Immunization with a multi-antigen targeted DNA vaccine eliminates chemoresistant pancreatic cancer by disrupting tumor-stromal cell crosstalk.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is characterised by limited responses to chemoimmunotherapy attributed to highly desmoplastic tumor microenvironment. Disrupting the tumor-stromal cell crosstalk is considered as an improved PDAC treatment strategy, whereas little progress has been made due to poor understanding of its underlying mechanism. Here, we examined the cellular role of melanoma associated antigen A isoforms (MAGEA) in regulating tumor-stromal crosstalk mediated chemoresistance. METHODS: We used clinical samples to explore the correlation between MAGEA expression and patient prognosis in multiple cancers. We utilized cancer cell lines, patient derived organoids and orthotopic PDAC model to examine the function of MAGEA in chemoresistance. We performed biochemical, proteome profiler array and transcriptional analysis to uncover a mechanism that governs tumor-stromal crosstalk. We developed a multi-MAGEA antigen targeted DNA vaccine and tested its effect on PDAC tumor growth. RESULTS: We establish MAGEA as a regulator of the tumor-stromal crosstalk in PDAC. We provide strong clinical evidence indicating that high MAGEA expression, including MAGEA2, MAGEA3 and MAGEA10, correlates with worse chemotherapeutic response and poor prognosis in multiple cancers, while their expression is up-regulated in chemoresistant PDAC patient derived organoids and cancer cell lines. Mechanistically, MAGEA2 prohibits gemcitabine-induced JNK-c-Jun-p53 mediated cancer cell apoptosis, while gemcitabine stimulated pancreatic stellate cells secretes GDF15 to further enhance the gemcitabine resistance of MAGEA2 expressing cells by activating GFRAL-RET mediated Akt and ERK1/2 dependent survival pathway. Strikingly, immunization with a DNA vaccine that targeting multiple MAGEA antigens, including MAGEA2, MAGEA3 and MAGEA10, elicits robust immune responses against the growth of gemcitabine resistant tumors. CONCLUSIONS: These findings suggest that targeting MAGEA-mediated paracrine regulation of chemoresistance by immunotherapy can be an improved pancreatic cancer treatment strategy.

Ceramide kinase confers tamoxifen resistance in estrogen receptor-positive breast cancer by altering sphingolipid metabolism.

Dysregulated sphingolipid metabolism contributes to ER+ breast cancer progression and therapeutic response, whereas its underlying mechanism and contribution to tamoxifen resistance (TAMR) is unknown. Here, we establish sphingolipid metabolic enzyme CERK as a regulator of TAMR in breast cancer. Multi-omics analysis reveals an elevated CERK driven sphingolipid metabolic reprogramming in TAMR cells, while high CERK expression associates with worse patient prognosis in ER+ breast cancer. CERK overexpression confers tamoxifen resistance and promotes tumorigenicity in ER+ breast cancer cells. Knocking out CERK inhibits the orthotopic breast tumor growth of TAMR cells while rescuing their tamoxifen sensitivity. Mechanistically, the elevated EHF expression transcriptionally up-regulates CERK expression to prohibit tamoxifen-induced sphingolipid ceramide accumulation, which then inhibits tamoxifen-mediated repression on PI3K/AKT dependent cell proliferation and its driven p53/caspase-3 mediated apoptosis in TAMR cells. This work provides insight into the regulation of sphingolipid metabolism in tamoxifen resistance and identifies a potential therapeutic target for this disease.

p53-dependent elimination of aneuploid mitotic offspring by entosis.

Entosis was proposed to promote aneuploidy and genome instability by cell-in-cell mediated engulfment in tumor cells. We reported here, in epithelial cells, that entosis coupled with mitotic arrest functions to counteract genome instability by targeting aneuploid mitotic progenies for engulfment and elimination. We found that the formation of cell-in-cell structures associated with prolonged mitosis, which was sufficient to induce entosis. This process was controlled by the tumor suppressor p53 (wild-type) that upregulates Rnd3 expression in response to DNA damages associated with prolonged metaphase. Rnd3-compartmentalized RhoA activities accumulated during prolonged metaphase to drive cell-in-cell formation. Remarkably, this prolonged mitosis-induced entosis selectively targets non-diploid progenies for internalization, blockade of which increased aneuploidy. Thus, our work uncovered a heretofore unrecognized mechanism of mitotic surveillance for entosis, which eliminates newly born abnormal daughter cells in a p53-dependent way, implicating in the maintenance of genome integrity.

Mechanical Ring Interfaces between Adherens Junction and Contractile Actomyosin to Coordinate Entotic Cell-in-Cell Formation.

Entosis is a cell-in-cell (CIC)-mediated death program. Contractile actomyosin (CA) and the adherens junction (AJ) are two core elements essential for entotic CIC formation, but the molecular structures interfacing them remain poorly understood. Here, we report the characterization of a ring-like structure interfacing between the peripheries of invading and engulfing cells. The ring-like structure is a multi-molecular complex consisting of adhesive and cytoskeletal proteins, in which the mechanical sensor vinculin is highly enriched. The vinculin-enriched structure senses mechanical force imposed on cells, as indicated by fluorescence resonance energy transfer (FRET) analysis, and is thus termed the mechanical ring (MR). The MR actively interacts with CA and the AJ to help establish and maintain polarized actomyosin that drives cell internalization. Vinculin depletion leads to compromised MR formation, CA depolarization, and subsequent CIC failure. In summary, we suggest that the vinculin-enriched MR, in addition to CA and AJ, is another core element essential for entosis.

PCDH7 Inhibits the Formation of Homotypic Cell-in-Cell Structure.

Though homotypic cell-in-cell (hoCIC) structures are implicated in the development and progression of multiple human tumors, the molecular mechanisms underlying their formation remain poorly understood. We found that the expression of Protocadherin-7 (PCDH7), an integral membrane protein, was negatively associated with the formation of hoCIC structures. Overexpression of PCDH7 efficiently inhibits, while its depletion significantly enhances, hoCIC formation, which was attributed to its regulation on intercellular adhesion and contractile actomyosin as well. Via directly interacting with and inactivating PP1α, a protein phosphatase that dephosphorylates pMLC2, PCDH7 increases the level of pMLC2 leading to enhanced actomyosin at the intercellular region and compromised hoCIC formation. Remarkably, PCDH7 enhanced anchorage-independent cell growth in a hoCIC-dependent manner. Together, we identified PCDH7 as the first trans-membrane protein that inhibits hoCIC formation to promote tumor growth.

Subtype-Based Prognostic Analysis of Cell-in-Cell Structures in Early Breast Cancer.

Though current pathological methods are greatly improved, they provide rather limited functional information. Cell-in-cell structures (CICs), arising from active cell-cell interaction, are functional surrogates of complicated cell behaviors within heterogeneous cancers. In light of this, we performed the subtype-based CIC profiling in human breast cancers by the "EML" multiplex staining method, and accessed their values as prognostic factors by Cox univariate, multivariate, and nomogram analysis. CICs were detected in cancer specimens but not in normal breast tissues. A total of five types of CICs were identified with one homotypic subtype (91%) and four heterotypic subtypes (9%). Overall CICs (oCICs) significantly associated with patient overall survival (OS) (P = 0.011) as an independent protective factor (HR = 0.423, 95% CI, 0.227-0.785; P = 0.006). Remarkably, three CICs subtypes (TiT, TiM, and MiT) were also independent prognostic factors. Among them, higher TiT, from homotypic cannibalism between tumor cells, predicted longer patient survival (HR = 0.529, 95% CI, 0.288-0.973; P = 0.04) in a way similar to that of oCICs and that (HR = 0.524, 95% CI, 0.286-0.962; P = 0.037) of heterotypic TiM (tumor cell inside macrophage); conversely, the presence of MiT (macrophage inside tumor cell) predicted a death hazard of 2.608 (95% CI, 1.344-5.063; P = 0.05). Moreover, each CIC subtype tended to preferentially affect different categories of breast cancer, with TiT (P < 0.0001) and oCICs (P = 0.008) targeting luminal B (Her2+), TiM (P = 0.011) targeting HR- (Her2+/HR- and TNBC), and MiT targeting luminal A (P = 0.017) and luminal B (Her-) (P = 0.006). Furthermore, nomogram analysis suggested that CICs impacted patient outcomes in contributions comparable (for oCICs, TiT, and TiM), or even superior (for MiT), to TNM stage and breast cancer subtype, and incorporating CICs improved nomogram performance. Together, we propose CICs profiling as a valuable way for prognostic analysis of breast cancer and that CICs and their subtypes, such as MiT, may serve as a type of novel functional markers assisting clinical practices.