Key Points: DIAPH1 (mDia1) is involved in both Rho-mediated actin polymerization and microtubule assembly and stability during proplatelet formation.

The circadian clock coordinates biological and physiological functions to day/night cycles. The perturbation of the circadian clock increases cancer risk and affects cancer progression. Here, we studied how BMAL1 knockdown (BMAL1-KD) by shRNA affects the epithelial–mesenchymal transition (EMT), a critical early event in the invasion and metastasis of colorectal carcinoma (CRC). In corresponding to a gene set enrichment analysis, which showed a significant enrichment of EMT and invasive signatures in BMAL1_high CRC patients as compared to BMAL1_low CRC patients, our results revealed that BMAL1 is implicated in keeping the epithelial–mesenchymal equilibrium of CRC cells and influences their capacity of adhesion, migration, invasion, and chemoresistance. Firstly, BMAL1-KD increased the expression of epithelial markers (E-cadherin, CK-20, and EpCAM) but decreased the expression of Twist and mesenchymal markers (N-cadherin and vimentin) in CRC cell lines. Finally, the molecular alterations after BMAL1-KD promoted mesenchymal-to-epithelial transition-like changes mostly appeared in two primary CRC cell lines (i.e., HCT116 and SW480) compared to the metastatic cell line SW620. As a consequence, migration/invasion and drug resistance capacities decreased in HCT116 and SW480 BMAL1-KD cells. Together, BMAL1-KD alerts the delicate equilibrium between epithelial and mesenchymal properties of CRC cell lines, which revealed the crucial role of BMAL1 in EMT-related CRC metastasis and chemoresistance.

(1) Background: Triple-negative breast cancer (TNBC) is a distinct subgroup of breast cancer presenting a high level of recurrence, and neo-adjuvant chemotherapy is beneficial in its therapy management. Anti-PD-L1 immunotherapy improves the effect of neo-adjuvant therapy in TNBC. (2) Methods: Immune-modulation and ferroptosis-related R-packages were developed for integrative omics analyses under ferroptosis-inducer treatments: TNBC cells stimulated with ferroptosis inducers (GSE173905, GSE154425), single cell data (GSE191246) and mass spectrometry on breast cancer stem cells. Clinical association analyses were carried out with breast tumors (TCGA and METABRIC cohorts). Protein-level validation was investigated through protein atlas proteome experiments. (3) Results: Erastin/RSL3 ferroptosis inducers upregulate CD274 in TNBC cells (MDA-MB-231 and HCC38). In breast cancer, CD274 expression is associated with overall survival. Breast tumors presenting high expression of CD274 upregulated some ferroptosis drivers associated with prognosis: IDO1, IFNG and TNFAIP3. At the protein level, the induction of Cd274 and Tnfaip3 was confirmed in breast cancer stem cells under salinomycin treatment. In a 4T1 tumor treated with cyclophosphamide, the single cell expression of Cd274 was found to increase both in myeloid- and lymphoid-infiltrated cells, independently of its receptor Pdcd1. The CD274 ferroptosis-driver score computed on a breast tumor transcriptome stratified patients on their prognosis: low score was observed in the basal subgroup, with a higher level of recurrent risk scores (oncotypeDx, ggi and gene70 scores). In the METABRIC cohort, CD274, IDO1, IFNG and TNFAIP3 were found to be overexpressed in the TNBC subgroup. The CD274 ferroptosis-driver score was found to be associated with overall survival, independently of TNM classification and age diagnosis. The tumor expression of CD274, TNFAIP3, IFNG and IDO1, in a biopsy of breast ductal carcinoma, was confirmed at the protein level (4) Conclusions: Ferroptosis inducers upregulate PD-L1 in TNBC cells, known to be an effective target of immunotherapy in high-risk early TNBC patients who received neo-adjuvant therapy. Basal and TNBC tumors highly expressed CD274 and ferroptosis drivers: IFNG, TNFAIP3 and IDO1. The CD274 ferroptosis-driver score is associated with prognosis and to the risk of recurrence in breast cancer. A potential synergy of ferroptosis inducers with anti-PD-L1 immunotherapy is suggested for recurrent TNBC.

Everolimus (EV), a rapamycin analogue mTOR inhibitor, is used in the clinic to treat Estrogen positive (ER⁺) breast cancer in order to avoid the resistance to hormonotherapy. Here, we investigated whether EV efficacy varied according to administration timing by using the ER⁺ breast cancer cell line MCF-7 as model system. Our results showed that instead of apoptosis, EV induced a G0/G1 phase blockage of MCF-7 cells. Following serum shock, MCF-7 cells displayed a statistically significant 24h rhythm of mammalian target of Rapamycin (mTOR) activity, but perturbed circadian clock genes oscillations. Interestingly, the different delivery schedule of EV presented different efficacy in G0/G1 phase blockage in serum shocked MCF-7 cells. Moreover, serum shock induced also a circadian-like oscillation in expression or activity of several important G1 phase progression proteins, such as Cyclin D1 and phosphorylated Retinoblastoma protein (RB). Inhibition mTOR activity by EV reduced Cyclin D1 and Cyclin D3 protein level as well as RB phosphorylation level. Taken together, the results indicated that serum shock synchronization induced a circadian oscillation in mTOR activity in MCF-7 cells, which rhythmically regulated the synthesis or phosphorylation of key G1 progression proteins, such as Cyclin D1 and phosphorylated RB, ultimately resulting in different G0/G1 blockage efficiency according to different EV administration timing.

Dysregulation of the circadian timing system (CTS) frequently appears during colorectal cancer (CRC) progression. In order to better understand the role of the circadian clock in CRC progression, this study evaluated in vitro how knockdown of a core circadian protein BMAL1 (BMAL1-KD) influenced the behavior of two primary human CRC cell lines (HCT116 and SW480) and a metastatic CRC cell line (SW620). Unexpectedly, BMAL1-KD induced CRC cell-type specific responses rather than the same phenomenon throughout. First, BMAL1-KD increased AKT/mTOR activation in each CRC cell line, but to different extents. Second, BMAL1-KD-induced P53 activation varied with cell context. In a wild type P53 background, HCT116 BMAL1-KD cells quickly underwent apoptosis after shBMAL1 lentivirus transduction, while surviving cells showed less P53 but increased AKT/mTOR activation, which ultimately caused higher proliferation. In the presence of a partially functional mutant P53, SW480 BMAL1-KD cells showed moderate P53 and mTOR activation simultaneously with cell senescence. With a moderate increased AKT but unchanged mutant P53 activation, SW620 BMAL1-KD cells grew faster. Thus, under different CRC cellular pathological contexts, BMAL1 knockdown induced relatively equal effects on AKT/mTOR activation but different effects on P53 activation, which finally triggered different CRC cell fates.

(1) Background: Breast cancer is a frequent heterogeneous disorder diagnosed in women and causes a high number of mortality among this population due to rapid metastasis and disease recurrence. Ferroptosis can inhibit breast cancer cell growth, improve the sensitivity of chemotherapy and radiotherapy, and inhibit distant metastases, potentially impacting the tumor microenvironment. (2) Methods: Through data mining, the ferroptosis/extracellular matrix remodeling literature text-mining results were integrated into the breast cancer transcriptome cohort, taking into account patients with distant relapse-free survival (DRFS) under adjuvant therapy (anthracyclin + taxanes) with validation in an independent METABRIC cohort, along with the MDA-MB-231 and HCC338 transcriptome functional experiments with ferroptosis activations (GSE173905). (3) Results: Ferroptosis/extracellular matrix remodeling text-mining identified 910 associated genes. Univariate Cox analyses focused on breast cancer (GSE25066) selected 252 individual significant genes, of which 170 were found to have an adverse expression. Functional enrichment of these 170 adverse genes predicted basal breast cancer signatures. Through text-mining, some ferroptosis-significant adverse-selected genes shared citations in the domain of ECM remodeling, such as TNF, IL6, SET, CDKN2A, EGFR, HMGB1, KRAS, MET, LCN2, HIF1A, and TLR4. A molecular score based on the expression of the eleven genes was found predictive of the worst prognosis breast cancer at the univariate level: basal subtype, short DRFS, high-grade values 3 and 4, and estrogen and progesterone receptor negative and nodal stages 2 and 3. This eleven-gene signature was validated as regulated by ferroptosis inductors (erastin and RSL3) in the triple-negative breast cancer cellular model MDA-MB-231. (4) Conclusions: The crosstalk between ECM remodeling-ferroptosis functionalities allowed for defining a molecular score, which has been characterized as an independent adverse parameter in the prognosis of breast cancer patients. The gene signature of this molecular score has been validated to be regulated by erastin/RSL3 ferroptosis activators. This molecular score could be promising to evaluate the ECM-related impact of ferroptosis target therapies in breast cancer.