Microbiota enterotoxigenic Bacteroides fragilis-secreted BFT-1 promotes breast cancer cell stemness and chemoresistance through its functional receptor NOD1

Tumor-resident microbiota in breast cancer promotes cancer initiation and malignant progression.However,targeting microbiota to improve the effects of breast cancer therapy has not been investigated in detail.Here,we evaluated the microbiota composition of breast tumors and found that enterotoxigenic Bacteroides fragilis(ETBF)was highly enriched in the tumors of patients who did not respond to taxane-based neoadjuvant chemotherapy.ETBF,albeit at low biomass,secreted the toxic protein BFT-1 to promote breast cancer cell stemness and chemoresistance.Mechanistic studies showed that BFT-1 directly bound to NOD1 and stabilized NOD1 protein.NOD1 was highly expressed on ALDH+breast cancer stem cells(BCSCs)and cooperated with GAK to phosphorylate NUMB and promote its lysosomal degradation,thereby activating the NOTCH1-HEY1 signaling pathway to increase BCSCs.NOD1 inhibition and ETBF clearance increase the chemosensitivity of breast cancer by impairing BCSCs.

PMN-MDSCs modulated by CCL20 from cancer cells promoted breast cancer cell stemness through CXCL2-CXCR2 pathway

Our previous studies have showed that C-C motif chemokine ligand 20(CCL20)advanced tumor progression and enhanced the chemoresistance of cancer cells by positively regulating breast cancer stem cell(BCSC)self-renewal.However,it is unclear whether CCL20 affects breast cancer progression by remodeling the tumor microenvironment(TME).Here,we observed that polymorphonuclear myeloid-derived suppressor cells(PMN-MDSCs)were remarkably enriched in TME of CCL20-overexpressing cancer cell orthotopic allograft tumors.Mechanistically,CCL20 activated the differentiation of granulocyte-monocyte progenitors(GMPs)via its receptor C-C motif chemokine receptor 6(CCR6)leading to the PMN-MDSC expansion.PMN-MDSCs from CCL20-overexpressing cell orthotopic allograft tumors(CCL20-modulated PMN-MDSCs)secreted amounts of C-X-C motif chemokine ligand 2(CXCL2)and increased ALDH+BCSCs via activating CXCR2/NOTCH1/HEY1 signaling pathway.Furthermore,C-X-C motif chemokine receptor 2(CXCR2)antagonist SB225002 enhanced the docetaxel(DTX)effects on tumor growth by decreasing BCSCs in CCL20high-expressing tumors.These findings elucidated how CCL20 modulated the TME to promote cancer development,indicating a new therapeutic strategy by interfering with the interaction between PMN-MDSCs and BCSCs in breast cancer,especially in CCL20high-expressing breast cancer.

Development of a novel method for rapid cloning of shRNA vectors, which successfully knocked down CD44 in mesenchymal triple-negative breast cancer cells

Dear Editor,Since the discovery of short hairpin RNA(shRNA)vec-tor-mediated RNA interference(RNAi),this technology has been widely used in cancer research for its specific-ity,potency,and convenience.However,researchers may find it costly to purchase commercial vectors from bio-companies or time-and labor-consuming to construct their own shRNA vectors using traditional method by inserting annealed duplex into digested vectors.Despite intensive efforts to accelerate shRNA vector cloning in laboratories,the development of a reliable,rapid,conven-ient,and cost-effective method is still in great demand.To this end,we developed a novel method named SuperSH(Super rapid cloning of shRNA vector)for the effective and rapid construction of shRNA-expressing vectors based on high-performance DNA polymerase and seamless cloning technique[1](Additional file 1:Fig-ure S1a;the detailed methods can be found in Additional file 1).In our SuperSH method,the shRNA sequences are introduced into the vector via a pair of polymerase chain reaction(PCR)primers rather than via annealed duplex.In detail,the 3′ends of the primers are designed to bind the template to initiate a PCR to amplify the vector back-bone,and the 5′portions are designed to introduce the sequences of interest as well as to form a short homol-ogous arm for subsequent recombination via seamless cloning[1].