Prashanth Gokare

Current treatment of early breast cancer: adjuvant and neoadjuvant therapy

Breast cancer is the most commonly diagnosed cancer in women. The latest world cancer statistics calculated by the International Agency for Research on Cancer (IARC) revealed that 1,677,000 women were diagnosed with breast cancer in 2012 and 577,000 died. The TNM classification of malignant tumor (TNM) is the most commonly used staging system for breast cancer. Breast cancer is a group of very heterogeneous diseases. The molecular subtype of breast cancer carries important predictive and prognostic values, and thus has been incorporated in the basic initial process of breast cancer assessment/diagnosis. Molecular subtypes of breast cancers are divided into human epidermal growth factor receptor 2 positive (HER2 +), hormone receptor positive (estrogen or progesterone +), both positive, and triple negative breast cancer. By virtue of early detection via mammogram, the majority of breast cancers in developed parts of world are diagnosed in the early stage of the disease. Early stage breast cancers can be completely resected by surgery. Over time however, the disease may come back even after complete resection, which has prompted the development of an adjuvant therapy. Surgery followed by adjuvant treatment has been the gold standard for breast cancer treatment for a long time. More recently, neoadjuvant treatment has been recognized as an important strategy in biomarker and target evaluation. It is clinically indicated for patients with large tumor size, high nodal involvement, an inflammatory component, or for those wish to preserve remnant breast tissue. Here we review the most up to date conventional and developing treatments for different subtypes of early stage breast cancer.

Sorafenib and Quinacrine Target Anti-Apoptotic Protein MCL1: A Poor Prognostic Marker in Anaplastic Thyroid Cancer (ATC).

Purpose and Experimental Design: Anaplastic thyroid cancer (ATC) comprises approximately 2% of all thyroid cancers, and its median survival rate remains poor. It is responsible for more than one third of thyroid cancer–related deaths. ATC is frequently resistant to conventional therapy, and NFκB signaling has been proposed to be a feature of the disease. We aimed to assess the activity of the antimalaria drug quinacrine known to target NFκB signaling in combination with the clinically relevant kinase inhibitor sorafenib in ATC cells. The presence of NFκB-p65/RELA and its target MCL1 was demonstrated in ATC by meta-data gene set enrichment analysis and IHC. We assessed the responses of a panel of human ATC cell lines to quinacrine and sorafenib in vitro and in vivo. Results: We detected increased expression of NFκB-p65/RELA and MCL1 in the nucleus of a subset of ATC compared with non-neoplastic thyroid. ATC cells were found to respond with additive/synergistic tumor cell killing to the combination of sorafenib plus quinacrine in vitro, and the drug combination improves survival of immunodeficient mice injected orthotopically with ATC cells as compared with mice administered either compound alone or doxorubicin. We also demonstrate that the combination of sorafenib and quinacrine is well tolerated in mice. At the molecular level, quinacrine and sorafenib inhibited expression of prosurvival MCL1, pSTAT3, and dampened NFκB signaling. Conclusions: The combination of quinacrine and sorafenib targets emerging molecular hallmarks of ATC and shows promising results in clinically relevant models for the disease. Further testing of sorafenib plus quinacrine can be conducted in ATC patients. Clin Cancer Res; 22(24); 6192–203. ©2016 AACR.

Application of 3D tumoroid systems to define immune and cytotoxic therapeutic responses based on tumoroid and tissue slice culture molecular signatures

We have developed 3D-tumoroids and tumor slice in vitro culture systems from surgical tumor specimens derived from patients with colorectal cancer (CRC) or lung cancer to evaluate immune cell populations infiltrating cultured tissues. The system incorporates patients peripherally and tumor-derived immune cells into tumoroid in vitro cultures to evaluate the ability of the culture to mimic an immunosuppressive tumor microenvironment (ITM). This system enables analysis of tumor response to standard therapy within weeks of surgical resection. Here we show that tumoroid cultures from a CRC patient are highly sensitive to the thymidylate synthase inhibitor 5-fluorouracil (adrucil) but less sensitive to the combination of nucleoside analog trifluridine and thymidine phosphorylase inhibitor tipiracil (Lonsurf). Moreover, re-introduction of isolated immune cells derived from surrounding and infiltrating tumor tissue as well as CD45+ tumor infiltrating hematopoietic cells displayed prolonged (>10 days) survival in co-culture. Established tumor slice cultures were found to contain both an outer epithelial and inner stromal cell compartment mimicking tumor structure in vivo. Collectively, these data suggest that, 3D-tumoroid and slice culture assays may provide a feasible in vitro approach to assess efficacy of novel therapeutics in the context of heterogeneous tumor-associated cell types including immune and non-transformed stromal cells. In addition, delineating the impact of therapeutics on immune cells, and cell types involved in therapeutic resistance mechanisms may be possible in general or for patient-specific responses.

Targeting of Chk2 as a countermeasure to dose-limiting toxicity triggered by topoisomerase-II (TOP2) poisons

The DNA damage response (DDR) gene cell cycle checkpoint kinase 2 (Chk2) triggers programmed cell death and lethal radiation-induced toxicity in mice in vivo. However, it is not well established to what extent targeting of Chk2 may protect from dose-limiting toxicities (DLT) inflicted by mainstay cancer chemotherapy. We screened different classes of chemotherapy in wild type and Chk2-deficient cells. Here we show that loss of Chk2 protect from cell death in vitro and lethal toxicity in vivo following treatment with topoisomerase II (TOP2)–inhibitors whereas no such protection was observed following treatment with topoisomerase I (TOP1) inhibitors. Furthermore, through combined in silico and functional screens of the Diversity Set II (NCI/NTP) chemical library we identified the carbanilide-derivative NSC105171, also known as ptu-23, as a novel Chk2 inhibitor (Chk2i). Indeed, NSC105171 can be administered safely to mice to countermeasure etoposide-induced toxicity. Incorporation of Chk2i into chemotherapy protocols employing TOP2-inhibitors may be an effective strategy to prevent DLTs without interfering with treatment.

Agonists of the TRAIL Death Receptor DR5 Sensitize Intestinal Stem Cells to Chemotherapy-Induced Cell Death and Trigger Gastrointestinal Toxicity

The combination of TRAIL death receptor agonists and radiochemotherapy to treat advanced cancers continues to be investigated in clinical trials. We previously showed that normal cells with a functional DNA damage response (DDR) upregulate the expression of death-inducing receptor DR5/TRAILR2/TNFRSF10B in a p53-dependent manner that sensitizes them to treatment with DR5 agonists. However, it is unclear if targeting DR5 selectively sensitizes cancer cells to agonist treatment following exposure to DNA-damaging chemotherapy, and to what extent normal tissues are targeted. Here, we show that the combined administration of the DR5 agonistic monoclonal antibody (mAb) and chemotherapy to wild-type mice triggered synergistic gastrointestinal toxicities (GIT) that were associated with the death of Lgr5(+) crypt base columnar stem cells in a p53- and DR5-dependent manner. Furthermore, we confirmed that normal human epithelial cells treated with the human DR5-agonistic mAb and chemotherapeutic agents were also greatly sensitized to cell death. Interestingly, our data also indicated that genetic or pharmacologic targeting of Chk2 may counteract GIT without negatively affecting the antitumor responses of combined DR5 agonist/chemotherapy treatment, further linking the DDR to TRAIL death receptor signaling in normal cells. In conclusion, the combination of DR5-targeting agonistic mAbs with DNA damaging chemotherapy may pose a risk of developing toxicity-induced conditions, and the effects of mAb-based strategies on the dose-limiting toxicity of chemotherapy must be considered when establishing new combination therapies.

Bcl-2 Protein Targeting by the p53/p21 Complex—Letter

Kim and colleagues reported a p53/p21 complex regulates cancer cell invasion and apoptosis by targeting the Bcl-2 family ([1][1]). Interaction of overexpressed p53 and p21 proteins in p53-null H1299 cells correlated with decreased cell invasion. No coimmunoprecipitation experiments documenting

Novel and Emerging Targeted Therapies of Colorectal Cancer

The survival rate of patients with colorectal cancer (CRC) is steadily increasing over the past decade. However, CRC continue to be one of the leading causes of cancer-related fatality in the United States. Current targeted strategies offer limited clinical benefits and the overall survival rate for CRC remains low. Improved understanding of the molecular changes associated with CRC that control growth factor signaling and evasion of cell death allow for the development of improved targeted therapy. This review aims to discuss some of the emerging therapies aimed to target CRC.

PIGN gene expression aberration is associated with genomic instability and leukemic progression in acute myeloid leukemia with myelodysplastic features

Previous studies have linked increased frequency of glycosylphosphatidylinositol-anchor protein (GPI-AP) deficiency with genomic instability and the risk of carcinogenesis. However, the underlying mechanism is still not clear. A randomForest analysis of the gene expression array data from 55 MDS patients (GSE4619) demonstrated a significant (p = 0.0007) correlation (Pearson r =-0.4068) between GPI-anchor biosynthesis gene expression and genomic instability, in which PIGN, a gene participating in GPI-AP biosynthesis, was ranked as the third most important in predicting risk of MDS progression. Furthermore, we observed that PIGN gene expression aberrations (increased transcriptional activity but diminished to no protein production) were associated with increased frequency of GPI-AP deficiency in leukemic cells during leukemic transformation/progression. PIGN gene expression aberrations were attributed to partial intron retentions between exons 14 and 15 resulting in frameshifts and premature termination which were confirmed by examining the RNA-seq data from a group of AML patients (phs001027.v1.p1). PIGN gene expression aberration correlated with the elevation of genomic instability marker expression that was independent of the TP53 regulatory pathway. Suppression/elimination of PIGN protein expression caused a similar pattern of genomic instability that was rescued by PIGN restoration. Finally, we found that PIGN bound to the spindle assembly checkpoint protein, MAD1, and regulated its expression during the cell cycle. In conclusion, PIGN gene is crucial in regulating mitotic integrity to maintain chromosomal stability and prevents leukemic transformation/progression.

MMR-deficiency and BRCA2/EGFR/NTRK mutations

Colorectal cancer (CRC) is the third most common cancer type in the developed world and a leading cause of cancer-related death. When detected at early stages, surgical interventions can lead to cure (92%, Stage I), and there is a better prognosis in Stages II and III as compared to stage IV [1]. Yet, molecular heterogeneity of tumors in early stage cancers may lead to variable clinical outcomes. This in general remains an area of active investigation in search of prognostic and predictive factors. There are some prognostic factors in stage II disease that confer high risk including tumor size, lymphovascular or perineural invasion, obstruction or perforation, and in stage III disease the spread to a majority of the lymph nodes examined is associated with worse outcomes. While most of CRCs arise via genetic changes that turn on different driver genes; a small fraction of about ~15% of cancers arise as a consequence of microsatellite instability (MSI) [2]. MSI-High (MSI-H) status of a CRC is the signature of a deficient or impaired DNA mismatch repair (dMMR) system in tumor cells and encompasses insertions or deletions in form short tandem repeats or “microsatellites” distributed in the genome as well as an increased mutation frequency. Most MSI-H CRC tumors arise due to the somatic inactivation of MLH1, MSH2, MSH6 and PSM2 genes. MSI-H CRCs also harbor mutations and genetic alterations in marker genes such as BRAF, MRE11A and TGF-β type II receptors [3] which may provide tailored treatment options for MSI-H patients, who otherwise poorly respond to adjuvant 5-FU chemotherapy. In a recent study, we [4] identified BRCA2, EGFR and NRTK mutations to be strongly associated with the MSI-H phenotype in CRC. The novel findings shed light on potentially actionable therapeutic vulnerabilities in MSI-H colorectal cancer. Using a combination of bioinformatics and predictive modelling approaches, we showed that a large fraction (~42%) of stage II MSI-H CRC patients have a higher frequency of BRCA2 mutations as compared to 6% of non-MSI-H patients. We characterized BRCA2 somatic mutations and found 75 unique mutations in 46% of MSI-H CRC patients, with the majority of the mutations being missense. Of note, the N-terminal and C-terminal of BRCA2 showed frequent mutations in the MSI-H CRC cohort. While C-terminal mutations affect the Editorial

Abstract PR03: P53 inhibits the expression of the pyrimidine catabolic gene Dihydropyrimidine dehydrogenase (DPYD)

Fluorouracil (5-FU) a widely used chemotherapeutic drug whose unpredictable pharmacokinetics is controlled by the pyrimidine catabolic gene dihydropyrimidine dehydrogenase (DPYD), that has recently also been shown to be a gatekeeper of the epithelial-to-mesenchymal transition (EMT) in breast cancer. Relatively little is known about the transcriptional control of DPYD and here we show for the first time an interaction between p53 and DPYD (involved in catabolism of pyrimidines as well as 5-FU) where p53 represses both the base-line expression of DPYD and that following 5-FU administration in vitro and in vivo. This mechanism affects the catabolic conversion of 5-FU to 5-FUH2 in mice in vivo. Using an in-silico approach we also identified several putative p53 binding sites (P53DBS) in and around ~20Kb upstream and downstream of the mouse DPYD gene. In-vivo ChIP from mice livers identified a key p53DBS binding site downstream (chr3: 119451237-11941257) of the gene to which p53 binds to at about 1.8 ± 0.05 fold over untreated control following a single IV bolus of 5-FU (150 mg/kg bw). Interestingly DPYD mRNA and protein levels were decreased by 1.8 and 1.5 fold respectively (P Citation Format: Prashanth Ravishankar Gokare, Niklas Finnberg, Jenny Dai, Wafik El-Deiry. P53 inhibits the expression of the pyrimidine catabolic gene Dihydropyrimidine dehydrogenase (DPYD). [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr PR03.