Rack1 mediates tyrosine phosphorylation of Anxa2 by Src and promotes invasion and metastasis in drug-resistant breast cancer cells.

BACKGROUND: Acquirement of resistance is always associated with a highly aggressive phenotype of tumor cells. Recent studies have revealed that Annexin A2 (Anxa2) is a key protein that links drug resistance and cancer metastasis. A high level of Anxa2 in cancer tissues is correlated to a highly aggressive phenotype. Increased Anxa2 expression appears to be specific in many drug-resistant cancer cells. The functional activity of Anxa2 is regulated by tyrosine phosphorylation at the Tyr23 site. Nevertheless, the accurate molecular mechanisms underlying the regulation of Anxa2 tyrosine phosphorylation and whether phosphorylation is necessary for the enhanced invasive phenotype of drug-resistant cells remain unknown. METHODS: Small interfering RNAs, small molecule inhibitors, overexpression, loss of function or gain of function, rescue experiments, Western blot, wound healing assays, transwell assays, and in vivo metastasis mice models were used to investigate the functional effects of Rack1 and Src on the tyrosine phosphorylation of Anxa2 and the invasion and metastatic potential of drug-resistant breast cancer cells. The interaction among Rack1, Src, and Anxa2 in drug-resistant cells was verified by co-immunoprecipitation assay. RESULTS: We demonstrated that Anxa2 Tyr23 phosphorylation is necessary for multidrug-resistant breast cancer invasion and metastasis. Rack1 is required for the invasive and metastatic potential of drug-resistant breast cancer cells through modulating Anxa2 phosphorylation. We provided evidence that Rack1 acts as a signal hub and mediates the interaction between Src and Anxa2, thereby facilitating Anxa2 phosphorylation by Src kinase. CONCLUSIONS: Our findings suggest a convergence point role of Rack1/Src/Anxa2 complex in the crosstalk between drug resistance and cancer aggressiveness. The interaction between Anxa2 and Rack1/Src is responsible for the association between drug resistance and invasive/metastatic potential in breast cancer cells. Thus, our findings provide novel insights on the mechanism underlying the functional linkage between drug resistance and cancer aggressiveness.

Tyr23 phosphorylation of Anxa2 enhances STAT3 activation and promotes proliferation and invasion of breast cancer cells.

PURPOSE: Overexpression of Annexin A2 (Anxa2) is positively correlated with breast cancer progression, drug resistance, and poor prognosis of patients with breast cancer. Tyr23 Phosphorylation by Src-family tyrosine kinase is an important post-translational modification of Anxa2. This modification regulates the subcellular localization and functions of Anxa2 and has significant effects on cell proliferation, migration, and invasion. This study aims at revealing the association of Anxa2-Tyr23 phosphorylation in Anxa2-mediated acceleration of breast cancer progression and their elaborate molecular mechanisms. METHODS: Cell biological function experiments were performed to determine the effects of Anxa2-Tyr23 Phosphorylation on breast cancer cell proliferation and invasion in vitro and metastasis in vivo. The interaction of Tyr23 phosphorylated Anxa2 and STAT3 was verified by co-immunoprecipitation assay. Related mRNA and protein expression levels of cyclin D1 and MMP2/9 and phosphorylation level of STAT3 were detected. RESULTS: Anxa2-Tyr23 phosphorylation is necessary for proliferation, invasion, and metastasis of breast cancer cells in vitro and in vivo. Tyr23 phosphorylated Anxa2 binds and enhances the sensitivity of STAT3 activation in response to IL-6, thereby increasing the protein and mRNA expression levels of cyclin D1 and MMP2/9 which are STAT3 key target genes and serve pivotal regulatory functions in cell proliferation and invasion, respectively. CONCLUSION: Our findings further confirmed the regulatory role of Anxa2 and revealed the direct relationship between Anxa2-Tyr23 phosphorylation and activation of STAT3. Moreover, this study provides novel insights into the function of Anxa2-Tyr23 phosphorylation in signal transduction for further understanding of the mechanism through which Anxa2 promotes the progression of breast cancer.

Drug-resistant cancer cell-derived exosomal EphA2 promotes breast cancer metastasis via the EphA2-Ephrin A1 reverse signaling.

Tumor metastasis induced by drug resistance is a major challenge in successful cancer treatment. Nevertheless, the mechanisms underlying the pro-invasive and metastatic ability of drug resistance remain elusive. Exosome-mediated intercellular communications between cancer cells and stromal cells in tumor microenvironment are required for cancer initiation and progression. Recent reports have shown that communications between cancer cells also promote tumor aggression. However, little attention has been regarded on this aspect. Herein, we demonstrated that drug-resistant cell-derived exosomes promoted the invasion of sensitive breast cancer cells. Quantitative proteomic analysis showed that EphA2 was rich in exosomes from drug-resistant cells. Exosomal EphA2 conferred the invasive/metastatic phenotype transfer from drug-resistant cells to sensitive cells. Moreover, exosomal EphA2 activated ERK1/2 signaling through the ligand Ephrin A1-dependent reverse pathway rather than the forward pathway, thereby promoting breast cancer progression. Our findings indicate the key functional role of exosomal EphA2 in the transmission of aggressive phenotype between cancer cells that do not rely on direct cell-cell contact. Our study also suggests that the increase of EphA2 in drug-resistant cell-derived exosomes may be an important mechanism of chemotherapy/drug resistance-induced breast cancer progression.

Comprehensive Analysis of Splicing Factor and Alternative Splicing Event to Construct Subtype-Specific Prognosis-Predicting Models for Breast Cancer.

Recent evidence suggests that splicing factors (SFs) and alternative splicing (AS) play important roles in cancer progression. We constructed four SF-risk-models using 12 survival-related SFs. In Luminal-A, Luminal-B, Her-2, and Basal-Like BRCA, SF-risk-models for three genes (PAXBP1, NKAP, and NCBP2), four genes (RBM15B, PNN, ACIN1, and SRSF8), three genes (LSM3, SNRNP200, and SNU13), and three genes (SRPK3, PUF60, and PNN) were constructed. These models have a promising prognosis-predicting power. The co-expression and protein-protein interaction analysis suggest that the 12 SFs are highly functional-connected. Pathway analysis and gene set enrichment analysis suggests that the functional role of the selected 12 SFs is highly context-dependent among different BRCA subtypes. We further constructed four AS-risk-models with good prognosis predicting ability in four BRCA subtypes by integrating the four SF-risk-models and 21 survival-related AS-events. This study proposed that SFs and ASs were potential multidimensional biomarkers for the diagnosis, prognosis, and treatment of BRCA.

Subtype-specific risk models for accurately predicting the prognosis of breast cancer using differentially expressed autophagy-related genes.

Emerging evidence suggests that the dysregulation of autophagy-related genes (ARGs) is coupled with the carcinogenesis and progression of breast cancer (BRCA). We constructed three subtype-specific risk models using differentially expressed ARGs. In Luminal, Her-2, and Basal-like BRCA, four- (BIRC5, PARP1, ATG9B, and TP63), three- (ITPR1, CCL2, and GAPDH), and five-gene (PRKN, FOS, BAX, IFNG, and EIF4EBP1) risk models were identified, which all have a receiver operating characteristic > 0.65 in the training and testing dataset. Multivariable Cox analysis showed that those risk models can accurately and independently predict the overall survival of BRCA patients. Comprehensive analysis showed that the 12 identified ARGs were correlated with the overall survival of BRCA patients; six of the ARGs (PARP1, TP63, CCL2, GAPDH, FOS, and EIF4EBP1) were differentially expressed between BRCA and normal breast tissue at the protein level. In addition, the 12 identified ARGs were highly interconnected and displayed high frequency of copy number variation in BRCA samples. Gene set enrichment analysis suggested that the deactivation of the immune system was the important driving force for the progression of Basal-like BRCA. This study demonstrated that the 12 ARG signatures were potential multi-dimensional biomarkers for the diagnosis, prognosis, and treatment of BRCA.

SHP2 promotes proliferation of breast cancer cells through regulating Cyclin D1 stability via the PI3K/AKT/GSK3ß signaling pathway.

Objective: The tyrosine phosphatase SHP2 has a dual role in cancer initiation and progression in a tissue type-dependent manner. Several studies have linked SHP2 to the aggressive behavior of breast cancer cells and poorer outcomes in people with cancer. Nevertheless, the mechanistic details of how SHP2 promotes breast cancer progression remain largely undefined. Methods: The relationship between SHP2 expression and the prognosis of patients with breast cancer was investigated by using the TCGA and GEO databases. The expression of SHP2 in breast cancer tissues was analyzed by immunohistochemistry. CRISPR/Cas9 technology was used to generate SHP2-knockout breast cancer cells. Cell-counting kit-8, colony formation, cell cycle, and EdU incorporation assays, as well as a tumor xenograft model were used to examine the function of SHP2 in breast cancer proliferation. Quantitative RT-PCR, western blotting, immunofluorescence staining, and ubiquitination assays were used to explore the molecular mechanism through which SHP2 regulates breast cancer proliferation. Results: High SHP2 expression is correlated with poor prognosis in patients with breast cancer. SHP2 is required for the proliferation of breast cancer cells in vitro and tumor growth in vivo through regulation of Cyclin D1 abundance, thereby accelerating cell cycle progression. Notably, SHP2 modulates the ubiquitin-proteasome-dependent degradation of Cyclin D1 via the PI3K/AKT/GSK3ß signaling pathway. SHP2 knockout attenuates the activation of PI3K/AKT signaling and causes the dephosphorylation and resultant activation of GSK3ß. GSK3ß then mediates phosphorylation of Cyclin D1 at threonine 286, thereby promoting the translocation of Cyclin D1 from the nucleus to the cytoplasm and facilitating Cyclin D1 degradation through the ubiquitin-proteasome system. Conclusions: Our study uncovered the mechanism through which SHP2 regulates breast cancer proliferation. SHP2 may therefore potentially serve as a therapeutic target for breast cancer.

Rack1 mediates Src binding to drug transporter P-glycoprotein and modulates its activity through regulating Caveolin-1 phosphorylation in breast cancer cells.

The failure of chemotherapy and the emergence of multidrug resistance (MDR) are the major obstacles for effective therapy in locally advanced and metastatic breast cancer. Overexpression of the drug transporter P-glycoprotein (P-gp) in cancer cells is one of the main causes of MDR due to its ability to efflux anticancer drugs out of cells. Although the signaling node that regulates the expression of P-gp has been intensively investigated; the regulatory mechanism underlying P-gp transport activity remains obscure. Herein, we reported that Rack1 and tyrosine kinase Src confer drug resistance through modulating the transport function of P-gp without altering its protein level. We provide evidences that Rack1 and Src regulate P-gp activity by modulating caveolin-1 (Cav1) phosphorylation. Importantly, Rack1 acts as a signaling hub and mediates Src binding to P-gp, thereby facilitating the phosphorylation of Cav1 by Src and abolishing the inhibitory effect of Cav1 on P-gp. Taken together, our results demonstrate the pivotal roles of Rack1 and Src in modulating P-gp activity in drug-resistant cells. Our findings also provide novel insights into the mechanism regulating P-gp transport activity. Rack1 may represent a new target for the development of effective therapies for reversing drug resistance.