MLIP and its Influence on Key Oncogenic Pathways: Implications for Cancer Therapeutics

1. Introduction

Eukaryotic cells maintain cellular homeostasis through an extensive array of sensory mechanisms to respond and adapt to both intrinsic and extrinsic stimuli and insults. This involves the regulation of various cellular processes, including metabolism, DNA repair, and cell cycle progression. Disruptions to cellular homeostasis can lead to the development of various diseases, including cancer. Cancer is a devasting disease with metabolic remodeling and dysregulated homeostasis as distinctive feature of cancer ¹.

Cancer is characterized by uncontrolled cell growth and division, leading to the formation of tumors². This process is the result of a disruption in the delicate balance between cell proliferation and cell death³, which is normally maintained by cellular homeostasis. Several signaling molecules and pathways have been identified as pro-oncogenic and have therefore been targeted for the therapeutic treatment of cancer⁴.

In cancer cells, however, mutations or changes in the genes encoding proteins in this pathway can cause hyperactivation, resulting in uncontrolled cell growth and resistance to apoptosis (programmed cell death). For examples, the phosphoinositide 3-kinase/Protein kinase B (PI3K/Akt) / mammalian target of rapamycin (mTOR) pathway is tightly regulated in normal cells, ensuring a balance between cell growth and death. however, two Tumor suppressors p53 (p53) and Forkhead box O family (FOXO), downstream of the PI3K/Akt/mTOR pathway, respectively, are critical integrators of genomic and metabolic stresses ^5–8. Both p53 and FOXO are stress-activated transcription factors that promote a pro-survival adaptive response to insult. Specifically, p53 stimulates DNA repair in response to DNA damage and FOXO regulates metabolic remodeling to maintain metabolic homeostasis. The loss of p53 and FOXO normal function is associated with tumorigenesis in a wide variety of tissues. Because of the importance of the PI3K/Akt/mTOR pathway to the propagation of tumorigenesis, a number of specific inhibitors targeting different components of this pathway have been developed.

Muscle enriched A-type Lamin-Interacting Protein (MLIP) is a novel protein of unknown structure and function, that is required for proper cardiac and skeletal muscle adaptation to stress ^9–14. MLIP is a crucial mediator of cardiac adaptation through its interaction with the Akt/mTOR pro-survival pathway ¹¹, FOXO1 ¹⁴ and p53 ¹¹. Detailed comparative pathway analysis based on global gene expression differences between normal and MLIP deficient hearts has now revealed MLIP as a modulator of both p53 and FOXO activity. Given MLIP’s interaction with PI3K/Akt pathway, p53, and FOXO ^15,16. this review explores the role MLIP may play in tumor formation, progression and the potential of MLIP as a new therapeutic target.

2. MLIP expression in cancer

Limited research has focused on elucidating the role of MLIP in the initiation and/or progression of cancer. Our investigation identified two primary types of cancers where MLIP's role was emphasized: breast cancer and esophageal cancer (Table 1).

Breast cancer and esophageal cancer represent significant global health challenges, with the former being one of the most prevalent cancers among women and the latter noted for its particularly low survival rates ^17,18. The genetic underpinnings of these cancers are complex, and though substantial progress has been made in identifying key genetic risk factors, however a significant proportion of the genetic risk remains unexplained. Recent research has begun to shed light on this gap, with a particular focus on the role of copy number variants (CNVs) and differentially expressed genes. One gene that has emerged as a potential key player in both breast and esophageal cancer is the MLIP gene.

Expression of MLIP in different types of cancer

Breast cancer stands as one of the prevalent malignancies affecting women, with around 1 million new cases and over 400,000 reported deaths annually worldwide. In the year 2023, an estimated 297,790 women and 2,800 men are projected to be diagnosed with breast cancer¹⁹. While single nucleotide polymorphisms and mutations contribute to approximately 49% of the genetic risk associated with breast cancer^20,21, Kumaran and colleagues (2017) sought to uncover the remaining 51% by identifying germline Copy Number Variants (CNVs) linked to breast cancer²². Whole genome CNV genotyping was performed on 422 cases and 348 controls. Two hundred CNVs were identified to be associated with breast cancer of which 21 CNV regions overlapped with 22 genes. MLIP was identified as 1 of 6 genes associated breast cancer risk and recurrence-free survival ²². Specifically, Kumaran and colleagues reported a loss in MLIP CNVs was associated with significant reduction of breast cancer risk and recurrence- free survival with a reported hazard ratio of 0.62 [0.4–0.94] ¹⁸.

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer that is defined by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression. These receptors are commonly used as targets for breast cancer treatment, and the absence of these receptors in TNBC makes it more difficult to treat. Zhang and colleagues performed RNA-seq on 30 TNBC patient tumors, 15 of which had lymph node metastasis while the other 15 showed no lymph node metastasis ²³. Differential gene expression analysis was performed in order to determine the key genes involved in the progression and oncogenesis of TNBC ²³. The analysis revealed 2953 genes with differential expression in breast cancer tumors compared to normal control tissues and 975 genes with differential expression between 15 patients with lymph node metastasis and 15 patients without. A subset of 117 genes exhibited differential expression in both sets among those with and without lymph node metastasis in triple-negative breast cancer (TNBC), implying their involvement in TNBC oncogenesis and progression. MLIP, among the 117 genes of interest, was found to be upregulated in TNBC and exhibited a negative association with the cytotoxicity of CD8+ T cells²³.

Esophageal cancer is one of the most common malignancies, ranking 7th in global morbidity and 6th in cancer-related mortality. The 5-year overall survival rate is only about 15–20%, although progress has been made in diagnosis and treatment ¹⁷. To further define prognostic mRNAs of esophageal cancer, functional enrichment analyses of lncRNA, mRNA, and miRNA of 81 tumors and 11 normal controls was performed. MLIP was identified as one of 7 risk RNAs for esophageal cancer with a hazard ratio of 1.67 (1.22-2.29, p<0.001) ²⁴.

Finally, according to the recently found role of MLIP in cancer (Table 1), it has been suggested as a potential biomarker for triple-negative breast cancer, and esophageal cancer. However, more research is needed to fully understand MLIP's role in these cancers and its potential as a therapeutic target or diagnostic tool.

3. Molecular relationship of MLIP with pro-survival/oncogenic pathways and tumor suppressors

The intricate network of cellular signaling pathways that govern cell growth, proliferation, survival, and metabolism is often dysregulated in various cancer types, contributing to tumorigenesis and disease progression. Central to this network are the PI3K/Akt/mTOR, FOXO1, AMPK, p53, and Lamin A/C pathways, each playing critical roles in maintaining cellular homeostasis and responding to stress signals. Recently, MLIP has emerged as a key regulator within these pathways, influencing a variety of cellular processes and potentially playing a role in both cancer pathogenesis and cardiac disorders.

Primary role of AMPK function and dysfunction in cancer

Adenosine monophosphate-activated protein kinase (AMPK) serves as a pivotal enzyme governing cellular energy balance. Its primary function involves detecting shifts in cellular energy status, particularly reductions in ATP, and initiating processes that generate ATP while concurrently inhibiting ATP-consuming processes. AMPK functions as a heterotrimeric complex, comprising catalytic α subunits and regulatory β and γ subunits. The γ subunit accommodates binding sites for AMP and ATP, enabling AMPK to sense alterations in the AMP/ATP ratio and self-activate during energy depletion. AMPK activation triggers diverse downstream effects, including heightened glucose uptake, fatty acid oxidation, and mitochondrial biogenesis, along with diminished protein synthesis, lipogenesis, and gluconeogenesis. AMPK also influences autophagy, cell growth, proliferation, and inflammation^25,26. In response to stressors causing ATP depletion, such as hypoxia and glucose deprivation, AMPK activity is heightened ^25,26. Additionally, stimulating AMPK in skeletal muscle enhances glucose uptake and fatty acid oxidation while reducing lipid accumulation and inflammation²⁷. These findings, combined with other research, collectively underscore the crucial role of AMPK in governing energy metabolism and cellular function.

The precise function of AMPK in cancer cells is complicated and relies on the specific context of AMPK activation. In certain instances, AMPK activation can serve as a tumor suppressor by restraining cell growth, curbing proliferation, and encouraging apoptosis. However, in other scenarios, AMPK activation might support the survival of tumor cells by facilitating metabolic adaptation to the unique conditions of the tumor microenvironment. Hence, targeting AMPK activation could be a problematic or promising approach for cancer treatment (Figure 1) ²⁸. Additionally, research indicates that combining AMPK activation with other anticancer therapies like chemotherapy or radiation has the potential to augment their effectiveness ^28,29.

The study by Cattin et al. in 2015 sheds light on the molecular mechanisms underlying the reduced glucose uptake observed in MLIP-deficient cardiac tissues¹¹. In MLIP-deficient hearts, AMPK was reported to undergo dephosphorylation at AMPK alpha-Thr-172, a crucial step leading to the deactivation of the AMPK complex and subsequently resulting in decreased glucose uptake compared to normal cardiac tissues¹¹. Remarkably, this deactivation of AMPK occurred despite similar activity in Liver kinase B1 (LKB1), the kinase responsible for AMPK activation³⁰, indicating an LKB1-independent inactivation of AMPK in MLIP-deficient hearts.

Interactions between MLIP and AMPK may hold implications for cancer biology. AMPK, recognized as a metabolic tumor suppressor, hampers cell growth and proliferation during low energy conditions, thereby impeding the uncontrolled cell growth characteristic of cancer³¹. Consequently, the observed reduction in AMPK activation in the absence of MLIP might potentially elevate the risk of unregulated cell growth and proliferation, contributing to oncogenesis. Furthermore, the decline in AMPK levels in MLIP-deficient cardiac tissues led to the heightened activation of the Akt/mTOR pathway^11,32. This pathway significantly influences cell growth, proliferation, and survival, and its dysregulation is commonly observed in various types of cancers. These findings suggest that MLIP could potentially modulate these crucial pathways, thereby influencing cancer biology^11,31,32. However, it is crucial to acknowledge that these observations were made specifically in cardiac tissue, and it remains uncertain whether similar mechanisms would apply to other tissues or cancer cells. Additional research is required to directly investigate the involvement of MLIP in cancer biology.

The PI3K/Akt/mTOR pathway and MLIP

The PI3K/Akt/mTOR pathway is a key signaling pathway that regulates various cellular processes, including cell growth, proliferation, survival, and metabolism. Dysregulation of these pathways is commonly observed in many types of cancer, and its activation has been shown to contribute to cancer development and progression (Figure 2) ^33–36. In cancer cells, the PI3K/Akt/mTOR pathway can become activated through several mechanisms, including mutation of genes encoding components of the pathway, activation of upstream growth factor receptors, and loss of negative regulators of the pathway³⁴. Activation of the pathway can lead to increased cell proliferation, survival, and resistance to cell death signals, which contribute to tumor growth and progression. Targeting the PI3K/Akt/mTOR pathway (Table 2) has emerged as a promising strategy for cancer treatment^35,36. Several drugs that target components of the pathway are currently being developed and tested in preclinical and clinical studies, and some have shown promising results in certain types of cancer^33,36. However, targeting this pathway can also have side effects, and there is ongoing research to develop more effective and selective therapies that minimize toxicity while maximizing anti-cancer activity^33,35,36.

⁸The documented association between MLIP and the PI3K/AKT/mTOR signaling pathway is evident in research findings that highlight MLIP's direct impact on this pathway. Specifically, the absence of MLIP leads to the selective hyperactivation of the Akt/mTOR signaling pathway in cardiac cells (Figure 3)¹¹. Conversely, MLIP overexpression results in the inhibition of this pathway. The study demonstrates that the hyperactivation of Akt/mTOR occurs in cardiac cells when MLIP is absent¹¹. These results suggest that a deficiency in MLIP may potentially contribute to an accelerated aging phenomenon within cardiac cells, heightening susceptibility to tumor development⁸

Role of MLIP in FOXO1 signaling

FOXO genes are a subgroup of the forkhead family of transcription factors that play a critical role in regulating various cellular processes, including cell cycle control, DNA repair, apoptosis, and oxidative stress response ^37–40. Dysregulation of FOXO gene expression or activity has been reported to be associated with development and progression of cancer ⁴¹. There are four members of the FOXO family in mammals: FOXO1, FOXO3, FOXO4, and FOXO6. Among these, FOXO1 and FOXO3 are the most well-studied in the context of cancer (Table 3).

In normal cells, FOXO1 and FOXO3 are often activated in response to cellular stress, leading to the expression of target genes that promote cell cycle arrest, DNA repair, and apoptosis. This helps to prevent the development of cancer by eliminating cells with damaged DNA⁴². However, in cancer cells, the activity of FOXO1 and FOXO3 is often dysregulated ^43–45. In tumors, FOXO expression or activity is often suppressed to promote cell proliferation and survival, or alternatively FOXO may be activated to promote cell migration and invasion ^43–45.

FOXO1 has been found to play a role in the regulation of estrogen receptor (ER) signaling. In breast cancer, the loss of FOXO1 activity has been associated with resistance to endocrine therapy, while overexpression of FOXO1 has been shown to sensitize breast cancer cells to endocrine therapy ⁴⁶. Likewise, in prostate cancer, FOXO3 has been identified as a participant in the control of androgen receptor signaling⁴⁵. Reduced FOXO3 activity has been linked to resistance against androgen deprivation therapy, whereas increased FOXO3 expression has demonstrated the ability to enhance the sensitivity of prostate cancer cells to this therapy ⁴⁶

Transcripts of Foxo-1 have been demonstrated to contribute to cardiac remodeling ^6,47. FOXO1 acts as an inhibitor of calcineurin-mediated adverse cardiac remodeling, which promotes hypertrophic responses and contributes to heart failure ^6,47. Notably, the deletion of MLIP has also been linked to the downregulation of the FOXO1 pathway ^11,14. This suggests that the transcription factor FOXO-1 operates as a downstream signal of MLIP ¹¹

Although the precise mechanism through which MLIP increases FOXO-1 expression remains unknown, FOXO-1 is acknowledged for its involvement in cell cycle arrest, apoptosis, and tumor suppression, implying a potential role of MLIP in cancer pathogenesis. The activation of FOXO1 prompts the transcription of the cyclin-dependent kinase inhibitor p27^KIP1 while suppressing the transcription of cyclin D1 and D2. Both effects result in cell cycle arrest at G1. The loss of one allele of FOXO may render cells susceptible to dysregulated cell cycle events, triggering tumor formation. Activation of MLIP may mitigate the impact of FOXO haploinsufficiency on tumorigenesis ⁴⁰

P53 and MLIP

The p53 gene functions as a crucial tumor suppressor, actively preventing cancer development by regulating various cellular processes, including DNA repair, cell cycle arrest, apoptosis, and senescence^7,8,48–51. In response to DNA damage, p53 is activated, enabling it to pause the cell cycle for DNA repair or initiate apoptosis to eliminate damaged cells. In cancer, the p53 gene is frequently mutated or deleted, resulting in the loss of its tumor suppressor function^52,53. Mutations in p53 represent one of the most prevalent genetic alterations in cancer, with up to 50% of all human cancers exhibiting p53 mutations^54,55. The functional loss of p53 contributes to cancer development and progression by allowing the proliferation of damaged cells, facilitating the accumulation of additional genetic changes that can lead to cancer formation.

Beyond its role in DNA damage response, p53 also participates in the regulation of cellular metabolism^56–58. P53 has been demonstrated to influence the expression of genes involved in glycolysis, oxidative phosphorylation^59,60, and fatty acid metabolism^56,61. P53 loss or mutation can contribute to the metabolic rewiring commonly observed in cancer cells⁶². MLIP deficient hearts were found to have increased activation of p53¹¹, indicating that MLIP-deficient hearts may be experiencing genotoxic and/or metabolic stress. However, the activation of p53 is triggered by other genes and is crucial for its role as a tumor suppressor. The specific mass of p53 is less significant than the quantity of activated p53, as only the activated form can bind to DNA and initiate the expression of its target genes⁸. This implies a potential alternative function of MLIP, wherein it may promote tumor formation by inhibiting p53, a critical tumor suppressor gene. Alternatively, MLIP inhibition might impact p53 function by influencing other genes associated with p53 activation. Investigating such a role could provide novel insights into the impact of MLIP on cancer through potential manipulation of p53 function.

4. Conclusion, MLIP as a potential therapeutic target

MLIP is an emerging factor implicated in the regulation of key signaling pathways that govern cell growth, proliferation, survival, and metabolism, which are often dysregulated in cancer. Through its interactions with the PI3K/Akt/mTOR pathway, MLIP appears to exert an inhibitory effect¹¹. Overexpression of MLIP leads to the downregulation of this pathway, while its loss results in the pathway's overactivation^11,14,63,64. This implies that MLIP might act as a suppressor of cell growth and proliferation, two key processes that are often hyperactivated in cancer. Therefore, therapies aimed at enhancing MLIP expression or its regulatory effect on the PI3K/Akt/mTOR pathway might be beneficial for inhibiting cancer progression.

Moreover, MLIP appears to be involved in the regulation of FOXO1 signaling¹⁴, a pathway that plays a critical role in cell cycle control, apoptosis, and DNA repair - processes that are crucial for maintaining genomic integrity and preventing tumorigenesis. Dysregulation of FOXO1 signaling is often associated with cancer progression. Given that the deletion of MLIP leads to a downregulation of the FOXO1 pathway, and overexpression of MLIP is likely to have the opposite effect, therapeutics aimed at enhancing MLIP function or expression could potentially restore the normal function of FOXO1 signaling, thereby inhibiting cancer development and progression.

Additionally, MLIP's interactions with p53¹¹, a well-known tumor suppressor gene, further underscore its potential as a therapeutic target. Given that MLIP deficient cardiomyocytes showed an increased expression of p53, it is plausible to hypothesize that MLIP could play a role in the regulation of p53, and by extension, cell cycle control and apoptosis.

However, it is essential to remember that the exact mechanisms of MLIP in these signaling pathways are not fully understood, and further research is necessary to establish MLIP as a therapeutic target. Furthermore, it's crucial to understand the potential off-target effects and safety profile of any MLIP-targeting therapies due to MLIP's role in non-cancerous cells and processes, such as cardiac function. In summary, the modulation of MLIP's function or its interactions with key signaling pathways presents a promising approach for the development of novel cancer therapeutics.