Triple Negative Breast Cancers (TNBCs) are heterogeneous and aggressive tumors with a median overall survival of less than two years. Despite the availability of new drugs, the prognosis remains poor, implicating a more aggressive clinical course in the metastatic setting. This study investigated the effects of metronomic treatment (mCHT) with 5-fluorouracil (5-FU) plus vinorelbine (VNR) on spheroids derived from two different TNBC cell lines (BT-549 and MDA-MB-231) and a patient-derived primary cell line (MS-186). mCHT significantly reduced spheroid growth and altered spheroid architecture, with a pronounced effect in second-generation spheroids, enriched in self-renewing cancer stem cells (CSCs). Expression of CSC-related markers (CD44, CD133, NOTCH-1, and MYC) was more significantly altered-both at the mRNA and protein levels-by mCHT than by standard treatment (STD). In MS-186-derived spheroids, mCHT downregulated EZH2 and STAT3, key regulators of CSC maintenance, and reduced H3K27ac, suggesting a global epigenetic reprogramming. Unlike STD, which partially and transiently reduced stemness markers, mCHT achieved sustained suppression, indicating preferential targeting of therapy-resistant CSCs. These results indicate mCHT as a promising strategy for specifically aiming at the CSC-like compartment in TNBC, underscoring a therapeutic approach that reprograms key epigenetic networks and overcomes resistance to treatment.

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by the absence of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor amplification/overexpression 2 (HER2). TNBC is biologically heterogeneous with different clinical behaviors, frequently related to short disease-free survival (DFS) and overall survival (OS). There is still a lack of specific targeted therapy for TNBC, so finding an effective treatment for patients is strictly required. In this scenario, metronomic therapy (mCHT, low doses of drugs without interruptions) could be effective.

Background: Breast cancer accounts for approximately 30% of all cancers diagnosed in women across all age groups, of which 70% are of the ER-positive (ER+) subtype. Despite the significant progress made in recent years in treating the disease, a large percentage of patients develop resistance to endocrine therapy limiting its benefits. In ER+ advanced breast cancer, the use of cyclin 4/6-dependent kinase inhibitors (CDK4/6) in combination with hormonal therapy has had considerable importance. Despite the improvement in disease management that CDK4/6 inhibitors offer to patients with ER+ breast cancer, not all patients respond to these drugs, and most patients initially responding to CDK4/6 inhibitors develop acquired resistance. However, there remains an unmet need to develop new therapeutic approaches and drugs for targeted therapies for this subtype of advanced breast cancer.

A group of 27 patients diagnosed with metastatic triple-negative breast cancer (mTNBC) was randomly distributed into two groups and underwent different lines of metronomic treatment (mCHT). The former group (N 14) received first-line mCHT and showed a higher overall survival rate than the second group (N 13), which underwent second-line mCHT. Analysis of one patient still alive from the first group, diagnosed with mTNBC in 2019, showed a complete metabolic response (CMR) after a composite approach implicating first-line mCHT followed by second-line epirubicin and third-line nab-paclitaxel, and was chosen for subsequent molecular characterization. We found altered expression in the cancer stemness-associated gene NOTCH-1 and its corresponding protein. Additionally, we found changes in the expression of oncogenes, such as MYC and AKT, along with their respective proteins. Overall, our data suggest that a first-line treatment with mCHT followed by MTD might be effective by negatively regulating stemness traits usually associated with the emergence of drug resistance.

Supplementary Figure 2 - PDF file 42K, GSK3B silencing by siRNA abolishes drug resistance of p53-null colon carcinoma cell lines

Supplementary Figure 4 - PDF file 458K, Caspases are not activated after 5FU treatment in GSK3B-silenced HCT116p53KO

Supplementary Table 1 - PDF file 22K, Known genetic alterations of the different colon carcinoma cell lines used in the paper

Constitutively active kinases play a crucial role in carcinogenesis, and their inhibition is a common target for molecular tumor therapy. We recently discovered the expression of two oncogenic isoforms of Bruton’s Tyrosine Kinase (BTK) in head and neck squamous cell carcinoma (HNSCC), Btk-p80 and BTK-p65. However, the precise role of BTK in HNSCC remains unclear. Analyses of a tissue microarray containing benign and malignant as well as inflammatory tissue samples of the head and neck region revealed the preferential expression of BTK-p80 in malignant tissue, whereas BTK-p65 expression was confirmed in over 80% of analyzed metastatic head and neck tumor cases. Therefore, processes associated with metastasis, like cancer stem cell (CSC) enrichment and the epithelial–mesenchymal transition (EMT), which in turn depend on an appropriate cytokine milieu, were analyzed. Treatment of HNSCC-derived cell lines cultured under 3D conditions with the BTK inhibitor AVL-292 caused reduced sphere formation, which was accompanied by reduced numbers of ALDH1A1+ CSCs as well as biological changes associated with the EMT. Moreover, we observed reduced NF-κB expression as well as altered NF-κB dependent pro-tumorigenic and EMT-associated cytokine release of IL-6, IFNγ, and TNFα when BTK activity was dampened. Therefore, an autocrine regulation of the oncogenic BTK-dependent process in HNSCC can be suggested, with BTK inhibition expected to be an effective treatment option for HNSCC.

Supplementary Figure 1 - PDF file 450K, Identification and validation of the genes supporting drug 4 resistance by shRNA-mediated phenotype screen

More than a decade ago, it was recognized that one the hallmarks of cancer is the major reprogramming of cellular metabolism in order to sustain continuous cell growth and proliferation.1Hanahan D. Weinberg R.A. Hallmarks of cancer: the next generation.Cell. 2011; 144: 646-674Summary Full Text Full Text PDF PubMed Scopus (45612) Google Scholar Since then, it has been demonstrated that cancer cells strictly depend on specific metabolites in several different experimental models. Moreover, cancer cells are especially vulnerable to nutrient deprivation and this liability can be exploited therapeutically.2Garcia-Bermudez J. Williams R.T. Guarecuco R. Birsoy K. Targeting extracellular nutrient dependencies of cancer cells.Mol Metab. 2020; 33: 67-82Crossref PubMed Scopus (41) Google Scholar Accordingly, it has been proved that - in mouse cancer models - dietary intervention ameliorates the response of cancer cells to chemo- and targeted therapy, radiotherapy, immunotherapy and hormone therapy while increasing the resistance of normal cells to anti-cancer treatments.3Nencioni A. Caffa I. Cortellino S. Longo V.D. Fasting and cancer: molecular mechanisms and clinical application.Nat Rev Cancer. 2018; 18: 707-719Crossref PubMed Scopus (237) Google Scholar Two types of dietary intervention have been experimentally tested, fasting and cycles of low-calories fast-mimicking diets (FMDs), the latter having proven to be feasible and safe also in human phase I/II clinical trials.4Valdemarin F. Caffa I. Persia A. et al.Safety and feasibility of fasting-mimicking diet and effects on nutritional status and circulating metabolic and inflammatory factors in cancer patients undergoing active treatment.Cancers. 2021; 18: 707-719Google Scholar Thus, several trials are ongoing to test the combination of periodic FMDs with standard treatments in patients with different types of cancer. Encouragingly, Ligorio and colleagues reported exceptional tumor responses to FMD combined with standard anticancer therapies in the NCT03340935 trial.5Ligorio F. Fucà G. Provenzano L. et al.Exceptional tumour responses to fasting-mimicking diet combined with standard anticancer therapies: a sub-analysis of the NCT03340935 trial.Eur J Cancer. 2022; 172: 300-310Summary Full Text Full Text PDF PubMed Scopus (10) Google Scholar Given that after repeated cycles of therapy, cancer cells develop the ability to escape drug-induced cytotoxicity, a major issue with any anti-cancer treatment is that it eventually become ineffective, leading to cancer recurrence. A growing body of evidence suggests that the reservoir for drug resistance and relapse is a sub-population of drug-tolerant persister (DTP) cells, i.e., cells with reduced drug sensitivity and decreased cell proliferation that eventually will reconstitute the tumor tissue.6Dhanyamraju P.K. Schell T.D. Amin S. Robertson G.P. Drug-tolerant persister cells in cancer therapy resistance.Cancer Res. 2022; 82: 2503-2514Crossref PubMed Scopus (10) Google Scholar In the recent issue of eBioMedicine, Liu and colleagues explored the effects of FMD on in vivo and in vitro models of colorectal cancer (CRC) and reported a dramatic reduction of the tumor masses in mice undergoing two cycles of FMD while being treated with a combination of 5-fluorouracil (5-FU) and oxaliplatin (OXA), the standard-of-care (SOC) therapeutic approach for CRC patients.7Liu X. Peng S. Tang G. et al.Fasting-mimicking diet synergizes with ferroptosis against quiescent, chemotherapy-resistant cells.eBioMedicine. 2023; 90104496https://doi.org/10.1016/j.ebiom.2023.104496Summary Full Text Full Text PDF Scopus (2) Google Scholar Interestingly, even though the combination FMD+5-FU/OXA was highly synergic in reducing tumor size compared to the single treatments, the necrotic/apoptotic areas were not expanded compared with chemotherapy alone and tumor tissue showed decreased proliferation markers, meaning that the dietary intervention acted by preventing cells from proliferating rather than killing them. In fact, transcriptomic and metabolomic analysis, and retention experiments demonstrated that cells entered a low proliferative state under fasting conditions. In addition, the most upregulated genes in FMD-treated cells were genes related to stemness and chemoresistance. Overall, the results were suggestive of FMD-inducing DTP cells. Using primary cells from CRC specimens, the authors also demonstrated that about 10% enter a persister state to evade the cytotoxicity of 5-FU/OXA and once the drugs were removed they were able to regrow and re-acquire sensitivity to 5-FU/OXA. Notably, DTP cells from fasting-mimicking cultures died twice less than those grown under standard conditions indicating that FMD effectively induced DTP cells acting as a reservoir for drug resistance. These results are particularly relevant from the clinical point of view and represent a caveat, given that several trials are ongoing to test FMDs in combination with anti-cancer therapies. In fact, it might be predicted that even in the case of the most favorable reduction of the tumor mass there would be also a parallel development of a pool of DTP cells that, in the long term, could reconstitute the tumor. Remarkably, Liu and colleagues also pinpoint a potential strategy to circumvent this obstacle by showing that nutrient-deprivation-induced quiescent cells and tumors - at variance with their counterparts cultured and fed normally - are exquisitely sensitive to ferroptosis inducers because they enter an autophagic state. The results after 3 weeks of treatment and 3 cycles of FMD - when the control group had to be euthanized - showed that FMD+5-FU/OXA halved the tumor mass and the addition of a ferroptosis inducer reduced the tumor to ¼ of the control, a ratio that could be observed also after 4 weeks. These initial data are very encouraging even though only performing long-term experiments (survival curve) will allow us to define the precise therapeutic advantage of administering a SOC therapy combined with ferroptosis inducers under an FMD. In addition, these studies should be repeated in different cancer models to understand if this approach might be generalized or if it pertains only to CRC. Despite these limitations, the results of this study are particularly relevant because they might open novel possibilities for circumventing the problem of drug resistance. Several compounds - either in clinical trials or already in the clinic for treating other pathologies - have been characterized also as ferroptosis inducers. Among them, the antibiotic salinomycin – and its derivative ionomycin – the anti-malaria artemisinin and its derivatives, the tyrosine kinase inhibitor sorafenib, the anti-malaria chloroquine in combination with the anticancer drug temozolomide, the anti-inflammatory sulfasalazine, the superparamagnetic iron oxide nanoparticle ferumoxytol, approved for use in the treatment of iron deficiency anemia.8Yang Y. Li X. Wang T. et al.Emerging agents that target signaling pathways in cancer stem cells.J Hematol Oncol. 2020; 13: 60Crossref PubMed Scopus (90) Google Scholar If the results obtained by Liu and colleagues will be confirmed on the long term and in other tumors, it can be envisaged that the addition and the re-purposing of these drugs to chemotherapy protocols performed under FMD would allow a better therapeutic result in cancer patients leading to prolonged, if not definitive, remissions. EG and MGC contributed equally in writing the paper. Both Authors read and approved the final manuscript. Authors have nothing to declare. Fasting-mimicking diet synergizes with ferroptosis against quiescent, chemotherapy-resistant cellsOur results suggest that ferroptosis could improve the antitumor activity of FMD + chemotherapy and highlight a potential therapeutic opportunity to avoid DTP cells-driven tumor relapse and therapy failure. Full-Text PDF Open Access

Supplementary Figure 3 - PDF file 33K, GSK3B depletion does not abolish the resistance of HCT116p53KO cells to targeted drugs

Hintergrund Die Bruton-Tyrosin-Kinase (BTK) gehört zur Tec-Familie der Nicht-Rezeptor-Tyrosin-Kinasen und wurde ursprünglich als exklusiv in Zellen hämatopoetischen Ursprungs exprimiert angesehen. Neben der 77 kDa BTK-Isoform, die in Immunzellen exprimiert wird, wurde vor kurzem die Expression neuer BTK-Isoformen der Größe 80 und 65 kDa bei mehreren soliden Tumorentitäten beschrieben. Daher untersuchen wir, ob diese BTK-Isoformen auch in HNSCC exprimiert werden und welche molekularen Konsequenzen dies für die Tumorentstehung hat.

Supplementary Data - PDF file 93K, Supplementary Materials and Methods and Supplementary Legend to Figures

Supplementary Figure 6 - PDF file 2812K, GSK3B is activated in colon carcinoma samples

Here, we describe the expression of Bruton's Tyrosine Kinase (BTK) in head and neck squamous cell carcinoma (HNSCC) cell lines as well as in primary HNSCC samples. BTK is a kinase initially thought to be expressed exclusively in cells of hematopoietic origin. Apart from the 77 kDa BTK isoform expressed in immune cells, particularly in B cells, we identified the 80 kDa and 65 kDa BTK isoforms in HNSCC, recently described as oncogenic. Importantly, we revealed that both isoforms are products of the same mRNA. By investigating the mechanism regulating oncogenic BTK-p80/p65 expression in HNSSC versus healthy or benign tissues, our data suggests that the epigenetic process of methylation might be responsible for the initiation of BTK-p80/p65 expression in HNSCC. Our findings demonstrate that chemical or genetic abrogation of BTK activity leads to inhibition of tumor progression in terms of proliferation and vascularization in vitro and in vivo. These observations were associated with cell cycle arrest and increased apoptosis and autophagy. Together, these data indicate BTK-p80 and BTK-p65 as novel HNSCC-associated oncogenes. Owing to the fact that abundant BTK expression is a characteristic feature of primary and metastatic HNSCC, targeting BTK activity appears as a promising therapeutic option for HNSCC patients.

Supplementary Table 3 - PDF file 74K, Correlation between activated GSK3B and patients outcome

Supplementary Table 2 - PDF file 70K, Cohort of patients characterization

In the last decade data piled up indicating that BTK - for twenty years considered as a "private matter" of bone marrow-derived cells - it is expressed and plays important and different roles also outside of the hematopoietic compartment and, most notably, in tumor cells. Initial evidence that BTK plays a critical role in B cell-derived malignancies prompted the chase for specific inhibitors, the forefather of which entered the clinic in a record time and paved the way for an ever increasing number of new molecules to be trialed. The growing interests in BTK also led to the discovery that, in solid tumors, two novel isoforms are mainly expressed and actionable liabilities for target therapy. Remarkably, the different isoforms appear to be involved in different signaling pathways which will have to be attentively specified in order to define the area of therapeutic intervention. In this perspective we briefly summarize the progress made in the last decade in studying BTK and its isoforms in cancer cells and define the open questions to be addressed in order to get the most benefits from its targeting for therapeutic purposes.

EDITORIAL article Front. Cell Dev. Biol., 26 April 2022Sec. Signaling https://doi.org/10.3389/fcell.2022.909655

High-dose standard-of-care chemotherapy is the only option for triple-negative breast cancer (TNBC) patients, which eventually die due to metastatic tumors. Recently, metronomic chemotherapy (mCHT) showed advantages in treating TNBCs leading us to investigate the anti-metastatic and anti-angiogenic potential of metronomic 5-Fluorouracil plus Vinorelbine (5-FU+VNR) on endothelial cells (ECs) and TNBCs in comparison to standard treatment (STD). We found that 10-fold lower doses of 5-FU+VNR given mCHT vs. STD inhibits cell proliferation and survival of ECs and TNBC cells. Both schedules strongly affect ECs migration and invasion, but in TNBC cells mCHT is significantly more effective than STD in impairing cell migration and invasion. The two treatments disrupt FAK/VEGFR/VEGF signaling in both ECs and TNBC cells. mCHT, and to a much lesser extent STD treatment, induces apoptosis in ECs, whereas it switches the route of cell death from apoptosis (as induced by STD) to autophagy in TNBC cells. mCHT-treated TNBCs-derived conditioned medium also strongly affects ECs' migration, modulates different angiogenesis-associated proteins, and hampers angiogenesis in matrix sponge in vivo. In conclusion, mCHT administration of 5-FU+VNR is more effective than STD schedule in controlling cell proliferation/survival and migration/invasion of both ECs and TNBC cells and has a strong anti-angiogenic effect. Our data suggest that the stabilization of tumor growth observed in TNBC patients treated with mCHT therapy schedule is likely due not only to direct cytotoxic effects but also to anti-metastatic and anti-angiogenic effects.