ALTering Cancer by Triggering Telomere Replication Stress through the Stabilization of Promoter G-Quadruplex in SMARCAL1.

Most of the human cancers are dependent on telomerase to extend the telomeres. But ∼10% of all cancers use a telomerase-independent, homologous recombination mediated pathway called alternative lengthening of telomeres (ALT). Due to the poor prognosis, ALT status is not being considered yet in the diagnosis of cancer. No such specific treatment is available to date for ALT positive cancers. ALT positive cancers are dependent on replication stress to deploy DNA repair pathways to the telomeres to execute homologous recombination mediated telomere extension. SMARCAL1 (SWI/SNF related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like 1) is associated with the ALT telomeres to resolve replication stress thus providing telomere stability. Thus, the dependency on replication stress regulatory factors like SMARCAL1 made it a suitable therapeutic target for the treatment of ALT positive cancers. In this study, we found a significant downregulation of SMARCAL1 expression by stabilizing the G-quadruplex (G4) motif found in the promoter of SMARCAL1 by potent G4 stabilizers, like TMPyP4 and BRACO-19. SMARCAL1 downregulation led toward the increased localization of PML (promyelocytic leukemia) bodies in ALT telomeres and triggered the formation of APBs (ALT-associated promyelocytic leukemia bodies) in ALT positive cell lines, increasing telomere replication stress and DNA damage at a genomic level. Induction of replication stress and hyper-recombinogenic phenotype in ALT positive cells mediated by G4 stabilizing molecules already highlighted their possible application as a new therapeutic window to target ALT positive tumors. In accordance with this, our study will also provide a valuable insight toward the development of G4-based ALT therapeutics targeting SMARCAL1.

Breast cancer stem cells generate immune-suppressive T regulatory cells by secreting TGFß to evade immune-elimination.

Cancer stem cells (CSCs), being the primary contributors in tumor initiation, metastasis, and relapse, ought to have seminal roles in evasion of immune surveillance. Tumor-promoting CD4+CD25+FOXP3+ T-regulatory cells (Tregs) have been described to abolish host defense mechanisms by impeding the activities of other immune cells including effector T cells. However, whether CSCs can convert effector T cells to immune-suppressive Treg subset, and if yes, the mechanism underlying CSC-induced Treg generation, are limitedly studied. In this regard, we observed a positive correlation between breast CSC and Treg signature markers in both in-silico and immunohistochemical analyses. Mirroring the conditions during tumor initiation, low number of CSCs could successfully generate CD4+CD25+FOXP3+ Treg cells from infiltrating CD4+ T lymphocytes in a contact-independent manner. Suppressing the proliferation potential as well as IFNγ production capacity of effector T cells, these Treg cells might be inhibiting antitumor immunity, thereby hindering immune-elimination of CSCs during tumor initiation. Furthermore, unlike non-stem cancer cells (NSCCs), CSCs escaped doxorubicin-induced apoptosis, thus constituting major surviving population after three rounds of chemotherapy. These drug-survived CSCs were also able to generate CD4+CD25+FOXP3+ Treg cells. Our search for the underlying mechanism further unveiled the role of CSC-shed immune-suppressive cytokine TGFß, which was further increased by chemotherapy, in generating tumor Treg cells. In conclusion, during initiation as well as after chemotherapy, when NSCCs are not present in the tumor microenvironment, CSCs, albeit present in low numbers, generate immunosuppressive CD4+CD25+FOXP3+ Treg cells in a contact-independent manner by shedding high levels of immune-suppressive Treg-polarizing cytokine TGFß, thus escaping immune-elimination and initiating the tumor or causing tumor relapse.

Tumor-associated macrophages: an effective player of the tumor microenvironment.

Cancer progression is primarily caused by interactions between transformed cells and the components of the tumor microenvironment (TME). TAMs (tumor-associated macrophages) make up the majority of the invading immune components, which are further categorized as anti-tumor M1 and pro-tumor M2 subtypes. While M1 is known to have anti-cancer properties, M2 is recognized to extend a protective role to the tumor. As a result, the tumor manipulates the TME in such a way that it induces macrophage infiltration and M1 to M2 switching bias to secure its survival. This M2-TAM bias in the TME promotes cancer cell proliferation, neoangiogenesis, lymphangiogenesis, epithelial-to-mesenchymal transition, matrix remodeling for metastatic support, and TME manipulation to an immunosuppressive state. TAMs additionally promote the emergence of cancer stem cells (CSCs), which are known for their ability to originate, metastasize, and relapse into tumors. CSCs also help M2-TAM by revealing immune escape and survival strategies during the initiation and relapse phases. This review describes the reasons for immunotherapy failure and, thereby, devises better strategies to impair the tumor-TAM crosstalk. This study will shed light on the understudied TAM-mediated tumor progression and address the much-needed holistic approach to anti-cancer therapy, which encompasses targeting cancer cells, CSCs, and TAMs all at the same time.

FOXP3/HAT1 Axis Controls Treg Infiltration in the Tumor Microenvironment by Inducing CCR4 Expression in Breast Cancer.

Infiltrating T-regulatory cells in the tumor microenvironment is a key impediment to immunotherapy and is linked to a poor prognosis. We found that tumor-infiltrating Tregs express a higher expression of the chemokine receptor CCR4 than peripheral Tregs in breast cancer patients. CCL22 and CCL17 are released by tumor cells and tumor-associated macrophages, attracting CCR4+ Tregs to the tumor site. The Treg lineage-specific transcription factor FOXP3 changes the CCR4 promoter epigenetically in conjunction with HAT1 to provide a space for FOXP3 binding and activation of the CCR4 gene. To increase CCR4 expression in Tregs, the FOXP3/HAT1 axis is required for permissive (K23 and K27) or repressive (K14 and K18) acetylation of histone-3. In murine breast and melanoma tumor models, genetic ablation of FOXP3 reduced CCR4+ Treg infiltration and tumor size while also restoring anti-tumor immunity. Overexpression of FOXP3, on the other hand, increased CCR4+ Treg infiltration, resulting in a decreased anti-tumor immune response and tumor progression. These findings point to FOXP3 playing a new role in the tumor microenvironment as a transcriptional activator of CCR4 and a regulator of Treg infiltration.

SMAR1 repression by pluripotency factors and consequent chemoresistance in breast cancer stem-like cells is reversed by aspirin.

The high abundance of drug efflux pumps in cancer stem cells (CSCs) contributes to chemotherapy resistance. The transcriptional regulator SMAR1 suppresses CSC expansion in colorectal cancer, and increased abundance of SMAR1 is associated with better prognosis. Here, we found in breast tumors that the expression of SMAR1 was decreased in CSCs through the cooperative interaction of the pluripotency factors Oct4 and Sox2 with the histone deacetylase HDAC1. Overexpressing SMAR1 sensitized CSCs to chemotherapy through SMAR1-dependent recruitment of HDAC2 to the promoter of the gene encoding the drug efflux pump ABCG2. Treating cultured CSCs or 4T1 tumor-bearing mice with the nonsteroidal anti-inflammatory drug aspirin restored SMAR1 expression and ABCG2 repression and enhanced tumor sensitivity to doxorubicin. Our findings reveal transcriptional mechanisms regulating SMAR1 that also regulate cancer stemness and chemoresistance and suggest that, by restoring SMAR1 expression, aspirin might enhance chemotherapeutic efficacy in patients with stem-like tumors.

Pyridoxine enhances chemo-responsiveness of breast cancer stem cells via redox reconditioning.

A plethora of molecular strategies are employed by breast cancer stem cells (bCSCs) to evade chemotherapy-induced death signals, redox modulation being a crucial factor among those. Here, we observed that bCSCs are resistant to DNA damage and generate low ROS upon doxorubicin (Dox) treatment. Further exploration revealed inherently high NEIL2, a base excision repair (BER) enzyme that plays a key regulatory role in repairing DNA damage, in bCSCs. However, its role in modulating the redox status of bCSCs remains unexplored. In addition, Dox not only upregulates NEIL2 in bCSCs at both transcriptional and translational levels but also declines p300-induced acetylation thus activating NEIL2 and providing a protective effect against the stress inflicted by the genotoxic drug. However, when the redox status of bCSCs is altered by inducing high ROS, apoptosis of the resistant population is accomplished. Subsequently, when NEIL2 is suppressed in bCSCs, chemo-sensitization of the resistant population is enabled by redox reconditioning via impaired DNA repair. This signifies a possibility of therapeutically disrupting the redox balance in bCSCs to enhance their chemo-responsiveness. Our search for an inhibitor of NEIL2 revealed that vitamin B6, i.e., pyridoxine (PN), hinders NEIL2-mediated transcription-coupled repair process by not only decreasing NEIL2 expression but also inhibiting its association with RNA Pol II, thus stimulating DNA damage and triggering ROS. As a consequence of altered redox regulation, bCSCs become susceptible towards Dox, which then induces apoptosis via caspase cascade. These findings signify that PN enhances chemo-responsiveness of bCSCs via redox reconditioning.