Huey-Jen Lin

Breast cancer is one of the most devastating malignancies in women worldwide. A growing body of evidence has linked neoplastic growth, invasion, metastasis, immune escape, and therapeutic resistance to infiltrating tumor-associated macrophages. In a breast cancer mass, macrophages are largely polarized to two main subtypes, M1 and M2, albeit with continuum intermediates, based on their immunological behaviors, gene signatures, and functional roles. While the former portrays proinflammatory and anti-cancer effects, the latter elicits the opposite impacts. M2 macrophages have gained rising attention as they are largely involved in fostering an immune-suppressive, cancer-promoting landscape and are imperative for malignant features across breast cancer subtypes. Through a positive feedback paracrine loop, M2 macrophages can be enriched by a plethora of dysregulated oncogenic signaling mediators, exemplified by CSF1/CSF1R, STAT3, IL-6, YAP, PI3K, PDK1, and AKT. These modulators could be released from or activated by surrounding malignant cells, fibroblasts, secreted extracellular vesicles, cell fragments generated after chemotherapies, hypoxia, dysregulated immune checkpoint pathways or oncometabolites. This review aims to discern the molecular cues fortifying M2 subpopulations. Moreover, recent advances in single-cell sequencing, spatial, and computational approaches have refined the understanding of TAM heterogeneity, while clinical translation remains limited by low therapeutic specificity, compensatory signaling, and differences between murine and human macrophage biology. Future therapeutic regimens should include strategies aimed at correcting aberrations that favor M2 polarization and are justified with divergences between humans and mice.

Mounting evidence links the phenomenon of enhanced recruitment of tumor-associated macrophages towards cancer bulks to neoplastic growth, invasion, metastasis, immune escape, matrix remodeling, and therapeutic resistance. In the context of cancer progression, naïve macrophages are polarized into M1 or M2 subtypes according to their differentiation status, gene signatures, and functional roles. While the former render proinflammatory and anticancer effects, the latter subpopulation elicits an opposite impact on pancreatic ductal adenocarcinoma. M2 macrophages have gained increasing attention as they are largely responsible for molding an immune-suppressive landscape. Through positive feedback circuits involving a paracrine manner, M2 macrophages can be amplified by and synergized with neighboring neoplastic cells, fibroblasts, endothelial cells, and non-cell autonomous constituents in the microenvironmental niche to promote an advanced disease state. This review delineates the molecular cues expanding M2 populations that subsequently convey notorious clinical outcomes. Future therapeutic regimens shall comprise protocols attempting to abolish environmental niches favoring M2 polarization; weaken cancer growth typically assisted by M2; promote the recruitment of tumoricidal CD8⁺ T lymphocytes and dendritic cells; and boost susceptibility towards gemcitabine as well as other chemotherapeutic agents.

Breast cancer is the most common malignancy among females in western countries, where women have an overall lifetime risk of >10% for developing invasive breast carcinomas. It is not a single disease but is composed of distinct subtypes associated with different clinical outcomes and is highly heterogeneous in both the molecular and clinical aspects. Although tumor initiation is largely driven by acquired genetic alterations, recent data suggest microenvironment-mediated immune evasion may play an important role in neoplastic progression. Beyond surgical resection, radiation, and chemotherapy, additional therapeutic options include hormonal deactivation, targeted-signaling pathway treatment, DNA repair inhibition, and aberrant epigenetic reversion. Yet, the fatality rate of metastatic breast cancer remains unacceptably high, largely due to treatment resistance and metastases to brain, lung, or bone marrow where tumor bed penetration of therapeutic agents is limited. Recent studies indicate the development of immune-oncological therapy could potentially eradicate this devastating malignancy. Evidence suggests tumors express immunogenic neoantigens but the immunity towards these antigens is frequently muted. Established tumors exhibit immunological tolerance. This tolerance reflects a process of immune suppression elicited by the tumor, and it represents a critical obstacle towards successful antitumor immunotherapy. In general, immune evasive mechanisms adapted by breast cancer encompasses down-regulation of antigen presentations or recognition, lack of immune effector cells, obstruction of anti-tumor immune cell maturation, accumulation of immunosuppressive cells, production of inhibitory cytokines, chemokines or ligands/receptors, and up-regulation of immune checkpoint modulators. Together with altered metabolism and hypoxic conditions, they constitute a permissive tumor microenvironment. This article intends to discern representative incidents and to provide potential innovative therapeutic regimens to reinstate tumoricidal immunity.

Pancreatic ductal adenocarcinoma is a fatal malignancy with a five-year survival rate lower than 7%, and most patients dying within six months of diagnosis. The factors that contribute to the aggressiveness of the disease include, but are not limited to: late diagnosis, prompt metastasis to adjacent vital organs, poor response, and resistance to anticancer treatments. This malignancy is uniquely associated with desmoplastic stroma that accounts for 80% of tumor mass. Understanding the biology of stroma can aid the discovery of innovative strategies for eradicating this lethal cancer in the future. This review highlights the critical components in the stroma and how they interact with the cancer cells to convey the devastating tumor progression.

Background: The oncogenic roles contributed by the Akt/PKB kinase family remain controversial and presumably depend on cell context, but are perceived to be modulated by an interplay and net balance between various isoforms. This study is intended to decipher whether distinct Akt kinase isoforms exert either redundant or unique functions in regulating neoplastic features of breast cancer cells, including epithelial-mesenchymal transition (EMT), cell motility, and stem/progenitor cell expansion. Results: We demonstrate that overactivation of Akt signaling in nonmalignant MCF10A cells and in primary cultures of normal human mammary epithelial tissue results in previously unreported inhibitory effects on EMT, cell motility and stem/progenitor cell expansion. Importantly, this effect is largely redundant and independent of Akt isoform types. However, using a series of isogenic cell lines derived from MCF-10A cells but exhibiting varying stages of progressive tumorigenesis, we observe that this inhibition of neoplastic behavior can be reversed in epithelial cells that have advanced to a highly malignant state. In contrast to the tumor suppressive properties of Akt, activated Akt signaling in MCF10A cells can rescue cell viability upon treatment with cytotoxic agents. This feature is regarded as tumor-promoting. Conclusion: We demonstrate that Akt signaling conveys novel dichotomy effects in which its oncogenic properties contributes mainly to sustaining cell viability, as opposed to the its tumor suppressing effects, which are mediated by repressing EMT, cell motility, and stem/progenitor cell expansion. While the former exerts a tumor-enhancing effect, the latter merely acts as a safeguard by restraining epithelial cells at the primary sites until metastatic spread can be moved forward, a process that is presumably dictated by the permissive tumor microenvironment or additional oncogenic insults.

Breast cancer is the leading type of cancer affecting women. It is estimated that breast cancer accounts for over a quarter of all newly diagnosed cancer cases in women. Despite advances in chemotherapy and hormonal treatment, at the present time, metastatic breast cancer remains an incurable disease. Although a large number of chemotherapeutic agents have been developed that are able to cause the regression of metastatic breast cancers, these tumors almost always recur following chemotherapy treatment. It is possible that breast cancer stem cells or breast cancer initiating cells are responsible for the drug-resistance, metastasis, and recurrence of cancer. Constitutive activation of Signal Transducers and Activators of Transcription 3 (STAT3) signaling is frequently detected in many types of cancers, including breast cancer, and has emerged as an attractive molecular target for cancer treatment. However, whether or not STAT3 is activated in breast cancer stem cells and what role STAT3 signaling plays in cancer stem cells is still unknown. If STAT3 is activated in breast cancer stem cells, targeting STAT3 may offer a promising opportunity to eliminate breast cancer stem cells and prevent the recurrence of cancer. We examined STAT3 activation in breast cancer stem cells, which are characterized by an aldehyde dehydrogenase (ALDH)-positive (ALDH⁺) and ALDH⁺/CD44⁺/CD24⁻ subpopulations. Interestingly, we observed that the ALDH⁺ and ALDH⁺/CD44⁺/CD24⁻ subpopulations of breast cancer cells expressed higher levels of phosphorylated STAT3, an active form of STAT3, as compared to the ALDH⁻ and ALDH⁻/CD44⁺/CD24⁺ subpopulations and un-separated breast cancer cells, suggesting that STAT3 is activated in breast cancer stem cells. We also demonstrated that a novel STAT3 inhibitor, LLL12, inhibited STAT3 phosphorylation, cell viability, STAT3 downstream target gene expression, and induced apoptosis in breast cancer stem cells. In addition, the STAT3 inhibitors Stattic and LLL12 inhibited the tumorsphere forming capacity of breast cancer stem cells. Furthermore, in a mouse tumor model using breast cancer stem cells, we demonstrated that LLL12 can suppress tumor growth in vivo. This is the first report to demonstrate that persistent STAT3 phosphorylation is expressed in breast cancer stem cells and that this subpopulation of breast cancer stem cells is sensitive to STAT3 inhibitors. Our results suggest that STAT3 is a novel therapeutic target in breast cancer stem cells and inhibition of activated STAT3 in cancer stem cells may offer a more effective treatment for breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4296.

Growing body of evidence demonstrates that AKT signaling plays a pivotal role in orchestrating various neoplastic phenotypes of breast carcinomas. Although the question of how tumorigenic activities are attributed to the respective AKT isoforms (namely AKT1, AKT2, and AKT3) has not been well delineated, AKT1 seems to impede transwell migration as well as epithelial-mesenchymal transition (EMT), whereas AKT2 exerts an opposing effect. Yet, the role that AKT3 plays remains poorly understood. Moreover, activated PI3K/AKT signaling augments the stem/progenitor subpopulation, which is presumably influenced from enhanced EMT. However, these findings were merely inferred from the results of ablating PTEN (one of AKT antagonists) and implementing PI3K as well as AKT inhibitors, but without ectopic delivery of activated AKT. As such, we aimed to substantiate the aforementioned outcomes by overly expressing various constitutive AKT isoforms and then observing their respective effects on cell motility, EMT, and stem/progenitor cell enrichment. To our surprise, rather than exerting an activation as previously reported, overexpression of all three AKT isoforms in the noncancerous breast epithelial cell line MCF10A consistently resulted in an inhibitory effect on the expression of EMT hallmarks, including upregulation of E-cadherin as well as down-regulation of N-cadherin and Vimentin. Importantly, inclusion of an EMT-inducer namely TGF-beta exerted a relatively more prominent suppressive effect. In addition to MCF10A, a similar outcome was rendered in another breast cancer cell line, namely SUM159, indicating that AKT can exert comparable effects on both normal and neoplastic breast epithelia in an isoform-independent manner. Regarding stem cell enrichment, we observed that AKT activation surprisingly manifested negligible effects in both MCF10A and SUM159 cells, as opposed to an increase appraised by knocking-down PTEN. Furthermore, as we have reported that activated AKT signaling can impose epigenetic silencing on a tumor suppressor gene CST6 by promoter hypermethylation, whether or not and which AKT isoforms can exert epigenetic effects on motility, or on EMT, were evaluated. Interestingly, expression of EMT hallmarks can be partially restored by using epigenetic drugs (DAC and TSA); a finding thereby indicates the likelihood of epigenetic regulation. Together, we highlighted a novelty that activated AKT signaling unlikely promotes EMT or enriches stem cells. Our data will be further clinically validated in breast tissues procured from women and thus the effect of AKT on both non-cancerous and malignant primary mammary epithelia can be investigated. Future therapeutic regimens implementing AKT-inhibiting drugs should require extra precautions as they might exert opposite effects of reactivating EMT followed by enriching stem cell subpopulation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 291.

The constitutive activation of the Stat3 oncogene product and mutation of the p53 tumor suppressor are both frequently detected in human breast cancer. We sought to determine whether there is functional regulation of Stat3 by wild-type (wt) p53. We demonstrate that expression of wt p53, but not mutant p53, significantly diminished phosphorylation of Stat3, reduced Stat3 DNA binding activity, and inhibited Stat3-dependent transcriptional activity in breast cancer cells expressing constitutively active Stat3. Expression of wt p53 did not cause a reduction in the phosphorylation of three unrelated protein kinases in other signal transduction pathways, AKT, extracellular signal-regulated kinase (ERK)1, and ERK2 or a reduction of phosphorylation of epidermal growth factor receptor. Furthermore, the expression of the p53 downstream target, p21(WAF-1), did not have an inhibitory effect on Stat3 phosphorylation. Wt p53 also induced significant apoptosis in breast cancer cell lines that express constitutively active Stat3. Interestingly, the p53-dependent apoptosis occurred in the presence of high levels of phosphorylated AKT and ERK1/2. Therefore, these findings demonstrate a novel p53-dependent cellular process that regulates Stat3 phosphorylation and activity.