Targeting TNFR2 in Cancer: All Roads Lead to Rome.

TNF receptor 2 (TNFR2) has become one of the best potential immune checkpoints that might be targeted, mainly because of its vital role in tumor microenvironments (TMEs). Overexpression of TNFR2 in some tumor cells and essential function in immunosuppressive cells, especially regulatory T cells (Tregs), makes blocking TNFR2 an excellent strategy in cancer treatment; however, there is evidence showing that activating TNFR2 can also inhibit tumor progression in vivo. In this review, we will discuss drugs that block and activate TNFR2 under clinical trials or preclinical developments up till now. Meanwhile, we summarize and explore the possible mechanisms related to them.

Sabutoclax, pan-active BCL-2 protein family antagonist, overcomes drug resistance and eliminates cancer stem cells in breast cancer.

Misregulation of BCL-2 family of proteins renders a survival signal to withstand cytotoxic anticancer drugs and is often found in drug resistant cells. The drug resistance phenotype is also associated with an enhancement of cancer stem cell-like (CSC) characteristics. Thus, inhibition of anti-apoptotic BCL-2 family proteins has been proposed as a possible antineoplastic strategy, and BCL-2 inhibitors are currently being clinically trailed in patients with leukemia, lymphoma or non-small cell lung cancer. However, the effects of BCL-2 inhibitors on drug resistant breast cancer have not yet been elucidated. In the present study, the effect of sabutoclax, a pan-active BCL-2 protein family antagonist, on two chemoresistant breast cancer cell lines was assessed. We found that sabutoclax showed a significant cytotoxic activity on chemoresistant breast cancer cells both in vitro and in vivo. When chemotherapeutic agents were combined with sabutoclax, strong synergistic antiproliferative effects were observed. Sabutoclax induced the blockage of BCL-2, MCL-1, BCL-xL and BFL-1, which in turn led to caspase-3/7 and caspase-9 activation and modulation of Bax, Bim, PUMA and survivin expression. Furthermore, sabutoclax effectively eliminated the CSC subpopulation and reduced sphere formation of drug-resistant cells through down-regulation of the IL-6/STAT3 signaling pathway. A similar effect was observed in a small panel of nine breast tumors ex vivo. Our findings indicate that sabutoclax partially overcomes the drug resistance phenotype of breast cancer cells by reactivation of apoptosis, mediated by the inhibition of several anti-apoptotic BCL-2 family proteins, and eliminates CSCs by abolition of the IL-6/STAT3 pathway. This offers a strong rationale to explore the therapeutic strategy of using sabutoclax alone or in combination for chemotherapy-nonresponsive breast cancer patients.

HSP90 inhibitor AUY922 can reverse Fulvestrant induced feedback reaction in human breast cancer cells.

Hormone therapy has become one of the main strategies for breast cancer, however, many estrogen receptor (ER) positive patients end in tumor collapse due to initial or acquired resistance to hormone treatment, which includes Fulvestrant. Here we report that ErbB receptors and downstream PI3K/AKT and ERK pathway have been reactivated after treatment of Fulvestrant in ER positive MCF-7 and T47D cells, which are related to Fulvestrant resistance. HSP90 is a universally expressed chaperone protein and plays a vital role in both normal and cancer cells, HSP90 inhibitor AUY922 can reverse this feedback reactivation effect of Fulvestrant by targeting multiple proteins related in ErbB receptors, PI3K/AKT and ERK pathway, which is much better than single targeting inhibitors. We also consolidate these effects in human fresh breast tumors. Combination of AUY922 and Fulvestrant may become a promising therapy strategy in breast cancer treatment.

The combination of NVP-BKM120 with trastuzumab or RAD001 synergistically inhibits the growth of breast cancer stem cells in vivo.

Deregulation of the phosphatidylinositol-3-kinase (PI3K)/Akt signalling pathway is common in breast cancer and is frequently associated with resistance to both traditional chemotherapy and targeted drugs. There is a growing body of evidence indicating that a small subpopulation of self-renewing cells, the so called cancer stem cells (CSC), are responsible for the growth of drug resistant secondary tumors. As many CSCs have upregulated the PI3K/Akt signalling pathway, preclinical and clinical studies are addressing the inhibition of this axis to target drug resistance. We evaluated the susceptibility of breast CSCs to NVP-BKM120 (BKM120), a new generation of PI3K-specific inhibitor, when used individually or in combination with trastuzumab or RAD001 both in vitro and in vivo. For this, a stem-like cell population (SC) was enriched from breast cancer cell lines after mammosphere cultures. We demonstrated that BKM120 inhibits growth, generation of drug-resistant derivatives and SC formation in a panel of four breast cancer cell lines: MCF-7, MDA-MB-231, SK-BR-3 and CAL51. Importantly, BKM120 inhibits the PI3K/Akt signalling pathway in SCs from these cell lines. When BKM120 was used in combination with trastuzumab, a targeted therapy to treat HER2-positive breast cancer, we found synergistic cell growth inhibition, generation of drug resistant cells as well as SC formation from SK-BR-3 cells. Importantly, SK-BR-3 xenograft-derived tumors showed marginal growth when the drug combination was used. We also found a similar synergistic anticancer effect of BKM120 in combination with RAD001, an mTOR inhibitor, when treating triple-negative breast cancer cells in vitro and in both MDA-MB-231 and CAL51- mouse xenografts. Moreover, mouse data indicate that these drug combinations are well tolerated and provide the proof-of-concept and rationale to initiate clinical trials in both HER2-positive and triple-negative breast cancer.

Epidermal growth factor receptor and AKT1 gene copy numbers by multi-gene fluorescence in situ hybridization impact on prognosis in breast cancer.

The epidermal growth factor receptor (EGFR)/PI3K/AKT signaling pathway aberrations play significant roles in breast cancer occurrence and development. However, the status of EGFR and AKT1 gene copy numbers remains unclear. In this study, we showed that the rates of EGFR and AKT1 gene copy number alterations were associated with the prognosis of breast cancer. Among 205 patients, high EGFR and AKT1 gene copy numbers were observed in 34.6% and 27.8% of cases by multi-gene fluorescence in situ hybridization, respectively. Co-heightened EGFR/AKT1 gene copy numbers were identified in 11.7% cases. No changes were found in 49.3% of patients. Although changes in EGFR and AKT1 gene copy numbers had no correlation with patients' age, tumor stage, histological grade and the expression status of other molecular makers, high EGFR (P = 0.0002) but not AKT1 (P = 0.1177) gene copy numbers correlated with poor 5-year overall survival. The patients with co-heightened EGFR/AKT1 gene copy numbers displayed a poorer prognosis than those with tumors with only high EGFR gene copy numbers (P = 0.0383). Both Univariate (U) and COX multivariate (C) analyses revealed that high EGFR and AKT1 gene copy numbers (P = 0.000 [U], P = 0.0001 [C]), similar to histological grade (P = 0.001 [U], P = 0.012 [C]) and lymph node metastasis (P = 0.046 [U], P = 0.158 [C]), were independent prognostic indicators of 5-year overall survival. These results indicate that high EGFR and AKT1 gene copy numbers were relatively frequent in breast cancer. Co-heightened EGFR/AKT1 gene copy numbers had a worse outcome than those with only high EGFR gene copy numbers, suggesting that evaluation of these two genes together may be useful for selecting patients for anti-EGFR-targeted therapy or anti-EGFR/AKT1-targeted therapy and for predicting outcomes.

Multi-gene fluorescence in situ hybridization to detect cell cycle gene copy number aberrations in young breast cancer patients.

Breast cancer is a disease of cell cycle, and the dysfunction of cell cycle checkpoints plays a vital role in the occurrence and development of breast cancer. We employed multi-gene fluorescence in situ hybridization (M-FISH) to investigate gene copy number aberrations (CNAs) of 4 genes (Rb1, CHEK2, c-Myc, CCND1) that are involved in the regulation of cell cycle, in order to analyze the impact of gene aberrations on prognosis in the young breast cancer patients. Gene copy number aberrations of these 4 genes were more frequently observed in young breast cancer patients when compared with the older group. Further, these CNAs were more frequently seen in Luminal B type, Her2 overexpression, and tiple-negative breast cancer (TNBC) type in young breast cancer patients. The variations of CCND1, Rb1, and CHEK2 were significantly correlated with poor survival in the young breast cancer patient group, while the amplification of c-Myc was not obviously correlated with poor survival in young breast cancer patients. Thus, gene copy number aberrations (CNAs) of cell cycle-regulated genes can serve as an important tool for prognosis in young breast cancer patients.

QM-FISH analysis of the genes involved in the G1/S checkpoint signaling pathway in triple-negative breast cancer.

This study was conducted to analyze copy number alterations (CNAs) of the genes involved in the G1/S checkpoint signaling pathway of triple-negative breast cancer (TNBC) and to evaluate their clinical value in the prognosis of TNBC. Quantitative multi-gene fluorescence in situ hybridization was used to study CNAs of the genes involved in the G1/S checkpoint signaling pathway, including cyclin d1 (CCND1), c-Myc, p21, cell-cycle-checkpoint kinase 2 gene, p16, retinoblastoma (Rb1), murine double minute 2 (Mdm2) and p53, in 60 TNBC samples and 60 non-TNBC samples. In comparison with the non-TNBC samples, CNAs of the genes involved in the G1/S checkpoint signaling pathway were more frequently observed in the TNBC samples (p = 0.000). Out of a total of eight genes, six (CCND1, c-Myc, p16, Rb1, Mdm2, and p53) exhibited significantly different CNAs between the TNBC group and the non-TNBC group. Univariate survival analysis revealed that the gene amplification of c-Myc (p = 0.008), Mdm2 (p = 0.020) and the gene deletion of p21 (p = 0.004), p16 (p = 0.015), and Rb1 (p = 0.028) were the independent predictive factor of 5-year OS for patients with TNBC. Cox multivariate analysis revealed that the gene amplification of c-Myc (p = 0.026) and the gene deletion of p21 (p = 0.019) and p16 (p = 0.034) were independent prognostic factors affecting the 5-year OS for TNBC. CNAs of the genes involved in the G1/S checkpoint signaling pathway presented a higher rate of incidence in TNBC than in non-TNBC, which could indicate one of the molecular mechanisms for the specific biological characteristics of TNBC. The genes c-Myc, p21, and p16 were correlated with the prognosis of TNBC and therefore may have potential clinical application values in the prognostic prediction of TNBC.