Biomarkers beyond BRCA: promising combinatorial treatment strategies in overcoming resistance to PARP inhibitors.

Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) exploit the concept of synthetic lethality and offer great promise in the treatment of tumors with deficiencies in homologous recombination (HR) repair. PARPi exert antitumor activity by blocking Poly(ADP-ribosyl)ation (PARylation) and trapping PARP1 on damaged DNA. To date, the U.S. Food and Drug Administration (FDA) has approved four PARPi for the treatment of several cancer types including ovarian, breast, pancreatic and prostate cancer. Although patients with HR-deficient tumors benefit from PARPi, majority of tumors ultimately develop acquired resistance to PARPi. Furthermore, even though BRCA1/2 mutations are commonly used as markers of PARPi sensitivity in current clinical practice, not all patients with BRCA1/2 mutations have PARPi-sensitive disease. Thus, there is an urgent need to elucidate the molecular mechanisms of PARPi resistance to support the development of rational effective treatment strategies aimed at overcoming resistance to PARPi, as well as reliable biomarkers to accurately identify patients who will most likely benefit from treatment with PARPi, either as monotherapy or in combination with other agents, so called marker-guided effective therapy (Mget). In this review, we summarize the molecular mechanisms driving the efficacy of and resistance to PARPi as well as emerging therapeutic strategies to overcome PARPi resistance. We also highlight the identification of potential markers to predict PARPi resistance and guide promising PARPi-based combination strategies.

Oncogenic signaling pathways associated with immune evasion and resistance to immune checkpoint inhibitors in cancer.

Immune checkpoint inhibitors (ICIs) are novel class of anti-cancer drugs that exhibit significant therapeutic effects even in patients with advanced-stage cancer. However, the efficacy of ICIs is limited due to resistance. Therefore, appropriate biomarkers to select patients who are likely to respond to these drugs as well as combination therapy to overcome the resistance are urgently necessary. Cancer is caused by various genetic alterations that lead to abnormalities in oncogenic signaling pathways. The aberrant oncogenic signaling pathways serve as not only prognostic and predictive biomarkers, but also targets for molecularly targeted therapy. Growing evidence shows that the aberrant oncogenic signaling pathways in cancer cells facilitate the resistance to ICIs by modulating the regulation of immune checkpoint and cancer immune surveillance. Indeed, it has been demonstrated that some molecular targeted therapies significantly improve the efficacy of ICIs in preclinical and clinical studies. In this review, we highlighted several oncogenic signaling pathways including receptor tyrosine kinases (RTKs), MAPK, PI3K-AKT-mTOR, JAK-STAT, Hippo, and Wnt pathways, and summarized the recent findings of the mechanisms underlying the regulation of cancer immunity and the ICI resistance induced by these aberrant oncogenic signaling pathways in cancer cells. Moreover, we discussed potential combination therapies with ICIs and molecularly targeted drugs to overcome the resistance and increase the efficacy of ICIs.

Estrogen promotes increased breast cancer cell proliferation and migration through downregulation of CPEB1 expression.

The polyadenylation element binding protein 1 (CPEB1) plays an important role in the regulation of poly(A) tail length at the 3'UTR of mRNA during transcription. Downregulation of CPEB1 expression, which is associated with the loss of mammary epithelial polarity, has been reported in breast cancer. CPEB1 downregulation leads to an increase in tumor aggressiveness of breast cancer. Breast cancer is also known to be responsive to the treatment with steroid hormones, which promotes cancer development and progression; however, the nature of these associations remains unclear. This study aimed to investigate whether estrogen and progesterone impacted CPEB1 expression in breast cancer in order to regulate cell proliferation and migration. MCF7 cell proliferation was increased in response to estrogen treatment, and estrogen application suppressed the expression of CPEB1 mRNA. Cells treated with estrogen or those depleted for CPEB1 by shRNA showed increased wound healing capacity compared with that of control cells in migration assay. Moreover, we found that CPEB1 level of expression in human breast cancer tissue was low compared with that in the healthy tissue. CPEB1 expression was downregulated in response to estrogen activity and in turn, that caused a significantly induced cell migration in breast cancer cells. This suggests that CPEB1 is one of the estrogen responsive genes, which stimulates breast cancer progression. Increasing and/or maintaining CPEB1 expression level has the potential to control breast cancer behavior.

MET Inhibitors Promote Liver Tumor Evasion of the Immune Response by Stabilizing PDL1.

BACKGROUND & AIMS: Inhibitors of MET have not produced satisfactory outcomes in trials of patients with liver cancer. We investigated the mechanisms of liver tumor resistance to MET inhibitors in mice. METHODS: We tested the effects of MET inhibitors tivantinib and capmatinib in the mouse hepatocellular carcinoma (HCC) cell line HCA-1 and in immune-competent and immunodeficient mice with subcutaneous tumors grown from this cell line. Tumors were collected from mice and tumor cells were analyzed by time-of-flight mass cytometry. We used short hairpin RNAs to weaken expression of MET in Hep3B, SK-HEP-1, HA59T, and HA22T liver cancer cell lines and analyzed cells by immunoblot, immunofluorescence, and immunoprecipitation assays. Mass spectrometry was used to assess interactions between MET and glycogen synthase kinase 3ß (GSK3B), and GSK3B phosphorylation, in liver cancer cell lines. C57/BL6 mice with orthotopic tumors grown from Hep1-6 cells were given combinations of capmatinib or tivantinib and antibodies against programmed cell death 1 (PDCD1; also called PD1); tumors were collected and analyzed by immunofluorescence. We analyzed 268 HCCsamples in a tissue microarray by immunohistochemistry. RESULTS: Exposure of liver cancer cell lines to MET inhibitors increased their expression of PD ligand 1 (PDL1) and inactivated cocultured T cells. MET phosphorylated and activated GSK3B at tyrosine 56, which decreased the expression of PDL1 by liver cancer cells. In orthotopic tumors grown in immune-competent mice, MET inhibitors decreased the antitumor activity of T cells. However, addition of anti-PD1 decreased orthotopic tumor growth and prolonged survival of mice compared with anti-PD1 or MET inhibitors alone. Tissue microarray analysis of HCC samples showed an inverse correlation between levels of MET and PDL1 and a positive correlation between levels of MET and phosphorylated GSK3B. CONCLUSIONS: In studies of liver cancer cell lines and mice with orthotopic tumors, MET mediated phosphorylation and activated GSK3B, leading to decreased expression of PDL1. Combined with a MET inhibitor, anti-PD1 and anti-PDL1 produced additive effect to slow growth of HCCs in mice.

Unlocking c-MET: A comprehensive journey into targeted therapies for breast cancer.

Breast cancer is the most common malignancy among women, posing a formidable health challenge worldwide. In this complex landscape, the c-MET (cellular-mesenchymal epithelial transition factor) receptor tyrosine kinase (RTK), also recognized as the hepatocyte growth factor (HGF) receptor (HGFR), emerges as a prominent protagonist, displaying overexpression in nearly 50% of breast cancer cases. Activation of c-MET by its ligand, HGF, secreted by neighboring mesenchymal cells, contributes to a cascade of tumorigenic processes, including cell proliferation, metastasis, angiogenesis, and immunosuppression. While c-MET inhibitors such as crizotinib, capmatinib, tepotinib and cabozantinib have garnered FDA approval for non-small cell lung cancer (NSCLC), their potential within breast cancer therapy is still undetermined. This comprehensive review embarks on a journey through structural biology, multifaceted functions, and intricate signaling pathways orchestrated by c-MET across cancer types. Furthermore, we highlight the pivotal role of c-MET-targeted therapies in breast cancer, offering a clinical perspective on this promising avenue of intervention. In this pursuit, we strive to unravel the potential of c-MET as a beacon of hope in the fight against breast cancer, unveiling new horizons for therapeutic innovation.

Advances and prospects of biomarkers for immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICIs) activate anti-cancer immunity by blocking T cell checkpoint molecules such as programmed death 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4). Although ICIs induce some durable responses in various cancer patients, they also have disadvantages, including low response rates, the potential for severe side effects, and high treatment costs. Therefore, selection of patients who can benefit from ICI treatment is critical, and identification of biomarkers is essential to improve the efficiency of ICIs. In this review, we provide updated information on established predictive biomarkers (tumor programmed death-ligand 1 [PD-L1] expression, DNA mismatch repair deficiency, microsatellite instability high, and tumor mutational burden) and potential biomarkers currently under investigation such as tumor-infiltrated and peripheral lymphocytes, gut microbiome, and signaling pathways related to DNA damage and antigen presentation. In particular, this review aims to summarize the current knowledge of biomarkers, discuss issues, and further explore future biomarkers.

MRCK as a Potential Target for Claudin-Low Subtype of Breast Cancer.

To find new molecular targets for triple negative breast cancer (TNBC), we analyzed a large-scale drug screening dataset based on breast cancer subtypes. We discovered that BDP-9066, a specific MRCK inhibitor (MRCKi), may be an effective drug against TNBC. After confirming the efficacy and specificity of BDP-9066 against TNBC in vitro and in vivo, we further analyzed the underlying mechanism of specific activity of BDP-9066 against TNBC. Comparing the transcriptome of BDP-9066-sensitive and -resistant cells, the activation of the focal adhesion and YAP/TAZ pathway were found to play an important role in the sensitive cells. Furthermore, YAP/TAZ is indeed repressed by BDP-9066 in the sensitive cells, and active form of YAP suppresses the effects of BDP-9066. YAP/TAZ expression and activity are high in TNBC, especially the Claudin-low subtype, consistent with the expression of focal adhesion-related genes. Interestingly, NF-κB functions downstream of YAP/TAZ in TNBC cells and is suppressed by BDP-9066. Furthermore, the PI3 kinase pathway adversely affected the effects of BDP-9066 and that alpelisib, a PI3 kinase inhibitor, synergistically increased the effects of BDP-9066, in PIK3CA mutant TNBC cells. Taken together, we have shown for the first time that MRCKi can be new drugs against TNBC, particularly the Claudin-low subtype.

Post-translational Modification of PD-1: Potential Targets for Cancer Immunotherapy.

Activation of effector T cells leads to upregulation of PD-1, which can inhibit T-cell activity following engagement with its ligand PD-L1. Post-translational modifications (PTM), including glycosylation, phosphorylation, ubiquitination, and palmitoylation, play a significant role in regulating PD-1 protein stability, localization, and interprotein interactions. Targeting PTM of PD-1 in T cells has emerged as a potential strategy to overcome PD-1-mediated immunosuppression in cancer and enhances antitumor immunity. The regulatory signaling pathways that induce PTM of PD-1 can be suppressed with small-molecule inhibitors, and mAbs can directly target PD-1 PTMs. Preliminary outcomes from exploratory studies suggest that focusing on the PTM of PD-1 has strong therapeutic potential and can enhance the response to anti-PD-1.

Membrane-bound trafficking regulates nuclear transport of integral epidermal growth factor receptor (EGFR) and ErbB-2.

Nuclear localization of multiple receptor-tyrosine kinases (RTKs), such as EGF receptor (EGFR), ErbB-2, FGF receptor (FGFR), and many others, has been reported by several groups. We previously showed that cell surface EGFR is trafficked to the nucleus through a retrograde pathway from the Golgi to the endoplasmic reticulum (ER) and that EGFR is then translocated to the inner nuclear membrane (INM) through the INTERNET (integral trafficking from the ER to the nuclear envelope transport) pathway. However, the nuclear trafficking mechanisms of other membrane RTKs, apart from EGFR, remain unclear. The purpose of this study was to compare the nuclear transport of EGFR family proteins with that of FGFR-1. Interestingly, we found that digitonin permeabilization, which selectively releases soluble nuclear transporters from the cytoplasm and has been shown to inhibit nuclear transport of FGFR-1, had no effects on EGFR nuclear transport, raising the possibility that EGFR and FGFR-1 use different pathways to be translocated into the nucleus. Using the subnuclear fractionation assay, we further demonstrated that biotinylated cell surface ErbB-2, but not FGFR-1, is targeted to the INM, associating with Sec61ß in the INM, similar to the nuclear trafficking of EGFR. Thus, ErbB-2, but not FGFR-1, shows a similar trafficking pathway to EGFR for translocation to the nucleus, indicating that at least two different pathways of nuclear transport exist for cell surface receptors. This finding provides a new direction for investigating the trafficking mechanisms of various nuclear RTKs.

PD-L1 and CD58 co-regulated by CMTM6 play yin and yang to shape anti-tumor immunity.

The CD58-CD2 axis regulates T cell-mediated cancer immunity, but little is known about the regulation of CD58. In two recent papers pubished in Cancer Cell, Miao et al. and Ho et al. define a mechanism of CD58 regulation by CMTM6 and show an unexpected yin-yang link between PD-L1 and CD58.

Herbal Compounds Dauricine and Isoliensinine Impede SARS-CoV-2 Viral Entry.

Targeting viral entry has been the focal point for the last 3 years due to the continued threat posed by SARS-CoV-2. SARS-CoV-2's entry is highly dependent on the interaction between the virus's Spike protein and host receptors. The virus's Spike protein is a key modulator of viral entry, allowing sequential cleavage of ACE2 at the S1/S2 and S2 sites, resulting in the amalgamation of membranes and subsequent entry of the virus. A Polybasic insertion (PRRAR) conveniently located at the S1/S2 site can also be cleaved by furin or by serine protease, TMPRSS2, at the cell surface. Since ACE2 and TMPRSS2 are conveniently located on the surface of host cells, targeting one or both receptors may inhibit receptor-ligand interaction. Here, we show that Dauricine and Isoliensinine, two commonly used herbal compounds, were capable of inhibiting SARS-CoV-2 viral entry by reducing Spike-ACE2 interaction but not suppressing TMPRSS2 protease activity. Further, our biological assays using pseudoviruses engineered to express Spike proteins of different variants revealed a reduction in infection rates following treatment with these compounds. The molecular modeling revealed an interconnection between R403 of Spike protein and both two compounds. Spike mutations at residue R403 are critical, and often utilized by ACE2 to gain cell access. Overall, our findings strongly suggest that Dauricine and Isoliensinine are effective in blocking Spike-ACE2 interaction and may serve as effective therapeutic agents for targeting SARS-CoV-2's viral entry.

Mechanisms regulating PD-L1 expression in cancers and associated opportunities for novel small-molecule therapeutics.

Antagonistic antibodies targeting the inhibitory immune-checkpoint receptor PD-1 or its ligand PD-L1 are used to treat a wide range of cancer types and can substantially improve patient survival. Nevertheless, strategies to overcome intrinsic and acquired resistance are required to respectively increase response rates and durations. PD-L1 is often upregulated in various malignancies, and emerging evidence suggests numerous underlying mechanisms involving distinct oncogenic signalling pathways. Thus, specific small-molecule inhibitors have the potential to simultaneously suppress not only a key oncogenic signalling pathway but also PD-L1 expression and/or activity in particular cancers, thereby presenting attractive candidate drugs for combination with existing immune-checkpoint inhibitors and/or other targeted agents. Herein, we summarize advances in understanding the mechanisms regulating PD-L1 expression at the transcriptional, post-transcriptional, translational and post-translational levels in cancers. We describe the roles of the diverse post-translational modifications of PD-L1, including phosphorylation, palmitoylation, glycosylation, acetylation and ubiquitination. Moreover, we discuss the potential use of small-molecule agents to modulate these mechanisms as well as of predictive biomarkers to stratify patients for optimal treatment, and provide our perspective on potential therapeutic strategies to circumvent resistance to conventional anti-PD-1/PD-L1 antibodies.

Shedding light on triple-negative breast cancer with Trop2-targeted antibody-drug conjugates.

Triple-negative breast cancer (TNBC) is well-known as the most aggressive subtype of breast cancer. Because TNBC does not express Her2, estrogen receptor, and progesterone receptors, there had been no effective U.S. Food and Drug Administration-approved targeted therapy for it until PARP inhibitors and two PD-1/PD-L1 monoclonal antibodies were approved for treatment of TNBC. Most recently, an antibody-drug conjugate (ADC), called sacituzumab govitecan (SG), was approved for the treatment of TNBC patients previously received chemotherapy with advanced disease. SG consists of an anti-trophoblast cell-surface antigen 2 (Trop2) antibody conjugated with a topoisomerase I inhibitor, SN-38, which is diffused out of the targeted Trop2 positive cancer cells and induces the bystander killing effect on surrounding cells regardless of their Trop2 expression status. In the Phase III clinical trial, TNBC patients treated with SG showed significantly longer progression-free and overall survival compared to those who were received chemotherapy. In the present review, we summarized the cellular function and signaling of Trop2, the mechanism of action of SG, and the clinical trials of SG that led to its quick approval for TNBC. In addition, we introduced the current ongoing clinical trials of SG as well as another Trop2 ADC, which has potential to overcome some disadvantages of SG.

Structural insights into EphA4 unconventional activation from prediction of the EphA4 and its complex with ribonuclease 1.

It has been shown that several ribonuclease (RNase) A superfamily proteins serve as ligands of receptor tyrosine kinases (RTKs), representing a new concept for ligand/receptor interaction. Moreover, recent studies indicate high clinical values for this type of ligand/RTK interactions. However, there is no structural report for this new family of ligand/receptor. In an attempt to understand how RNase and RTK may interact, we focused on the RNase1/ephrin type-A receptor 4 (EphA4) complex and predicted their structure by using the state-of-the-art machine learning method, AlphaFold and its derivative method, AF2Complex. In this model, electrostatic force plays an essential role for the specific ligand/receptor interaction. We found the R39 of RNase1 is the key residue for EphA4-binding and activation. Mutation on this residue causes disruption of an essential basic patch, resulting in weaker ligand-receptor association and leading to the loss of activation. By comparing the surface charge distribution of the RNase A superfamily, we found the positively charged residues on the RNase1 surface is more accessible for EphA4 forming salt bridges than other RNases. Furthermore, RNase1 binds to the ligand-binding domain (LBD) of EphA4, which is responsible for the traditional ligand ephrin-binding. Our model reveals the location of RNase1 on EphA4 partially overlaps with that of ephrin-A5, a traditional ligand of EphA4, suggesting steric hindrance as the basis by which the ephrin-A5 precludes interactions of RNase1 with EphA4. Together, our discovery of RNase1/EphA4 interface provides a potential treatment strategy by blocking the RNase1-EphA4 axis.

Targeting the ALK-CDK9-Tyr19 kinase cascade sensitizes ovarian and breast tumors to PARP inhibition via destabilization of the P-TEFb complex.

Poly(ADP-ribose) polymerase (PARP) inhibitors have demonstrated promising clinical activity in multiple cancers. However, resistance to PARP inhibitors remains a substantial clinical challenge. In the present study, we report that anaplastic lymphoma kinase (ALK) directly phosphorylates CDK9 at tyrosine-19 to promote homologous recombination (HR) repair and PARP inhibitor resistance. Phospho-CDK9-Tyr19 increases its kinase activity and nuclear localization to stabilize positive transcriptional elongation factor b and activate polymerase II-dependent transcription of HR-repair genes. Conversely, ALK inhibition increases ubiquitination and degradation of CDK9 by Skp2, an E3 ligase. Notably, combination of US Food and Drug Administration-approved ALK and PARP inhibitors markedly reduce tumor growth and improve survival of mice in PARP inhibitor-/platinum-resistant tumor xenograft models. Using human tumor biospecimens, we further demonstrate that phosphorylated ALK (p-ALK) expression is associated with resistance to PARP inhibitors and positively correlated with p-Tyr19-CDK9 expression. Together, our findings support a biomarker-driven, combinatorial treatment strategy involving ALK and PARP inhibitors to induce synthetic lethality in PARP inhibitor-/platinum-resistant tumors with high p-ALK-p-Tyr19-CDK9 expression.

The translocon Sec61beta localized in the inner nuclear membrane transports membrane-embedded EGF receptor to the nucleus.

Accumulating evidence indicates that endocytosis plays an essential role in the nuclear transport of the ErbB family members, such as epidermal growth factor receptor (EGFR) and ErbB-2. Nevertheless, how full-length receptors embedded in the endosomal membrane pass through the nuclear pore complexes and function as non-membrane-bound receptors in the nucleus remains unclear. Here we show that upon EGF treatment, the biotinylated cell surface EGFR is trafficked to the inner nuclear membrane (INM) through the nuclear pore complexes, remaining in a membrane-bound environment. We further find that importin ß regulates EGFR nuclear transport to the INM in addition to the nucleus/nucleoplasm. Unexpectedly, the well known endoplasmic reticulum associated translocon Sec61ß is found to reside in the INM and associate with EGFR. Knocking down Sec61ß expression reduces EGFR level in the nucleoplasm portion and accumulates it in the INM portion. Thus, the Sec61ß translocon plays an unrecognized role in the release of the membrane-anchored EGFR from the lipid bilayer of the INM to the nucleus. The newly identified Sec61ß function provides an alternative pathway for nuclear transport that can be utilized by membrane-embedded proteins such as full-length EGFR.

Ovarian progesterone suppresses depression and anxiety-like behaviors by increasing the Lactobacillus population of gut microbiota in ovariectomized mice.

Depression and anxiety, which are severe symptoms during menopause, are caused by ceased ovarian activity and declined serum progesterone levels. Studies have demonstrated that gut microbiota can regulate brain function and change the microbiota composition during the perimenopause period. This study investigated whether progesterone affects depressant and anxious behaviors via gut microbiota. In ovariectomized (OVX) mice, treatment with progesterone improved depressive and anxious behaviors, and gut microbiota composition was significantly changed. In particular, increased Lactobacillus spp. were observed in these mice. Reduction of microbiota by antibiotic treatment abolished the effect of progesterone on depression and anxiety. In addition, administration of Lactobacillus (L.) reuteri that was increased by progesterone also reduced the depressant behavior in OVX mice, and BDNF gene expression was elevated by progesterone treatment and L. reuteri administration in the hippocampus. Moreover, we found that progesterone stimulated the growth of L. reuteri in vitro. In summary, our findings indicate that progesterone reduces depression and anxiety through changes in gut microbiota composition, particularly by increasing the Lactobacillus spp. population.

TYRO3 induces anti-PD-1/PD-L1 therapy resistance by limiting innate immunity and tumoral ferroptosis.

Immune checkpoint blockade therapy has demonstrated promising clinical outcomes for multiple cancer types. However, the emergence of resistance as well as inadequate biomarkers for patient stratification have largely limited the clinical benefits. Here, we showed that tumors with high TYRO3 expression exhibited anti-programmed cell death protein 1/programmed death ligand 1 (anti-PD-1/PD-L1) resistance in a syngeneic mouse model and in patients who received anti-PD-1/PD-L1 therapy. Mechanistically, TYRO3 inhibited tumor cell ferroptosis triggered by anti-PD-1/PD-L1 and facilitated the development of a protumor microenvironment by reducing the M1/M2 macrophage ratio, resulting in resistance to anti-PD-1/PD-L1 therapy. Inhibition of TYRO3 promoted tumor ferroptosis and sensitized resistant tumors to anti-PD-1 therapy. Collectively, our findings suggest that TYRO3 could serve as a predictive biomarker for patient selection and a promising therapeutic target to overcome anti-PD-1/PD-L1 resistance.

Human ribonuclease 1 serves as a secretory ligand of ephrin A4 receptor and induces breast tumor initiation.

Human ribonuclease 1 (hRNase 1) is critical to extracellular RNA clearance and innate immunity to achieve homeostasis and host defense; however, whether it plays a role in cancer remains elusive. Here, we demonstrate that hRNase 1, independently of its ribonucleolytic activity, enriches the stem-like cell population and enhances the tumor-initiating ability of breast cancer cells. Specifically, secretory hRNase 1 binds to and activates the tyrosine kinase receptor ephrin A4 (EphA4) signaling to promote breast tumor initiation in an autocrine/paracrine manner, which is distinct from the classical EphA4-ephrin juxtacrine signaling through contact-dependent cell-cell communication. In addition, analysis of human breast tumor tissue microarrays reveals a positive correlation between hRNase 1, EphA4 activation, and stem cell marker CD133. Notably, high hRNase 1 level in plasma samples is positively associated with EphA4 activation in tumor tissues from breast cancer patients, highlighting the pathological relevance of the hRNase 1-EphA4 axis in breast cancer. The discovery of hRNase 1 as a secretory ligand of EphA4 that enhances breast cancer stemness suggests a potential treatment strategy by inactivating the hRNase 1-EphA4 axis.

High speed digital protein interaction analysis using microfluidic single molecule detection system.

The understanding of protein interaction dynamics is important for signal transduction research but current available techniques prove difficult in addressing this issue. Thus, using the microfluidic approach, we developed a digital protein analytical platform and methodology named MAPS (Microfluidic system Analyzing Protein in Single complex) that can measure the amount of target proteins and protein complexes at the digitally single molecule resolution. By counting protein events individually, this system can provide rough protein interaction ratios which will be critical for understanding signal transduction dynamics. In addition, this system only requires less than an hour to characterize the target protein sample, which is much quicker than conventional approaches. As a proof of concept, we have determined the interaction ratios of oncogenic signaling protein complexes EGFR/Src and EGFR/STAT3 before and after EGF ligand stimulation. To the best of our knowledge, this is the first time that the interaction ratio between EGFR and its downstream proteins has been characterized. The information from MAPS will be critical for the study of protein signal transduction quantitation and dynamics.