The DNA Damage Transducer RNF8 Facilitates Cancer Chemoresistance and Progression through Twist Activation

Abstract

Twist has been shown to cause treatment failure, cancer progression, and cancer-related death. However, strategies that directly target Twist are not yet conceivable. Here we reveal that K63-linked ubiquitination is a crucial regulatory mechanism for Twist activation. Through an E3 ligase screen and biochemical studies, we unexpectedly identified that RNF8 functions as a direct Twist activator by triggering K63-linked ubiquitination of Twist. RNF8-promoted Twist ubiquitination is required for Twist localization to the nucleus for subsequent EMT and CSC functions, thereby conferring chemoresistance. Our histological analyses showed that RNF8 expression is upregulated and correlated with disease progression, EMT features, and poor patient survival in breast cancer. Moreover, RNF8 regulates cancer cell migration and invasion and cancer metastasis, recapitulating the effect of Twist. Together, our findings reveal a previously unrecognized tumor-promoting function of RNF8 and provide evidence that targeting RNF8 is an appealing strategy to tackle tumor aggressiveness and treatment resistance.

Figure 1. Identification of RNF8 as a Twist activator through an E3 ligase screening

(A) BT549 TNBC cells were serum-starved and treated with or without EGF for various time periods; whole cell extracts were harvested for immunoprecipitation (IP) with Twist, followed by immunoblotting (IB) analysis. (B) BT549 cells were serum-starved, treated with EGF for 60 min. and harvested for IP with Twist, followed by IB analysis. (C) Schematic illustration of the strategy screening for E3 ligases that activate Twist. (D) Overlapping Candidate E3 ligases that regulate Twist activity in both HMLE and MCF7 cells are shown in the heat map. The experiment was independently performed for two times (n=3). (E) cBioPortal was used to access and to visualize the cancer genome atlas (TCGA) data for 107 cases of invasive carcinoma of basal-like breast cancer. Each column represents an individual sample. (F) Twist-overexpressing HMLE cells were infected with various doses of viruses containing shRNAs as indicated and were harvested for an E-cadherin luciferase reporter assay. Quantified results are presented as means ± SEM (n=3); **, p < 0.01. See also Figure S1 and Table S1-S3.

Figure 2. RNF8 regulates K63-linked ubiquitination and nuclear localization of Twist

(A) BT549 cells were serum-starved, treated with or without EGF for various time periods and harvested for co-IP assay followed by IB analysis. (B) In vivo ubiquitination assay in 293T cells transfected with Twist, Flag-tagged-RNF8 (Flag-RNF8) along with Histidine-tagged ubiquitin (His-Ub), His–Ub K48R, or His–Ub K63R constructs. Ni-nitrilotriacetic acid (NTA) indicates nickel bead precipitate; WCE indicates whole-cell extracts. (C) GST-Twist proteins were incubated with adenosine triphosphate, E1 and E2 (Ubc13/Uev1) along with or without purified Flag-RNF8 for in vitro Twist ubiquitination assay. (D) Confocal image analysis of Twist subcellular localization in BT549 cells with GFP or RNF8 knockdown. The arrow indicates the nuclear localization of Twist (upper). IB analysis of RNF8 expression and the quantification results are shown in the bottom of the graph. See also Figure S2.

Figure 3. K63-linked ubiquitination is essential for Twist nuclear transport and EMT

(A) In vivo ubiquitination assay in 293T cells transfected with His-Ub and Flag-Twist or various Flag-Twist mutants. (B) MCF7 cells were transfected with the indicated plasmids and harvested for an E-cadherin luciferase reporter assay. Quantified results are presented as means ± SEM (n=3); *, p < 0.05 and **, p < 0.01. (C) MCF7 cells transfected with mock, wild-type and the K38R mutant of Flag-Twist were fixed and stained with indicated antibodies, and subjected to confocal microscopy analysis. The arrow indicates the cells harboring ectopic expression of Flag-Twist wild-type or K38R mutant. (D) MCF7 cells stably expressing mock, Flag-Twist or various Flag-Twist mutants were harvested for IB analysis. (E) MCF7 cells transfected with mock, Flag-Twist wild-type or Flag-Twist K38R mutant were fixed and stained with indicated antibodies, and subjected to confocal microscopy analysis. Representative confocal images of nuclear and cytosolic Twist were shown in the left panel. The arrow indicates the cytosolic localization of Twist. Quantification results of nuclear and cytosolic Twist were shown in the right panel. See also Figure S3.

Figure 4. RNF8 regulates EMT and CSC self-renewal via promoting K63-linked ubiquitination of Twist

(A) BT549 cells with GFP or RNF8 knockdown were fixed and stained with indicated antibodies, and subjected to confocal microscopy analysis for expression and subcellular localization of E-cadherin and N-cadherin. (B) IB analysis for expression of Twist, N-cadherin and Vimentin in BT549 cells with GFP or RNF8 knockdown. (C) Mammosphere formation assay in BT549 cells with GFP or RNF8 knockdown. (D) Mammosphere formation assay in MDA-MB-231 cells with stable expression of vector alone, Flag-Twist wild-type or K38R mutant. (E) Mammosphere formation assay in BT549 cells with GFP, RNF8 knockdown or with RNF8 knockdown plus overexpression of Twist wild-type or K38R mutant. The quantified results are presented as means ± SEM (n=3); **, p < 0.01. Scale bar, 100 µm in (C, E) and 50 µm in (D). See also Figure S4.

Figure 5. RNF8 confers cancer chemoresistance through K63-linked ubiquitination of Twist

(A) Mammosphere formation assay in BT549 cells with GFP or RNF8 knockdown in response to doxorubicin (Dox). (B) Cell growth inhibition assay in BT549 and MDA-MB-231 cells with GFP or RNF8 knockdown in response to Dox. (C) GFP and RNF8-knockdown BT549 cells treated with vehicle or doxorubicin for 2 hrs were harvested for IP with Twist, followed by IB analysis to determine the level of K63-linked ubiquitination of Twist. (D) Mammosphere formation assay in MDA-MB-231 cells with stable expression of vector alone (pBabe), Twist wild-type and Twist K38R mutant in the presence of doxorubicin treatment. The quantified results are presented as means ± SEM (n=3); *, p < 0.05 and **, p < 0.01. Scale bar, 100 µm in (A) and 50 µm in (D). (E and F) Histological (E) and quantification (F) analyses of RNF8, Twist and E-cadherin in breast cancer patients with progressive or stable disease following receiving Dox. as a primary treatment (n=15 in each group). Scale bar represents 200 µm. The quantified results are presented as means ± SEM. See also Figure S5.

Figure 6. RNF8 is overexpressed in breast cancer and is positively correlated with poor disease outcomes

(A) Representative images of histological analyses of RNF8, Twist and E- cadherin expressions in breast cancer patients in low (stage I) and high (stage III) stages of tumor. Scale bar represents 200 µm. (B) Quantification analysis of RNF8 expressions in 202 cases of resected breast tumors. (C) Scatter plot analysis for the correlation between RNF8 expression versus Twist and E-cadherin expression levels was determined using the Pearson correlation coefficient analysis (n=202). LI represents labeling index. (D) Kaplan-Meier plot analysis of metastasis-free survival of 202 cases of breast cancer patients with low or high expression of RNF8. (E) Kaplan-Meier plot analysis of metastasis-free survival of 202 cases of breast cancer patients with low or high expression of RNF8, Twist and E-cadherin individually and concurrently. See also Figure S6, and Table S4-S6.

Figure 7. RNF8 regulates cancer cell migration, invasion and cancer metastasis as Twist does

(A and B) Transwell cell migration (A) and Matrigel cell invasion (B) assays in BT549 cells with GFP and RNF8 knockdown. (C) Matrigel cell invasion assay and IB analysis in MDA- MB-231 cells with GFP, RNF8 knockdown or with RNF8 knockdown plus expression of Flag- Twist wild-type and K38R mutant. The quantified results are presented as means ± SEM (n=3); *, p < 0.05 and **, p < 0.01. Scale bar, 100 µm. (D) MDA-MB-231-Luciferase cells with GFP, RNF8 or Twist knockdown were injected into nude mice, and the kinetics of breast cancer metastasis to the lung were measured and quantified using the IVIS bioluminescence system (n = 6 per group). Representative bioluminescent images are shown for days 0, 7 and 21. (E) Histological analysis of lung metastases derived from MDA-MB-231-Luciferase cells with GFP, RNF8 or Twist knockdown. Lung tissues were isolated 28 days after injection. H&E indicates haematoxylin and eosin stain; M indicates metastatic breast tumors. Scale bar, 100 µm. (F) A working model depicting the mechanism by which cancer exploits overexpressed RNF8 to drive chemoresistance and cancer metastasis via Twist activation. Ub indicates ubiquitination. See also Figure S7.