Pan-inhibition of the three H2S synthesizing enzymes restrains tumor progression and immunosuppression in breast cancer.

BACKGROUND: Hydrogen sulfide (H2S) is a significant endogenous mediator that has been implicated in the progression of various forms of cancer including breast cancer (BC). Cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST) are the three principal mammalian enzymes responsible for H2S production. Overexpression of CBS, CSE and 3MST was found to be associated with poor prognosis of BC patients. Moreover, H2S was linked to an immune-suppressive tumor microenvironment in BC. Recently it was observed that BC cells, in response to single or dual inhibition of H2S synthesizing enzymes, develop an escape mechanism by overexpressing alternative sources of H2S generation. Thus, the aim of this work is to escape the H2S compensatory mechanism by pan repressing the three enzymes using microRNAs (miRNAs) and to investigate their impact on the oncogenic and immunogenic profile of BC cells. METHODS: BC female patients (n = 25) were recruited. In-silico analysis was used to identify miRNAs targeting CBS, CSE, and 3MST. MDA-MB-231 cells were cultured and transfected using oligonucleotides. Total RNA was extracted using Biazol, reverse transcribed and quantified using qRT-PCR. H2S levels were measured using AzMc assay. BC hallmarks were assessed using trans-well migration, wound healing, MTT, and colony forming assays. RESULTS: miR-193a and miR-548c were validated by eight different bioinformatics software to simultaneously target CBS, CSE and 3MST. MiR-193a and miR-548c were significantly downregulated in BC tissues compared to their non-cancerous counterparts. Ectopic expression of miR-193a and miR-548c in MDA-MB-231 TNBC cells resulted in a marked repression of CBS, CSE, and 3MST transcript and protein levels, a significant decrease in H2S levels, reduction in cellular viability, inhibition of migration and colony forming ability, repression of immune-suppressor proteins GAL3 GAL9, and CD155 and upregulation of the immunostimulatory MICA and MICB proteins. CONCLUSION: This study sheds the light onto miR-193a and miR-548c as potential pan-repressors of the H2S synthesizing enzymes. and identifies them as novel tumor suppressor and immunomodulatory miRNAs in TNBC.

Dual targeting of H2S synthesizing enzymes; cystathionine ß-synthase and cystathionine γ-lyase by miR-939-5p effectively curbs triple negative breast cancer.

Introduction: Hydrogen sulfide (H2S) has been recently scrutinized for its critical role in aggravating breast cancer (BC) tumorigenicity. Several cancers aberrantly express H2S synthesizing enzymes; Cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE). However, their levels and interdependence in BC require further studies. Objectives: Firstly, this study aimed to demonstrate a comparative expression profile of H2S synthesizing enzymes in BC vs normal tissue. Moreover, to investigate the reciprocal relationship between CBS and CSE and highlight the importance of dual targeting. Finally, to search for a valid dual repressor of the H2S synthesizing enzymes that could cease H2S production and reduce TNBC pathogenicity. Methods: Pairwise analysis of tumor vs. normal tissues of 40 BC patients was carried out. The TNBC cell line MDA-MB-231 was transfected with oligonucleotides to study the H2S mediated molecular mechanisms. In silico screening was performed to identify dual regulator(s) for CBS and CSE. Gene expression analysis was performed using qRT-PCR and was confirmed on protein level using Western blot. TNBC hallmarks were evaluated using MTT, migration, and clonogenicity assays. H2S levels were detected using a AzMc fluorescent probe. Results: BC tissues exhibited elevated levels of both CBS and CSE. Interestingly, upon CBS knockdown, CSE levels increased compensating for H2S production in TNBC cells, underlining the importance of dually targeting both enzymes in TNBC. In silico screening suggested miR-939-5p as a regulator of both CBS and CSE with high binding scores. Low expression levels of miR-939-5p were found in BC tissues, especially the aggressive subtypes. Ectopic expression of miR-939-5p significantly repressed CBS and CSE transcript and protein levels, diminished H2S production and attenuated TNBC hallmarks. Moreover, it improved the immune surveillance potency of TNBC cells through up regulating the NKG2D ligands, MICB and ULBP2 and reducing the immune suppressive cytokine IL-10. Conclusion: This study sheds light on the reciprocal relationship between CBS and CSE and on the importance of their dual targeting, particularly in TNBC. It also postulates miR-939-5p as a potent dual repressor for CBS and CSE overcoming their redundancy in H2S production, a mechanism that can potentially attenuate TNBC oncogenicity and improves the immunogenic response.

Let-7a/cMyc/CCAT1/miR-17-5p Circuit Re-sensitizes Atezolizumab Resistance in Triple Negative Breast Cancer through Modulating PD-L1.

BACKGROUND: Triple negative breast cancer (TNBC) is an immunogenically hot tumor. The immune checkpoint blockades (ICBs) have been recently emerged as promising therapeutic candidates for several malignancies including TNBC. Yet, the development of innate and/or adaptive resistance by TNBC patients towards ICBs such as programmed death-ligand 1 (PD-L1) inhibitors (e.g. Atezolizumab) shed the light on importance of identifying the underlying mechanisms regulating PD-L1 in TNBC. Recently, it was reported that non-coding RNAs (ncRNAs) perform a fundamental role in regulating PD-L1 expression in TNBC. Hence, this study aims to explore a novel ncRNA axis tuning PD-L1 in TNBC patients and investigate its possible involvement in fighting Atezolizumab resistance. METHODS: In-silico screening was executed to identify ncRNAs that could potentially target PD-L1. Screening of PD-L1 and the nominated ncRNAs (miR-17-5p, let-7a and CCAT1 lncRNA) was performed in BC patients and cell lines. Ectopic expression and/or knockdown of respective ncRNAs were performed in MDA-MB-231. Cellular viability, migration and clonogenic capacities were evaluated using MTT, scratch assay and colony-forming assay, respectively. RESULTS: PD-L1 was upregulated in BC patients, especially in TNBC patients. PD-L1 is positively associated with lymph node metastasis and high Ki-67 in recruited BC patients. Let-7a and miR-17-5p were nominated as potential regulators of PD-L1. Ectopic expression of let-7a and miR-17-5p caused a noticeable reduction in PD-L1 levels in TNBC cells. In order to investigate the whole ceRNA circuit regulating PD-L1 in TNBC, intensive bioinformatic studies were performed. The lncRNA, Colon Cancer-associated transcript 1 (CCAT1), was reported to target PD-L1 regulating miRNAs. Results showed that CCAT1 is an upregulated oncogenic lncRNA in TNBC patients and cell lines. CCAT1 siRNAs induced a noticeable reduction in PD-L1 levels and a marked increase in miR-17-5p level, building up a novel regulatory axis CCAT1/miR-17-5p/PD-L1 in TNBC cells that was tuned by the let-7a/c-Myc engine. On the functional level, co-treatment of CCAT-1 siRNAs and let-7a mimics efficiently relieved Atezolizumab resistance in MDA-MB-231 cells. CONCLUSION: The present study revealed a novel PD-L1 regulatory axis via targeting let-7a/c-Myc/CCAT/miR-17-5p. Additionally, it sheds the light on the potential combinational role of CCAT-1 siRNAs and Let-7a mimics in relieving Atezolizumab resistance in TNBC patients.

MALAT-1: Immunomodulatory lncRNA hampering the innate and the adaptive immune arms in triple negative breast cancer.

BACKGROUND: Triple negative breast cancer (TNBC) is known as hot immunogenic tumor. Yet, it is one of the most aggressive BC subtypes. TNBC evolve several tactics to evade the immune surveillance phenomena, one of which is shedding of natural killer (NK) cells activating immune ligands such as MICA/B and/or by inducing the expression of the immune checkpoints such as PD-L1 and B7-H4. MALAT-1 is an oncogenic lncRNA. MALAT-1 immunogenic profile is not well investigated. AIM: The study aims at exploring the immunogenic role of MALAT-1 in TNBC patients and cell lines and to identify its molecular mechanism in altering both innate and adaptive immune cells present at the tumor microenvironment of TNBC METHODS: BC patients (n = 35) were recruited. Primary NK cells and cytotoxic T lymphocytes were isolated from normal individuals using the negative selection method. MDA-MB-231 cells were cultured and transfected by several oligonucleotides by lipofection technique. Screening of ncRNAs was performed using q-RT-PCR. Immunological functional analysis experiments were performed upon co-culturing primary natural killer cells and cytotoxic T lymphocytes using LDH assay. Bioinformatics analysis was performed to identify potential microRNAs targeted by MALAT-1. RESULTS: MALAT-1 expression was significantly upregulated in BC patinets with a profound expression in TNBC patients compared to their normal counterparts. Correlation analysis revealed a positive correlation between MALAT-1, tumor size and lymph node metastasis. Knocking down of MALAT-1 in MDA-MB-231 cells resulted in a significant induction of MICA/B, repression of PD-L1 and B7-H4 expression levels. Enhancement of cytotoxic activity of co-cultured NK and CD8+ cells with MALAT-1 siRNAs transfected MDA-MB-231 cells. In silico analysis revealed that miR-34a and miR-17-5p are potential targets to MALAT-1; accordingly, they were found to be downregulated in BC patients. Forcing the expression of miR-34a in MDA-MB-231 cells resulted in a significant induction in MICA/B levels. Ectopic expression of miR-17-5p in MDA-MB-231 cells significantly repressed the expression of PD-L1 and B7-H4 checkpoints. Validations of MALAT-1/miR-34a" and "MALAT-1/miR-17-5p axes were performed by a series of co-transfections and functional assessment of cytotoxic profile of primary immune cells. CONCLUSION: This study proposes a novel epigenetic alteration exerted by TNBC cells mainly by inducing the expression of MALAT-1 lncRNA. MALAT-1 mediates innate and adaptive immune suppression events partially via targeting miR-34a/MICA/B and miR-175p/PD-L1/B7-H4 axes in TNBC patients and cell lines.

Expression of GPR68, an Acid-Sensing Orphan G Protein-Coupled Receptor, in Breast Cancer.

Breast cancer (BC) is the most diagnosed cancer and the leading cause of global cancer incidence in 2020. It is quite known that highly invasive cancers have disrupted metabolism that leads to the creation of an acidic tumor microenvironment. Among the proton-sensing G protein-coupled receptors is GPR68. In this study, we aimed to explore the expression pattern of GPR68 in tissues from BC patients as well as different BC cell lines. METHODS: In-silico tools were used to assess the expression of GPR68 in BC patients. The expression pattern was validated in fresh and paraffin-embedded sections of BC patients using qPCR and immunohistochemistry (IHC), respectively. Also, in-silico tools investigated GPR68 expression in different BC cell lines. Validation of GPR68 expression was performed using qPCR and immunofluorescence techniques in four different BC cell lines (MCF-7, MDA-MB-231, BT-549 and SkBr3). RESULTS: GPR68 expression was found to be significantly increased in BC patients using the in-silico tools and validation using qPCR and IHC. Upon classification according to the molecular subtypes, the luminal subtype showed the highest GPR68 expression followed by triple-negative and Her2-enriched cells. However, upon validation in the recruited cohort, the triple-negative molecular subtype of BC patients showed the highest GPR68 expression. Also, in-silico and validation data revealed that the triple-negative breast cancer cell line MDA-MB-231 showed the highest expression of GPR68. CONCLUSION: Therefore, this study highlights the potential utilization of GPR68 as a possible diagnostic and/or prognostic marker in BC.

Lateral chest wall perforator flaps in partial breast reconstruction.

BACKGROUND: Breast conserving surgery (BCS) has been a standard procedure for the treatment of breast cancer instead of mastectomy whenever possible. Lateral chest wall perforator flaps are one of the volume replacement techniques that participate in increasing the rate of BCS especially in small- to moderate-sized breasts with good cosmetic outcome. In this study, we tried to evaluate the outcome of those flaps as an oncoplastic procedure instead of the conventional flaps. METHODS: This study included 26 patients who underwent partial mastectomy with immediate reconstruction using lateral chest wall perforator flaps in the period from October 2019 to November 2020. The operative time, techniques, and complications were recorded. The cosmetic outcome was assessed 3 months post-radiation therapy through a questionnaire and photographic assessment. RESULTS: Lateral intercostal artery perforator (LICAP), lateral thoracic artery perforator (LTAP) and combined flaps were performed in 24, 1, and 1 patients, respectively. The mean operative time was 129.6 ± 13.2 min. The flap length ranged from 10 to 20 cm and its width from 5 to 9 cm. Overall patients' satisfaction was observed to be 88.5% as either excellent or good and the photographic assessment was 96.2% as either excellent or good. CONCLUSIONS: Lateral chest wall perforator flaps are reliable and safe option for partial breast reconstruction with an acceptable aesthetic outcome. In the era of oncoplastic breast surgery, they deserve to gain attention especially with the advantages of some modifications added to the classic technique.

Tumor Mutation Burden Prediction Model in Egyptian Breast Cancer patients based on Next Generation Sequencing.

OBJECTIVES: This study aimed to identify the tumor mutation burden (TMB) value in Egyptian breast cancer (BC) patients. Moreover, to find the best TMB prediction model based on the expression of estrogen (ER), progesterone (PR), human epidermal growth factor receptor 2 (HER-2), and proliferation index Ki-67. METHODS: The Ion AmpliSeq Comprehensive Cancer Panel was used to determine TMB value of 58 Egyptian BC tumor tissues. Different machine learning models were used to select the optimal classification model for prediction of TMB level according to patient's receptor status. RESULTS: The measured TMB value was between 0 and 8.12/Mb. Positive expression of ER and PR was significantly associated with TMB ≤ 1.25 [(OR =0.35, 95% CI: 0.04-2.98), (OR = 0.17, 95% CI= 0.02-0.44)] respectively. Ki-67 expression positive was significantly associated with TMB >1.25 than those who were Ki-67 expression negative (OR = 9.33, 95% CI= 2.07-42.18). However, no significant differences were observed between HER2 positive and HER2 negative groups. The optimized logistic regression model was TMB = -27.5 -1.82 ER - 0.73 PR + 0.826 HER2 + 2.08 Ki-67. CONCLUSION: Our findings revealed that TMB value can be predicted based on the expression level of ER, PR, HER-2, and Ki-67.

The implications of a pathological complete response of the primary tumour after neoadjuvant chemotherapy for breast cancer on axillary surgery.

BACKGROUND: Management of the node-positive axilla after neoadjuvant chemotherapy is controversial. The aim of this study is to predict the group of patients who may require a less invasive approach for axillary management. One possible group are patients with pathological complete response of the primary after chemotherapy. RESULTS: A unicentral retrospective cohort study including all breast cancer patients with axillary node metastases at presentation who received neoadjuvant chemotherapy resulting in pathological complete response. Pathological complete response in the axillary lymph nodes was recorded. A correlation between the response in the primary tumour and the lymph nodes was assessed. A subgroup analysis was conducted for different biological groups. Complete response was seen in the axillary nodes in 80.5% of patients. Patients with lobular cancer were less likely to show a similar response in the axilla as the primary tumour (p = 0.077). A higher incidence of axillary response was observed in HER2-positive tumours (p = 0.082). All patients with grade 3 tumours achieved complete response in the axilla (p = 0.094). Patients with negative or weak positive hormone receptor status had a significantly higher rate of complete response in the axilla compared to strongly positive hormone receptor status (OR, 7.8; 95% CI, 1.7-34.5; p = 0.007). CONCLUSION: A less invasive axillary surgery may be safely recommended in selected group of node-positive patients after neoadjuvant chemotherapy when the primary tumour shows complete response. This group may include HER2-positive, ER-negative and grade 3 tumours. Less response is expected in ER-positive and lobular carcinoma even with complete response in the primary.

Skin sparing mastectomy: technique and suggested methods of reconstruction.

AIM: To demonstrate the feasibility and accessibility of performing adequate mastectomy to extirpate the breast tissue, along with en-block formal axillary dissection performed from within the same incision. We also compared different methods of immediate breast reconstruction used to fill the skin envelope to achieve the best aesthetic results. METHODS: 38 patients with breast cancer underwent skin-sparing mastectomy with formal axillary clearance, through a circum-areolar incision. Immediate breast reconstruction was performed using different techniques to fill in the skin envelope. Two reconstruction groups were assigned; group 1: Autologus tissue transfer only (n=24), and group 2: implant augmentation (n=14). AUTOLOGUS TISSUE TRANSFER: The techniques used included filling in the skin envelope using Extended Latissimus Dorsi flap (18 patients) and Pedicled TRAM flap (6 patients). AUGMENTATION WITH IMPLANTS: Subpectoral implants(4 patients), a rounded implant placed under the pectoralis major muscle to augment an LD reconstructed breast. LD pocket (10 patients), an anatomical implant placed over the pectoralis major muscle within a pocket created by the LD flap. No contra-lateral procedure was performed in any of the cases to achieve symmetry. RESULTS: All cases underwent adequate excision of the breast tissue along with en-block complete axillary clearance (when indicated), without the need for an additional axillary incision. Eighteen patients underwent reconstruction using extended LD flaps only, six had TRAM flaps, four had augmentation using implants placed below the pectoralis muscle along with LD flaps, and ten had implants placed within the LD pocket. Breast shape, volume and contour were successfully restored in all patients. Adequate degree of ptosis was achieved, to ensure maximal symmetry. CONCLUSIONS: Skin Sparing mastectomy through a circum-areolar incision has proven to be a safe and feasible option for the management of breast cancer in Egyptian women, offering them adequate oncologic control and optimum cosmetic outcome through preservation of the skin envelope of the breast when ever indicated. Our patients can benefit from safe surgery and have good cosmetic outcomeby applying different reconstructive techniques.