Triple-negative breast cancer (TNBC) is a particularly aggressive subtype of breast cancer. TNBC is a heterogenous subtype of breast cancer that is beginning to be refined by its molecular characteristics and clinical response to a targeted therapeutic approach. Until recently the backbone of therapy against TNBC has been cytotoxic chemotherapy. However, the breast oncology community is now seeing encouraging clinical activity from molecularly targeted approaches to TNBC. Recently, we have seen 3 newly approved targeted therapies for TNBC, including the PARP inhibitors olaparib and talazoparib for germline BRCA mutation associated breast cancer (gBRCAm-BC) and most recently the checkpoint inhibitor, atezolizumab in combination with nab-paclitaxel for programmed death-ligand 1 (PD-L1+) advanced TNBC. Improved biomarkers are needed to inform better patient selection for treatment with checkpoint inhibition. Higher response rates are seen when checkpoint inhibitors are combined with chemotherapy in the first-line setting and the use of these agents at an earlier stage of the disease does show promise. Antibody-drug conjugates are generating much excitement and may allow re-examination of prior cytotoxics that failed in development due to toxicity. Tumor sequencing is identifying potential molecular targets and ongoing studies are evaluating novel small molecule agents in this field such as AKT inhibition and many others. The treatment paradigm of chemotherapy as “one size fits all” approach for management of TNBC is changing based on molecular subtyping. Soon, the term TNBC may no longer be appropriate, as this heterogenous subtype of breast cancer is further refined by its molecular characteristics and clinical response to a targeted therapeutic approach.

Targeted Therapies for Triple-Negative Breast Cancer

Curcumin in cancer therapy: A novel adjunct for combination chemotherapy with paclitaxel and alleviation of its adverse effects.

Mutant p53 (R248Q) induces doxorubicin (ADM) resistance in hepatocellular carcinoma (HCC). Dihydroartemisinin (DHA) can synergistically enhance anticancer effect of many chemotherapeutic agents. However, whether DHA could increase therapeutic efficacy of ADM in p53 (R248Q)-expressing HCC cells remains unknown. In the present study, we established mutant p53 (R248Q)-expressing Hep3B cells to study the effect and mechanism of DHA on ADM resistance and the synergistic effect of DHA with ADM. We found that P-gp was highly expressed in p53 (R248Q)-expressing Hep3B cells. As a result, cells expressing p53 (R248Q) displayed higher cell viability and lower cell apoptosis level upon ADM treatment. Meanwhile, phosphorylation levels of ERK1/2 and p65 were elevated in p53 (R248Q)-expressing Hep3B cells. However, combination of DHA and ADM treatment decreased cell viability and elevated cell apoptosis level in p53 (R248Q)-expressing Hep3B cells. Molecular dynamics simulations showed that DHA had the potential to bind with mutant p53 (R248Q) protein. Furthermore, DHA treatment decreased P-gp expression and inhibited phosphorylation levels of ERK1/2 and p65 in p53 (R248Q)-expressing Hep3B cells. Finally, DHA treatment could significantly reduce ADM efflux in p53 (R248Q)-expressing cells. Our results indicate that DHA could decrease P-gp expression via inhibiting the p53 (R248Q)-ERK1/2-NF-κB signaling pathway, which eventually confers sensitization of p53 (R248Q)-expressing HCC cells to ADM. Our study provides evidence for the potential application of DHA and ADM combination in treatment of mutant p53 (R248Q)-harbored HCC.

/pdf/dihydroartemisinin-sensitizes-mutant-p53-r248q-expressing-5gnf31kb14.pdf

Dihydroartemisinin Sensitizes Mutant p53 (R248Q)-Expressing Hepatocellular Carcinoma Cells to Doxorubicin by Inhibiting P-gp Expression.

Breast cancer is a heterogeneous disease consisting of different biological subtypes, with differences in terms of incidence, response to diverse treatments, risk of disease progression, and sites of metastases. In the last years, several molecular targets have emerged and new drugs, targeting PI3K/Akt/mTOR and cyclinD/CDK/pRb pathways and tumor microenvironment have been integrated into clinical practice. However, it is clear now that breast cancer is able to develop resistance to these drugs and the identification of the underlying molecular mechanisms is paramount to drive further drug development. Autophagy is a highly conserved homeostatic process that can be activated in response to antineoplastic agents as a cytoprotective mechanism. Inhibition of autophagy could enhance tumor cell death by diverse anti-cancer therapies, representing an attractive approach to control mechanisms of drug resistance. In this manuscript, we present a review of autophagy focusing on its interplay with targeted drugs used for breast cancer treatment.

/pdf/targeting-autophagy-in-breast-cancer-1cmgpt3o2n.pdf

Targeting Autophagy in Breast Cancer

Targeting telomeres: advances in telomere maintenance mechanism-specific cancer therapies

Dihydroartemisinin (DHA) has been reported to possess anti-cancer activity against many cancers. However, the pharmacologic effect of DHA on HBV-positive hepatocellular carcinoma (HCC) remains unknown. Thus, the objective of the present study was to determine whether DHA could inhibit the proliferation of HepG2.2.15 cells and uncover the underlying mechanisms involved in the effect of DHA on HepG2.2.15 cells. We found that DHA effectively inhibited HepG2.2.15 HCC cell proliferation both in vivo and in vitro. DHA also reduced the migration and tumorigenicity capacity of HepG2.2.15 cells. Regarding the underlying mechanisms, results showed that DHA induced cellular senescence by up-regulating expression levels of proteins such as p-ATM, p-ATR, γ-H2AX, P53, and P21 involved in DNA damage response. DHA also induced autophagy (green LC3 puncta gathered together and LC3II/LC3I ratio increased through AKT-mTOR pathway suppression). Results also revealed that DHA-induced autophagy was not linked to senescence or cell death. TPP1 (telomere shelterin) overexpression could not rescue DHA-induced anticancer activity (cell proliferation). Moreover, DHA down-regulated TPP1 expression. Gene knockdown of TPP1 caused similar phenotypes and mechanisms as DHA induced phenotypes and mechanisms in HepG2.2.15 cells. These results demonstrate that DHA might inhibit HepG2.2.15 cells proliferation through inducing cellular senescence and autophagy. [BMB Reports 2019; 52(8): 520-524].

Dihydroartemisinin inhibits HepG2.2.15 proliferation by inducing cellular senescence and autophagy.

The PI3K/mTOR dual inhibitor NVP-BEZ235 stimulates mutant p53 degradation to exert anti-tumor effects on triple-negative breast cancer cells.

Dihydroartemisinin (DHA), a derivate of artemisinin, is an effective antimalarial agent. DHA has been shown to exert anticancer activities to numerous cancer cells in the past few years, while the exact molecular mechanisms remain to be elucidated, especially in esophageal cancer. Crystal violet assay was conducted to determine the cell viability of human esophageal cancer cell line Eca109 treated with DHA. Tumor-bearing nude mice were employed to evaluate the anticancer effect of DHA in vivo. Soft agar and crystal violet assays were used to measure the tumorigenicity of Eca109 cells. Flow cytometry was performed to evaluate ROS or cell cycle distribution. GFP-LC3 plasmids were delivered into Eca109 cells to visualize autophagy induced by DHA under a fluorescence microscope. The mRNA and protein levels of each gene were tested by qRT-PCR and western blot, respectively. Our results proved that DHA significantly reduced the viability of Eca109 cells in a dose- and time-dependent manner. Further investigation showed that DHA evidently induced cell cycle arrest at the G2/M phase in Eca109 cells. Mechanistically, DHA induced intracellular ROS generation and autophagy in Eca109 cells, while blocking ROS by an antioxidant NAC obviously inhibited autophagy. Furthermore, we found that telomere shelterin component TRF2 was down-regulated in Eca109 cells exposed to DHA through autophagy-dependent degradation, which could be rescued after autophagy was blocked by ROS inhibition. Moreover, the DNA damage response (DDR) was induced obviously in DHA treated cells. To further explore whether ROS or autophagy played a vital role in DHA induced cell cycle arrest, the cell cycle distribution of Eca109 cells was evaluated after ROS or autophagy blocking, and the results showed that autophagy, but not ROS, was essential for cell cycle arrest in DHA treated cells. Taken together, DHA showed anticancer effect on esophageal cancer cells through autophagy-dependent cell cycle arrest at the G2/M phase, which unveiled a novel mechanism of DHA as a chemotherapeutic agent, and the degradation of TRF2 followed by DDR might be responsible for this cell phenotype.

/pdf/autophagy-dependent-cell-cycle-arrest-in-esophageal-cancer-2u3sgezelr.pdf

Autophagy-dependent cell cycle arrest in esophageal cancer cells exposed to dihydroartemisinin.

Esophageal carcinoma is a malignancy that severely threatens human health, with a high incidence rate and a low 5-year survival rate. Resistance to chemotherapy frequently emerges during its treatment, partly due to the induction of autophagy. Therefore, targeting autophagy may be a promising therapeutic approach for the treatment of esophageal carcinoma. In the present study, it was investigated how chloroquine (CQ) can influence the growth ability and biological behaviors of EC109 esophageal squamous carcinoma cells in vitro, as well as the potential molecular mechanisms behind its activity. It was demonstrated that CQ could suppress the growth and proliferation of EC109 cells in a time- and dose-dependent manner; migration and colony formation abilities were also inhibited by CQ. Furthermore, subsequent to the exposure to CQ, the number of autophagosomes was clearly increased in EC109 cells overexpressing green fluorescent protein tagged-light chain (LC)3 when observed by fluorescence microscopy. Protein expression of the endogenous autophagy markers LC3-II and p62 was elevated subsequent to CQ treatment, whereas the expression of proteins from the protein kinase B/mechanistic target of rapamycin target of rapamycin pathway was inhibited. This suggested that CQ could induce the formation of autophagosomes in the initiation of autophagy, but inhibit the degradation of autophagosomes in a later stage of autophagy. The overall effect was that autophagic cell death was activated by CQ, as confirmed by flow cytometry. Overall, the anticancer effect of chloroquine on EC109 was revealed to be mediated through modulating autophagy, and this may produce promising therapeutic benefits for esophageal carcinoma.

/pdf/chloroquine-affects-autophagy-to-achieve-an-anticancer-51ds9jtix9.pdf

Chloroquine affects autophagy to achieve an anticancer effect in EC109 esophageal carcinoma cells in vitro.

Objective: To elucidate the oncogenic role of human telomerase reverse transcriptase (hTERT) in esophageal squamous cancer and unravel the therapeutic role and molecular mechanism of dihydroartemisinin (DHA) by targeting hTERT.

Methods: The expression of hTERT in esophageal squamous cancer and the patients prognosis were analyzed by bioinformatic analysis from TCGA database, and further validated with esophageal squamous cancer tissues in our cohort. The Cell Counting Kit-8 (CCK8) and colony formation assay were used to evaluate the proliferation of esophageal squamous cancer cell lines (Eca109, KYSE150, and TE1) after hTERT overexpression or treated with indicated concentrations of DHA. Transwell migration assay and scratch assay were employed to determine the migration abilities of cancer cells. Fluorescence microscopy and flow cytometry were conducted to measure the intracellular reactive oxygen species (ROS) levels in cancer cells after treated with DHA. Moreover, RT-PCR and Western blot were performed to test the alteration of associated genes on mRNA and protein level in DHA treated esophageal squamous cancer cell lines, respectively. Furthermore, tumor-bearing nude mice were employed to evaluate the anticancer effect of DHA in vivo.

Results: We found that hTERT was significantly upregulated in esophageal squamous cancer both from TCGA database and our cohort also. Overexpression of hTERT evidently promoted the proliferation and migration of esophageal squamous cancer cells in vitro. Moreover, DHA could significantly inhibit the proliferation and migration of esophageal cancer cell lines Eca109, KYSE150, and TE1 in vitro, and significantly down-regulate the expression of hTERT on both mRNA and protein level in a time- and dose-dependent manner as well. Further studies showed that DHA could induce intracellular ROS production in esophageal cancer cells and down-regulate SP1 expression, a transcription factor that bound to the promoter region of hTERT gene. Moreover, overexpression of SP1 evidently promoted the proliferation and migration of Eca109 and TE1 cells. Intriguingly, rescue experiments showed that inhibiting ROS by NAC alleviated the downregulation of SP1 and hTERT in cells treated with DHA. Furthermore, overexpression of SP1 or hTERT could attenuate the inhibition effect of DHA on the proliferation and migration of Eca109 cells. In tumor-bearing nude mice model, DHA significantly inhibited the growth of esophageal squamous cancer xenografts, and downregulated the expression of SP1 and hTERT protein, while no side effects were observed from heart, kidney, liver, and lung tissues by HE stain.

Conclusion: hTERT plays an oncogenic role in esophageal squamous cancer and might be a therapeutic target of DHA through regulating ROS/SP1 pathway.

/pdf/human-telomerase-reverse-transcriptase-as-a-therapeutic-5ch66xw25z.pdf

Jiang Zou

Papers

Dihydroartemisinin inhibits HepG2.2.15 proliferation by inducing cellular senescence and autophagy.

The PI3K/mTOR dual inhibitor NVP-BEZ235 stimulates mutant p53 degradation to exert anti-tumor effects on triple-negative breast cancer cells.

Autophagy-dependent cell cycle arrest in esophageal cancer cells exposed to dihydroartemisinin.

Chloroquine affects autophagy to achieve an anticancer effect in EC109 esophageal carcinoma cells in vitro.

Human Telomerase Reverse Transcriptase as a Therapeutic Target of Dihydroartemisinin for Esophageal Squamous Cancer