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Aims and Objective: This study aimed to compare hormone receptor (HR) status before and after neoadjuvant chemotherapy that is discordance in locally advanced breast cancer patients, which are amenable for surgery. The secondary objective was to study the correlation between tumor response and HR expression., Materials and Methods: The duration of the study was from August 2018 to December 2020. A total of 23 patients were selected as per certain inclusion criteria. American Society of Clinical Oncologys methodology was used to analyze estrogen receptor (ER) and progesterone receptor (PR) status of histopathology specimen. For study purposes, patients were classified into four groups after core biopsy of breast lump and after definitive surgery (post-NACT (neoadjuvant chemotherapy)) - Group A (ER+, PR+), Group B (ER+, PR-), Group C (ER-, PR+), and Group D (ER-, PR-)., Results: ER discordance was found to be (2/23) 8.69% (P value 0.76). PR discordance was (4/23) 17.39%. PR discordance was found to be higher than ER discordance. Changes in staining patterns in ERs were seen in 14 patients (93.33%). Changes in staining percentage in PRs were seen in eight patients (80%). It was found that both receptor-positive and negative diseases had an equal proportion of stable disease., Conclusion: From the study, it is noted that performing ER PR study twice (before and after chemotherapy) is necessary as discordance is noted and this may impact the further treatment strategy., Competing Interests: None

The overexpression and amplification of the protooncogene neu (ERBB2) play an important role in the development of aggressive breast cancer (BC) in humans. Ral-interacting protein (RLIP), a modular stress-response protein with pleiotropic functions, is overexpressed in several types of cancer, including BC. Here, we show that blocking RLIP attenuates the deleterious effects caused by the loss of the tumor suppressor p53 and inhibits the growth of human BC both in vitro and in vivo in MMTV-neu mice. In addition, we show that treatment with the diet-derived, RLIP-targeting chemotherapeutic 2'-hydroxyflavanone (2HF), alone or in combination with RLIP-specific antisense RNA or antibodies, significantly reduced the cumulative incidence and/or burden of mammary hyperplasia and carcinoma in MMTV-neu mice. 2HF treatment correlated with reduced tumor cell proliferation and increased apoptosis, and the average number of Ki67-positive (proliferating) cells was significantly lower in the tumors of 2HF-treated mice than in the tumors of control mice. Furthermore, targeting RLIP also resulted in the overexpression of E-cadherin and the infiltration of CD3 + T cells into mammary tumors. Taken together, these results underscore the translational potential of RLIP-targeting agents and provide a strong rationale to validate them in the clinic., (© 2021 Wiley Periodicals LLC.)

Breast cancer (BC) remains one of the major causes of cancer deaths in women. Over half of all BCs carry genetic defects in the gene encoding p53, a powerful tumor suppressor. P53 is known as the 'guardian of the genome' because it is essential for regulating cell division and preventing tumor formation. Ral-interacting protein (RLIP) is a modular protein capable of participating in many cellular functions. Blocking this stress-responsive protein, which is overexpressed during malignancy, enables BC cells to overcome the deleterious effects of p53 loss more effectively. In the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas9) system, a single-guide RNA (sgRNA) recognizes a specific DNA sequence and directs the endonuclease Cas9 to make a double-strand break, which enables editing of targeted genes. Here, we harnessed CRISPR/Cas9 technology to target the RLIP gene in BC cells. We screened sgRNAs using a reporter system and lentivirally delivered them, along with Cas9, to BC cells for validation. We then assessed the survival, proliferation, and tumorigenicity of BC cells in vitro and the growth of tumors in vivo after CRISPR-mediated knockdown of RLIP. Doxycycline-inducible expression of Cas9 in BC cells transduced with lentiviral vectors encoding the sgRNAs disrupted the RLIP gene, leading to inhibition of BC cell proliferation both in vitro and in vivo, with resected tumors showing reduced levels of the survival and proliferation markers Ki67, RLIP, pAkt, and survivin, the cell cycle protein CDK4, and the mesenchymal marker vimentin, as well as elevated levels of the differentiation protein E-cadherin and pro-apoptotic protein Bim. Inducible Cas9/sgRNA-transduced BC cells without doxycycline treatment did not exhibit altered cell survival or proliferation in vitro or in vivo. Our study provides proof-of-concept that the CRISPR/Cas9 system can be utilized to target RLIP in vitro and in vivo., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

SRY-related high-mobility group box 9 (SOX9) is an indispensable transcription factor that regulates multiple developmental pathways related to stemness, differentiation, and progenitor development. Previous studies have demonstrated that the SOX9 protein directs pathways involved in tumor initiation, proliferation, migration, chemoresistance, and stem cell maintenance, thereby regulating tumorigenesis as an oncogene. SOX9 overexpression is a frequent event in breast cancer (BC) subtypes. Of note, the molecular mechanisms and functional regulation underlying SOX9 upregulation during BC progression are still being uncovered. The focus of this review is to appraise recent advances regarding the involvement of SOX9 in BC pathogenesis. First, we provide a general overview of SOX9 structure and function, as well as its involvement in various kinds of cancer. Next, we discuss pathways of SOX9 regulation, particularly its miRNA-mediated regulation, in BC. Finally, we describe the involvement of SOX9 in BC pathogenesis via its regulation of pathways involved in regulating cancer hallmarks, as well as its clinical and therapeutic importance. In general, this review article aims to serve as an ample source of knowledge on the involvement of SOX9 in BC progression. Targeting SOX9 activity may improve therapeutic strategies to treat BC, but precisely inhibiting SOX9 using drugs and/or small peptides remains a huge challenge for forthcoming cancer research., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The Notch signaling pathway, which is highly conserved from sea urchins to humans, plays an important role in cell-differentiation, survival, proliferation, stem-cell renewal, and determining cell fate during development and morphogenesis. It is well established that signaling pathways are dysregulated in a wide-range of diseases, including human malignancies. Studies suggest that the dysregulation of the Notch pathway contributes to carcinogenesis, cancer stem cell renewal, angiogenesis, and chemo-resistance. Elevated levels of Notch receptors and ligands have been associated with cancer-progression and poor survival. Furthermore, the Notch signaling pathway regulates the transcriptional activity of key target genes through crosstalk with several other signaling pathways. Indeed, increasing evidence suggests that the Notch signaling pathway may serve as a therapeutic target for the treatment of several cancers, including breast cancer. Researchers have demonstrated the anti-tumor properties of Notch inhibitors in various cancer types. Currently, Notch inhibitors are being evaluated for anticancer efficacy in a number of clinical-trials. However, because there are multiple Notch receptors that can exhibit either oncogenic or tumor-suppressing roles in various cells, it is important that the Notch inhibitors are specific to particular receptors that are tumorigenic in nature. This review critically evaluates existing Notch inhibitory drugs and strategies and summarizes the previous discoveries, current understandings, and recent developments in support of Notch receptors as therapeutic targets in breast cancer., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Breast tumor metastasis is a leading cause of cancer-related deaths worldwide. Breast cancer (BC) cells frequently metastasize to the lungs, where they pose a formidable therapeutic challenge. In the current study, we evaluated the anti-proliferative and anti-metastatic effects of 2'-hydroxyflavanone (2HF) and RLIP inhibition in an array of triple-negative BC cell lines and an orthotopic mouse model of breast-to-lung metastasis. Compared to control treatment, RLIP inhibition reduced in-vitro cell viability and suppressed the migratory and invasive potential of BC cells. In-vitro studies showed that 2HF treatment reduced the expression of RLIP, KRAS, pERK, pSTAT3, and pP70S6K. Further, mice orthotopically implanted with lung-seeking luciferase-expressing TMD231 cells were treated with 2HF (50 mg/kg, b.w.), RLIP antisense (RAS; 5 mg/kg, b.w.), RLIP antibody (Rab; 5 mg/kg, b.w.) or a combination of 2HF + RAS + Rab. 2HF-, RAS-, and Rab-treated mice exhibited significantly lower primary tumor weight and reduced lung metastasis compared to control mice. Mice treated with a combination of 2HF + RAS + Rab exhibited no metastasis and significantly lower tumor weight than the single agent-treated mice. Collectively, our results suggest that 2HF has potential to be combined with RLIP inhibition/depletion to more effectively suppress primary breast tumor growth and metastasis to the lungs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Brain metastasis is an important cause of morbidity and mortality in cancer-patients. Breast tumor cells frequently metastasize to brain and initiate severe therapeutic complications. In the present study, we evaluated the anti-metastatic effects of 2'-hydroxyflavanone (2HF) alone and in combination with RLIP targeted therapy in a novel murine model of breast tumor metastasis. The MDA-MB231Br (brain-seeking) breast cancer (BC) cells stably-transfected with luciferase were injected into the left-ventricle of NSG mouse heart and the migration of cells to brain was monitored using a non-invasive bioluminescent imaging system. To evaluate the tumor growth suppressive effects, mice were given 2HF (50 mg/kg, b.w., alternate days orally), RLIP-antibody (Rab; 5 mg/kg, b.w., weekly i.p.) or combination of 2HF+Rab starting day1 after intra-cardiac injection. Our results reveal that 2HF and Rab significantly prevented the metastasis of BC cells to brain. Further, mice treated with combination of 2HF+Rab exhibited no metastasis as compared to either or the single agent-treated mice. This study for the first time demonstrates the anti-metastatic effects of 2HF and RLIP-inhibition in-vivo in a novel breast tumor metastasis model and provides the rationale for further clinical investigation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Consumption of citrus-fruits is associated with reduced incidence of breast cancer (BC), the most common cancer diagnosed in women across the globe. In this study, we investigated the anticancer potential of 2-Hydroxyflavanone (2HF) in BC. 2HF, a citrus-bioflavonoid, has demonstrated anticancer properties in various cancers, but its anticancer role in BC has not been well studied. We investigated the in vitro and in vivo growth inhibitory effects of 2HF in an array of BC lines and in xenograft mouse models of ER-positive and HER2-positive BC cells. Compared to control, 2HF treatment reduced cell viability and suppressed migratory and invasive potential of BC cells, while, no growth inhibitory effects were observed in non-tumorigenic breast epithelial cells. Further, 2HF inhibited the expression of RLIP76, a stress-defensive and anti-apoptotic protein, which is over-expressed in BC cells and simultaneously reduced proliferation of BC cells. Nude mice bearing MCF7 or SKBR3 BC cells xenografts treated with either 2HF or targeting RLIP76 by RLIP76-antisense or RLIP76-antibody treatment had significantly lower tumor-weight as compared to corresponding controls. In addition, Western-blotting and immunohistochemical analysis of tumor tissue from control and treatment group mice showed that 2HF decreased protein expression levels of RLIP76, and the decrease was similar to those seen following RLIP76-antisense treatment. Furthermore, 2HF decreased expression of Ki67, CD31, vimentin, inhibited phosphorylation of Akt and expression of survivin and Bcl2, and increased levels of Bax, E-cadherin, and cleaved-PARP. Therefore, our results indicate that 2HF may suppress BC growth in vitro and in vivo by targeting RLIP76, and may serve as a potential adjuvant treatment in BC patients., (© 2018 Wiley Periodicals, Inc.)

Breast cancer (BC) prevention and therapy in the context of life-style risk factors and biological drivers is a major focus of developmental therapeutics in oncology. Obesity, alcohol, chronic estrogen signaling and smoking have distinct BC precipitating and facilitating effects that may act alone or in combination. A spectrum of signaling events including enhanced oxidative stress and changes in estrogen-receptor (ER)-dependent and -independent signaling drive the progression of BC. Breast tumors modulate ERα/ERβ ratio, upregulate proliferative pathways driven by ERα and HER2 with a parallel loss and/or downregulation of tumor suppressors such as TP53 and PTEN which together impact the efficacy of therapeutic strategies and frequently lead to emergence of drug resistance. Natural phytochemicals modulate oxidative stress, leptin, integrin, HER2, MAPK, ERK, Wnt/β-catenin and NFκB signaling along with regulating ERα and ERβ, thereby presenting unique opportunities for both primary and combinatorial interventions in BC. In this regard, this article focuses on critical analyses of the evidence from multiple studies on the efficacy of natural phytochemicals in BC. In addition, areas in which the combinations of such effective natural phytochemicals with approved and/or developing anticancer agents can be translationally beneficial are discussed to derive evidence-based inference for addressing challenges in BC control and therapy., (© 2017 UICC.)

RALBP1 (RLIP76) is the major transporter of doxorubicin (DOX) in lung cancer cells, and that the difference in sensitivity of small cell lung cancer (SCLC) cells to DOX is due to differential phosphorylation by PKCalpha. Our recent studies have suggested that RALBP1 present in MCF-7 breast cancer cells has significantly lower specific activity for transport of DOX than wild-type recombinant protein, and its level of expression is significantly lower than that in lung cancer cells. In the present study, we have explored whether or not this is a generalized phenomenon for breast cancer, and have compared the relative contributions of RALBP1 and the ABC-family transporter, ABCG2 to total DOX transport activities in two SCLC (H1417 and H1618), two non-small cell lung cancer (NSCLC) (H358 and H520), and three breast cancer (T-47D, MDA-MB231, and MCF-7) cell lines. Results of these studies show lower protein expression and specific activity of RALBP1 in all three breast cancer cell lines as compared with lung cancer cell lines. Furthermore, we demonstrate that RALBP1 contributes only a minor fraction of DOX transport activity in breast cancer cell lines, suggesting that greater DOX sensitivity of breast cancer may be related to lower RALBP1 transporter activity and that the transport mechanisms involved in multidrug resistance of lung and breast cancer are distinct.