metastasis per irradiated region, only the most critical bone metastasis was analyzed according to the SINS system. Prior to RT, pain and neurologic deficits were present in 76% (n = 72) and 22% (n = 21) of irradiated lesions, respectively, and 68% of the irradiated lesions (n = 65) were assessed as unstable or potentially unstable prior to RT. SBM-related pain symptoms and neurologic deficits responded to RT in 63% and 47% of the treated lesions, respectively. Among patients still alive at 3 and 6 months after RT with potentially unstable or unstable SBM, a shift to a better stability class according to the SINS was observed in 20% and 33% of the irradiated SBM, respectively. Pathological fractures of SBM were frequently detected before the start of irradiation (43%; n = 41), but after RT, new fractures or increasing vertebral body sintering within the irradiated region occurred rarely (8%; n = 8). A pathological fracture before RT was negatively associated with stabilization 6 months after RT (OR 0.1, 95% CI 0.02–0.49, p = 0.004), while a Karnofsky performance score (KPS) ≥ 70% was associated positively with a stabilization effect through irradiation (OR 6.09, 95% CI 1.68–22.05, p = 0.006). Mean OS following first palliative RT was 10.7 months, and the KPS (≥70% vs. <70%) was shown to be a strong predictive factor for OS after RT (HR 0.197, 95% CI 0.11–0.35, p < 0.001). There was no significant difference in OS between patients with SCC and non-SCC. Palliative RT in symptomatic SBM of HNC provides sufficient symptom relief in the majority of patients, while only about one third of initially unstable SBM show re-stabilization after RT. Since patients in our multi-center cohort exhibited very limited OS, fractionation schemes should be determined depending on the patients’ performance status. Full article

Colorectal cancer, along with its high potential for recurrence and metastasis, is a major health burden. Uncovering proteins and pathways required for tumor cell growth is necessary for the development of novel targeted therapies. Ajuba is a member of the LIM domain family of proteins whose expression is positively associated with numerous cancers. Our data shows that Ajuba is highly expressed in human colon cancer tissue and cell lines. Publicly available data from The Cancer Genome Atlas shows a negative correlation between survival and Ajuba expression in patients with colon cancer. To investigate its function, we transduced SW480 human colon cancer cells, with lentiviral constructs to knockdown or overexpress Ajuba protein. The transcriptome of the modified cell lines was analyzed by RNA sequencing. Among the pathways enriched in the differentially expressed genes, were cell proliferation, migration and differentiation. We confirmed our sequencing data with biological assays; cells depleted of Ajuba were less proliferative, more sensitive to irradiation, migrated less and were less efficient in colony formation. In addition, loss of Ajuba expression decreased the tumor burden in a murine model of colorectal metastasis to the liver. Taken together, our data supports that Ajuba promotes colon cancer growth, migration and metastasis and therefore is a potential candidate for targeted therapy. Full article

HIV-1 Tat Interactive Protein 2 (HTATIP2) is a tumor suppressor, of which reduced or absent expression is associated with increased susceptibility to tumorigenesis and enhanced tumor invasion and metastasis. However, whether the absent expression of HTATIP2 is a tumor-promoting factor that acts through improving tumor adaptation to hypoxia is unclear. Here, we established a stable HTATIP2-knockdown A549 human lung adenocarcinoma cell line (A549shHTATIP2) using lentiviral-delivered HTATIP2-targeting short hairpin RNA (shRNA), employed a double subcutaneous xenograft model and incorporated photoacoustic imaging and metabolomics approaches to elucidate the impact of the absent HTATIP2 expression on tumor response to hypoxic stress. Results from the in vivo study showed that A549shHTATIP2 tumors exhibited accelerated growth but decreased intratumoral oxygenation and angiogenesis and reduced sensitivity to sorafenib treatment as compared with their parental counterparts. Moreover, results of the immunoblot and real-time PCR analyses revealed that the HIF2α protein and mRNA levels in vehicle-treated A549shHTATIP2 tumors were significantly increased (p < 0.01 compared with the parental control tumors). Despite the strong HIF2α-c-Myc protein interaction indicated by our co-immunoprecipitation data, the increase in the c-Myc protein and mRNA levels was not significant in the A549shHTATIP2 tumors. Nonetheless, MCL-1 and β-catenin protein levels in A549shHTATIP2 tumors were significantly increased (p < 0.05 compared with the parental control tumors), suggesting an enhanced β-catenin/c-Myc/MCL-1 pathway in the absence of HTATIP2 expression. The finding of significantly decreased E-cadherin (p < 0.01 compared with vehicle-treated A549shHTATIP2 tumors) and increased vimentin (p < 0.05 compared with sorafenib-treated A549 tumors) protein levels in A549shHTATIP2 tumors implicates that the absence of HTATIP2 expression increases the susceptibility of A549 tumors to sorafenib-activated epithelial-mesenchymal transition (EMT) process. Comparison of the metabolomic profiles between A549 and A549shHTATIP2 tumors demonstrated that the absence of HTATIP2 expression resulted in increased tumor metabolic plasticity that enabled tumor cells to exploit alternative metabolic pathways for survival and proliferation rather than relying on glutamine and fatty acids as a carbon source to replenish TCA cycle intermediates. Our data suggest a mechanism by which the absent HTATIP2 expression modulates tumor adaptation to hypoxia and promotes an aggressive tumor phenotype by enhancing the HIF2α-regulated β-catenin/c-Myc/MCL-1 signaling, increasing the susceptibility of tumors to sorafenib treatment-activated EMT process, and improving tumor metabolic plasticity. Full article

Leptomeningeal metastases (LM) from solid tumors represent an unmet need of increasing importance due to an early use of MRI for diagnosis and improvement of outcome of some molecular subgroups following targeted agents and immunotherapy. In this review, we first discussed factors limiting the efficacy of targeted agents in LM, such as the molecular divergence between primary tumors and CNS lesions and CNS barriers at the level of the normal brain, brain tumors and CSF. Further, we reviewed pathogenesis and experimental models and modalities, such as MRI (with RANO and ESO/ESMO criteria), CSF cytology and liquid biopsy, to improve diagnosis and monitoring following therapy. Efficacy and limitations of targeted therapies for LM from EGFR-mutant and ALK-rearranged NSCLC, HER2-positive breast cancer and BRAF-mutated melanomas are reported, including the use of intrathecal administration or modification of traditional cytotoxic compounds. The efficacy of checkpoint inhibitors in LM from non-druggable tumors, in particular triple-negative breast cancer, is discussed. Last, we focused on some recent techniques to improve drug delivery. Full article

The tumor microenvironment (TME) regulates essential tumor survival and promotion functions. Interactions between the cellular and structural components of the TME allow cancer cells to become invasive and disseminate from the primary site to distant locations, through a complex and multistep metastatic cascade. Tumor-associated M2-type macrophages have growth-promoting and immunosuppressive functions; mesenchymal cells mass produce exosomes that increase the migratory ability of cancer cells; cancer associated fibroblasts (CAFs) reorganize the surrounding matrix creating migration-guiding tracks for cancer cells. In addition, the tumor extracellular matrix (ECM) exerts determinant roles in disease progression and cancer cell migration and regulates therapeutic responses. The hypoxic conditions generated at the primary tumor force cancer cells to genetically and/or epigenetically adapt in order to survive and metastasize. In the circulation, cancer cells encounter platelets, immune cells, and cytokines in the blood microenvironment that facilitate their survival and transit. This review discusses the roles of different cellular and structural tumor components in regulating the metastatic process, targeting approaches using small molecule inhibitors, nanoparticles, manipulated exosomes, and miRNAs to inhibit tumor invasion as well as current and future strategies to remodel the TME and enhance treatment efficacy to block the detrimental process of metastasis. Full article

Metabolic adaptation is emerging as an important hallmark of cancer and metastasis. In the last decade, increasing evidence has shown the importance of metabolic alterations underlying the metastatic process, especially in breast cancer metastasis but also in colorectal cancer metastasis. Being the main cause of cancer-related deaths, it is of great importance to developing new therapeutic strategies that specifically target metastatic cells. In this regard, targeting metabolic pathways of metastatic cells is one of the more promising windows for new therapies of metastatic colorectal cancer, where still there are no approved inhibitors against metabolic targets. In this study, we review the recent advances in the field of metabolic adaptation of cancer metastasis, focusing our attention on colorectal cancer. In addition, we also review the current status of metabolic inhibitors for cancer treatment. Full article

Glycolysis is a crucial metabolic process in rapidly proliferating cells such as cancer cells. Phosphofructokinase-1 (PFK-1) is a key rate-limiting enzyme of glycolysis. Its efficiency is allosterically regulated by numerous substances occurring in the cytoplasm. However, the most potent regulator of PFK-1 is fructose-2,6-bisphosphate (F-2,6-BP), the level of which is strongly associated with 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase activity (PFK-2/FBPase-2, PFKFB). PFK-2/FBPase-2 is a bifunctional enzyme responsible for F-2,6-BP synthesis and degradation. Four isozymes of PFKFB (PFKFB1, PFKFB2, PFKFB3, and PFKFB4) have been identified. Alterations in the levels of all PFK-2/FBPase-2 isozymes have been reported in different diseases. However, most recent studies have focused on an increased expression of PFKFB3 and PFKFB4 in cancer tissues and their role in carcinogenesis. In this review, we summarize our current knowledge on all PFKFB genes and protein structures, and emphasize important differences between the isoenzymes, which likely affect their kinase/phosphatase activities. The main focus is on the latest reports in this field of cancer research, and in particular the impact of PFKFB3 and PFKFB4 on tumor progression, metastasis, angiogenesis, and autophagy. We also present the most recent achievements in the development of new drugs targeting these isozymes. Finally, we discuss potential combination therapies using PFKFB3 inhibitors, which may represent important future cancer treatment options. Full article

Residual metastasis is a major cause of cancer-associated death. Recent advances in understanding the molecular basis of the epithelial–mesenchymal transition (EMT) and the related cancer stem cells (CSCs) have revealed the landscapes of cancer metastasis and are promising contributions to clinical treatments. However, this rarely leads to practical advances in the management of cancer in clinical settings, and thus cancer metastasis is still a threat to patients. The reason for this may be the heterogeneity and complexity caused by the evolutional transformation of tumor cells through interactions with the host environment, which is composed of numerous components, including stromal cells, vascular cells, and immune cells. The reciprocal evolution further raises the possibility of successful tumor escape, resulting in a fatal prognosis for patients. To disrupt the vicious spiral of tumor–immunity aggravation, it is important to understand the entire metastatic process and the practical implementations. Here, we provide an overview of the molecular and cellular links between tumors’ biological properties and host immunity, mainly focusing on EMT and CSCs, and we also highlight therapeutic agents targeting the oncoimmune determinants driving cancer metastasis toward better practical use in the treatment of cancer patients. Full article

Distant organ metastases accounts for the majority of breast cancer deaths. Given the prevalence of breast cancer in women, it is imperative to understand the underlying mechanisms of its metastatic progression and identify potential targets for therapy. Since their discovery in 1993, microRNAs (miRNAs) have emerged as important regulators of tumour progression and metastasis in various cancers, playing either oncogenic or tumour suppressor roles. In the following review, we discuss the roles of miRNAs that potentiate four key areas of breast cancer metastasis—angiogenesis, epithelial-mesenchymal transition, the Warburg effect and the tumour microenvironment. We then evaluate the recent developments in miRNA-based therapies in breast cancer, which have shown substantial promise in controlling tumour progression and metastasis. Yet, certain challenges must be overcome before these strategies can be implemented in clinical trials. Full article

Metastatic cancer is one of the major causes of cancer-related mortalities. Metastasis is a complex, multi-process phenomenon, and a hallmark of cancer. Calcium (Ca²⁺) is a ubiquitous secondary messenger, and it has become evident that Ca²⁺ signalling plays a vital role in cancer. Ca²⁺ homeostasis is dysregulated in physiological processes related to tumour metastasis and progression—including cellular adhesion, epithelial–mesenchymal transition, cell migration, motility, and invasion. In this review, we looked at the role of intracellular and extracellular Ca²⁺ signalling pathways in processes that contribute to metastasis at the local level and also their effects on cancer metastasis globally, as well as at underlying molecular mechanisms and clinical applications. Spatiotemporal Ca²⁺ homeostasis, in terms of oscillations or waves, is crucial for hindering tumour progression and metastasis. They are a limited number of clinical trials investigating treating patients with advanced stages of various cancer types. Ca²⁺ signalling may serve as a novel hallmark of cancer due to the versatility of Ca²⁺ signals in cells, which suggests that the modulation of specific upstream/downstream targets may be a therapeutic approach to treat cancer, particularly in patients with metastatic cancers. Full article

Obesity is a modern health problem that has reached pandemic proportions. It is an established risk factor for carcinogenesis, however, evidence for the contribution of adipose tissue to the metastatic behavior of tumors is also mounting. Over 90% of cancer mortality is attributed to metastasis and metastatic tumor cells must communicate with their microenvironment for survival. Many of the characteristics observed in obese adipose tissue strongly mirror the tumor microenvironment. Thus in the case of prostate, pancreatic and breast cancer and esophageal adenocarcinoma, which are all located in close anatomical proximity to an adipose tissue depot, the adjacent fat provides an ideal microenvironment to enhance tumor growth, progression and metastasis. Adipocytes provide adipokines, fatty acids and other soluble factors to tumor cells whilst immune cells infiltrate the tumor microenvironment. In addition, there are emerging studies on the role of the extracellular vesicles secreted from adipose tissue, and the extracellular matrix itself, as drivers of obesity-induced metastasis. In the present review, we discuss the major mechanisms responsible for the obesity–metastatic link. Furthermore, understanding these complex mechanisms will provide novel therapies to halt the tumor–adipose tissue crosstalk with the ultimate aim of inhibiting tumor progression and metastatic growth. Full article

Metastasis represents a serious complication in the treatment of cancer. Flavonoids are plant secondary metabolites exerting various health beneficiary effects. The effects of flavonoids against cancer are associated not only with early stages of the cancer process, but also with cancer progression and spread into distant sites. Flavonoids showed potent anti-cancer effects against various cancer models in vitro and in vivo, mediated via regulation of key signaling pathways involved in the migration and invasion of cancer cells and metastatic progression, including key regulators of epithelial-mesenchymal transition or regulatory molecules such as MMPs, uPA/uPAR, TGF-β and other contributors of the complex process of metastatic spread. Moreover, flavonoids modulated also the expression of genes associated with the progression of cancer and improved inflammatory status, a part of the complex process involved in the development of metastasis. Flavonoids also documented clear potential to improve the anti-cancer effectiveness of conventional chemotherapeutic agents. Most importantly, flavonoids represent environmentally-friendly and cost-effective substances; moreover, a wide spectrum of different flavonoids demonstrated safety and minimal side effects during long-termed administration. In addition, the bioavailability of flavonoids can be improved by their conjugation with metal ions or structural modifications by radiation. In conclusion, anti-cancer effects of flavonoids, targeting all phases of carcinogenesis including metastatic progression, should be implemented into clinical cancer research in order to strengthen their potential use in the future targeted prevention and therapy of cancer in high-risk individuals or patients with aggressive cancer disease with metastatic potential. Full article