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In normal embryogenesis and homeostasis, the canonical WNT pathway is activated by secreted WNT ligands produced in highly controlled context-dependent manners and in precise amounts. WNT activity is transduced in the cytoplasm, inactivates the APC destruction complex, and results in the translocation of activate β-CATENIN to the nucleus, where it cooperates with DNA-binding TCF/LEF factors to regulate WNT-TCF targets and the ensuing genomic response

beyond the loss of activity of the APC destruction complex, for instance throughAPC mutation, phosphorylation of β-CATENIN at C-terminal sites is required for the full activation of WNT-TCF signaling and the ensuing WNT-TCF responses in cancer.

The WNT-TCF response blockade that we describe for low doses of Ivermectin suggests an action independent to the deregulation of chloride channels

involve the repression of the levels of C-terminally phosphorylated β-CATENIN forms and of CYCLIN D1, a critical target that is an oncogene and positive cell cycle regulator.

the Avermectin single-molecule derivative Selamectin, a drug widely used in veterinarian medicine (Nolan & Lok, 2012), is ten times more potent acting in the nanomolar range

Ivermectin also diminished the protein levels of CYCLIN D1, a direct TCF target and oncogene, in both HT29 and H358 tumor cells

Activated Caspase3 was used as a marker of apoptosis by immunohistochemistry 48 h after drug treatment. Selamectin and Ivermectin induced up to a sevenfold increase in the number of activated Caspase3+ cells in two primary (CC14 and CC36) and two cell line (DLD1 and Ls174T) colon cancer cell types (Fig​(Fig2C).2C). All changes were significative

The strong downregulation of the expression of the intestinal stem cell genesASCL2 andLGR5 (van der Flieret al, 2009; Scheperset al, 2012; Zhuet al, 2012b) by Ivermectin and Selamectin (Fig​(Fig2D)2D) raised the possibility that these drugs could affect WNT-TCF-dependent colon cancer stem cell behavior

Pre-established H358 tumors responded to Ivermectin showing a ˜ 50% repression of growth

Ivermectin hasin vivo efficacy against human colon cancer xenografts sensitive to TCF inhibition with no discernable side effects

Ivermectin (Campbellet al, 1983), an off-patent drug approved for human use, and related macrocyclic lactones, have WNT-TCF pathway response blocking and anti-cancer activities

these drugs block WNT-TCF pathway responses, likely acting at the level of β-CATENIN/TCF function, affecting β-CATENIN phosphorylation status.

anti-WNT-TCF activities of Ivermectin and Selamectin

Ivermectin has a well-known anti-parasitic activity mediated via the deregulation of chloride channels, leading to paralysis and death (Hibbs & Gouaux, 2011; Lynagh & Lynch, 2012). The same mode of action has been suggested to underlie the toxicity of Ivermectin for liquid tumor cells and the potentiation or sensitization effect of Avermectin B1 on classical chemotherapeutics

the specificity of the blockade of WNT-TCF responses we document, at low micromolar doses for Ivermectin and low nanomolar doses for Selamectin, indicate that the blockade of WNT-TCF responses and chloride channel deregulation are distinct modes of action

What is key then is to find a dose and a context where the use of Ivermectin has beneficial effects in patients, paralleling our results with xenografts in mice.

Cell toxicity appears at doses greater (> 10 μM for 12 h or longer or > 5 μM for 48 h or longer for Ivermectin) than those required to block TCF responses and induce apoptosis.

Our data point to a repression of WNT-β-CATENIN/TCF transcriptional responses by Ivermectin, Selamectin and related macrocylic lactones.

(i) The ability of Avermectin B1 to inhibit the activation of WNT-TCF reporter activity by N-terminal mutant (APC-insensitive) β-CATENIN as detected in our screen

(ii) The ability of Avermectin B1, Ivermectin, Doramectin, Moxidectin and Selamectin to parallel the modulation of WNT-TCF targets by dnTCF

(iii) The finding that the specific WNT-TCF response blockade by low doses of Ivermectin and Selamectin is reversed by constitutively active TCF

(iv) The repression of key C-terminal phospho-isoforms of β-CATENIN resulting in the repression of the TCF target and positive cell cycle regulator CYCLIN D1 by Ivermectin and Selamectin

(v) The specific inhibition ofin-vivo-TCF-dependent, but notin-vivo-TCF-independent cancer cells by Ivermectin in xenografts.

These results together with the reduction of the expression of the colon cancer stem cell markersASCL2 andLGR5 (e.g., Hirschet al, 2013; Ziskinet al, 2013) raise the possibility of an inhibitory effect of Ivermectin, Selamectin and related macrocyclic lactones on TCF-dependent cancer stem cells.

the capacity of cancer cells to form 3D spheroids in culture, as well as the growth of these, is also WNT-TCF-dependent (Kanwaret al, 2010) and they were also affected by Ivermectin treatment

If Ivermectin is specific, it should only block TCF-dependent tumor growth. Indeed, the sensitivity and insensitivity of DLD1 and CC14 xenografts to Ivermectin treatment, respectively, together with the desensitization to Ivermectin actionin vivo by constitutively active TCF provide evidence of the specificity of this drug to block an activated WNT-TCF pathway in human cancer.

Ivermectin has a good safety profile since onlyin-vivo-dnTCF-sensitive cancer xenografts are responsive to Ivermectin treatment, and we have not detected side effects in Ivermectin-treated mice at the doses used

previous work has shown that side effects from systemic treatments with clinically relevant doses in humans are rare (Yang, 2012), that birth defects were not observed after exposure of pregnant mothers (Pacquéet al, 1990) and that this drug does not cross the blood–brain barrier (Kokozet al, 1999). Similarly, only dogs with mutantABCB1 (MDR1) alleles leading to a broken blood–brain barrier show Ivermectin neurotoxicity (Mealeyet al, 2001; Orzechowskiet al, 2012)

Indications may include treatment for incurable β-CATENIN/TCF-dependent advanced and metastatic human tumors of the lung, colon, endometrium, and other organs.

Ivermectin, Selamectin, or related macrocyclic lactones could also serve as topical agents for WNT-TCF-dependent skin lesions and tumors such as basal cell carcinomas

they might also be useful as routine prophylactic agents, for instance against nascent TCF-dependent intestinal tumors in patients with familial polyposis and against nascent sporadic colon tumors in the general aging population

others studies suggested that baseline 18FDG uptake, which would avoid performing a second examination, could also be of interest to predict patient outcome, especially in ER+/HER2- BC

ER+/HER2- BC has less intense 18FDG uptake than some other phenotypes such as TN carcinoma

Patients with high baseline 18FDG tumor uptake are at higher risk of early recurrence

The 3-year EFS was 78.4% in patients with baseline tumor SUVmax > 8.3 (vs. 94.0% in those with SUVmax ≤ 8.3)

event-free survival (EFS)

The association between FDG uptake and tumor burden or stage has been well documented

advanced tumors tend to have higher FDG uptake (and thus higher SUV values)

the impact of the SUV on treatment outcome has been observed even within a given tumor stage

primary tumor SUVmax >10 predicted survival, independent of the tumor stage and diameter

FDG uptake not only reflects tumor burden/stage but also expresses, at least in part, some intrinsic biologic characteristic(s) of the tumor

SUVmax reflects the highest voxel value within the ROI or VOI. It is the most widely used parameter to measure metabolic tumor activity in oncologic FDG-PET/CT imaging

Several studies suggest that primary tumor baseline SUVmax also has predictive value in assessing the tumor burden, lymph node involvement and local extension

According to EORTC, a drop (delta between baseline and post-therapy) of 15–25% in SUVmax may represent a good treatment response

PERCIST criteria was proposed by the investigators at the Johns Hopkins Medical Institutions and suggested that a decrease in SUV normalized to lean body mass of at least 30% should be achieved before considering partial tumor response

The side effects were transient and returned to baseline following treatment

Tumors do not express IL-2 receptors and thus the antitumor activity was the result of IL-2 stimulation of immune cell

Patients with metastatic melanoma or metastatic renal cell cancer were uniquely responsive to high-dose IL-2 administration, and except for patients with advanced non-Hodgkin’s lymphomas (35) only rare responses were seen in patients with other tumor types

The underlying toxicity of IL-2 results from a capillary leak that leads to fluid extravasation into visceral organs that can compromise their function

Eighty-five percent of lung cancers are non-small-cell lung cancer (NSCLC)

Vitamin C inhibits GAPDH (a glycolytic enzyme) and depletes the cellular pool of glutathione, resulting in high ROS production and oxidative stress

DoxyR CSCs are between 4- to 10-fold more susceptible to the effects of Vitamin C

Doxycycline and Vitamin C may represent a new synthetic lethal drug combination for eradicating CSCs, by ultimately targeting both mitochondrial and glycolytic metabolism

inhibiting their propagation in the range of 100 to 250 µM

metabolic flexibility in cancer cells allows them to escape therapeutic eradication, leading to chemo- and radio-resistance

used doxycycline to pharmacologically induce metabolic inflexibility in CSCs, by chronically inhibiting mitochondrial biogenesis

This treatment resulted in a purely glycolytic population of surviving cancer cells

DoxyR cells are mainly glycolytic

MCF7 cells survive and develop Doxycycline-resistance, by adopting a purely glycolytic phenotype

Cancer stem cells (CSCs) are thought to be the “root cause” of tumor recurrence, distant metastasis and therapy-resistance

the conserved evolutionary similarities between aerobic bacteria and mitochondria, certain classes of antibiotics inhibit mitochondrial protein translation, as an off-target side-effect

Vitamin C was more potent than 2-DG; it inhibited DoxyR CSC propagation by > 90% at 250 µM and 100% at 500 µM

IC-50

DoxyR CSCs are between 4- to 10-fold more sensitive to Vitamin C than control MCF7 CSCs

Berberine, which is a naturally occurring antibiotic that also behaves as an OXPHOS inhibitor

treatment with Berberine effectively inhibited the propagation of the DoxyR CSCs by > 50% at 1 µM and > 80% at 10 µM.

Doxycycline, a clinically approved antibiotic, induces metabolic stress in cancer cells. This allows the remaining cancer cells to be synchronized towards a purely glycolytic phenotype, driving a form of metabolic inflexibility

Doxycycline-driven aerobic glycolysis

new synthetic lethal strategy for eradicating CSCs, by employing i) Doxycycline (to target mitochondria) and ii) Vitamin C (to target glycolysis)

Doxycycline inhibits mitochondrial biogenesis and OXPHOS,

hibits glycolytic metabolism by targeting and inhibiting the enzyme GAPDH

CSCs act as the main promoter of tumor recurrence and patient relapse

a metabolic shift from oxidative to glycolytic metabolism represents an escape mechanism for breast cancer cells chronically-treated with a mitochondrial stressor like Doxycycline, as mitochondrial dys-function leads to a stronger dependence on glucose

Vitamin C has been demonstrated to selectively kill cancer cells in vitro and to inhibit tumor growth in experimental mouse models

many of these actions have been attributed to the ability of Vitamin C to act as a glycolysis inhibitor, by targeting GAPDH and depleting the NAD pool

here we show that DoxyR CSCs are more vulnerable to the inhibitory effects of Vitamin C, at 4- to 10-fold lower concentrations, between 100 to 250 μM

concurrent use of Vitamin C, with standard chemotherapy, reduces tumor recurrence and patient mortality

after oral administration, Vitamin C plasma levels reach concentrations of ~70-220 μM

intravenous administration results in 30- to 70- fold higher plasma concentrations of Vitamin C

pro-oxidant activity results from Vitamin C’s action on metal ions, which generates free radicals and hydrogen peroxide, and is associated with cell toxicity

it has been shown that high-dose Vitamin C is more cytotoxic to cancer cells than to normal cells

This selectivity appears to be due to the higher catalase content observed in normal cells (~10-100 fold greater), as compared to tumor cells. Hence, Vitamin C may be regarded as a safe agent that selectively targets cancer cells

the concurrent use of Doxycycline and Vitamin C, in the context of this infectious disease, appeared to be highly synergistic in patients

Goc et al., 2016, showed that Doxycycline is synergistic in vitro with certain phytochemicals and micronutrients, including Vitamin C, in the in vitro killing of the vegetative spirochete form of Borrelia spp., the causative agent underlying Lyme disease

Doxycycline, an FDA-approved antibiotic, behaves as an inhibitor of mitochondrial protein translation

CSCs successfully escape from the anti-mitochondrial effects of Doxycycline, by assuming a purely glycolytic phenotype. Therefore, DoxyR CSCs are then more susceptible to other metabolic perturbations, because of their metabolic inflexibility

whereas the expression of SVCT-2 is ubiquitous

differential sensitivity to VC may result from variations in VC flow into cells, which is dependent on SVCT-2 expression.

high-dose VC significantly impaired both the tumorspheres initiation (Fig. 4d, e) and the growth of established tumorspheres derived from HCC cells (Fig. 4f, g) in a time-dependent and dose-dependent manner.

Hepatocellular carcinoma (HCC)

The antioxidant, N-acetyl-L-cysteine (NAC), preventing VC-induced ROS production (a ROS scavenger), completely restored the viability and colony formation among VC-treated cells

DNA double-strand damage was found following VC treatment

DNA damage was prevented by NAC

Interestingly, the combination of VC and cisplatin was even more effective in reducing tumor growth and weight

Consistent with the in vitro results, stemness-related genes expressions in tumor xenograft were remarkably reduced after VC or VC+cisplatin treatment, whereas conventional cisplatin therapy alone led to the increase of CSCs

VC is one of the numerous common hepatoprotectants.

Interestingly, at extracellular concentrations greater than 1 mM, VC induces strong cytotoxicity to cancer cells including liver cancer cells

we hypothesized that intravenous VC might reduce the risk of recurrence in HCC patients after curative liver resection.

Intriguingly, the 5-year disease-free survival (DFS) for patients who received intravenous VC was 24%, as opposed to 15% for no intravenous VC-treated patients

Median DFS time for VC users was 25.2 vs. 18 months for VC non-users

intravenous VC use is linked to improved DFS in HCC patients.

In this study, based on the elevated expression of SVCT-2, which is responsible for VC uptake, in liver CSCs, we revealed that clinically achievable concentrations of VC preferentially eradicated liver CSCs in vitro and in vivo

Additionally, we found that intravenous VC reduced the risk of post-surgical HCC progression in a retrospective cohort study.

Three hundred thirty-nine participants (55.3%) received 2 g intravenous VC for 4 or more days after initial hepatectomy

As the key protein responsible for VC uptake in the liver, SVCT-2 played crucial roles in regulating the sensitivity to ascorbate-induced cytotoxicity

we also observed that SVCT-2 was highly expressed in human HCC samples and preferentially elevated in liver CSCs

SVCT-2 might serve as a potential CSC marker and therapeutic target in HCC

CSCs play critical roles in regulating tumor initiation, relapse, and chemoresistance

we revealed that VC treatment dramatically reduced the self-renewal ability, expression levels of CSC-associated genes, and percentages of CSCs in HCC, indicating that CSCs were more susceptible to VC-induced cell death

as a drug for eradicating CSCs, VC may represent a promising strategy for treatment of HCC, alone or particularly in combination with chemotherapeutic drugs

In HCC, we found that VC-generated ROS caused genotoxic stress (DNA damage) and metabolic stress (ATP depletion), which further activated the cyclin-dependent kinase inhibitor p21, leading to G2/M phase cell cycle arrest and caspase-dependent apoptosis in HCC cells

we demonstrated a synergistic effect of VC and chemotherapeutic drug cisplatin on killing HCC both in vitro and in vivo

Intravenous VC has also been reported to reduce chemotherapy-associated toxicity of carboplatin and paclitaxel in patients,38 but the specific mechanism needs further investigation

Our retrospective cohort study also showed that intravenous VC use (2 g) was related to the improved DFS in HCC patients after initial hepatectomy

several clinical trials of high-dose intravenous VC have been conducted in patients with advanced cancer and have revealed improved quality of life and prolonged OS

high-dose VC was not toxic to immune cells and major immune cell subpopulations in vivo

high recurrence rate and heterogeneity

tumor progression, metastasis, and chemotherapy-resistance

SVCT-2 was highly expressed in HCC samples in comparison to peri-tumor tissues

high expression (grade 2+/3+) of SVCT-2 was in agreement with poorer overall survival (OS) of HCC patients (Fig. 1c) and more aggressive tumor behavior

SVCT-2 is enriched in liver CSCs

these data suggest that SVCT-2 is preferentially expressed in liver CSCs and is required for the maintenance of liver CSCs.

pharmacologic concentrations of plasma VC higher than 0.3 mM are achievable only from i.v. administration

The viabilities of HCC cells were dramatically decreased after exposure to VC in dose-dependent manner

VC and cisplatin combination further caused cell apoptosis in tumor xenograft

These results verify that VC inhibits tumor growth in HCC PDX models and SVCT-2 expression level is associated with VC response

qPCR and IHC analysis demonstrated that expression levels of CSC-associated genes and percentages of CSCs in PDXs dramatically declined after VC treatment, confirming the inhibitory role of VC in liver CSCs

Hyperthermia differs fundamentally from fever in that it elevates the core body temperature without changing the physiological set point

hyperthermia is induced by increasing the heat load and/or inactivating heat dissipation

mor cells [2]. Although significant cell killing could be achieved by heating cells or tissues to temperatures > 42°C for 1 or more hours, the application, measurement and consistency of this temperature range within the setting of cancer clinical trials

mild temperature hyperthermia (ie, within the fever-range, 39–41°C)

moderate hyperthermia (41°C)

Hsps are a family of stress-induced proteins

they are key regulators of cellular protein activity, turnover and trafficking

Hsps ensure appropriate post-translational protein folding, and are able to refold denatured proteins, or mark irreversibly damaged proteins for destruction

the ability of fever-range hyperthermia to induce reactive immunity against tumor antigens through DCs and NK-cells is likely mediated by Hsps

thermotolerance

Hsps support the malignant phenotype of cancer cells by not only affecting the cells’ survival, but also participating in angiogenesis, invasion, metastasis and immortalization mechanisms

Hsps released from stressed or dying cells activate dendritic cells (DCs), transforming them into mature APCs

In theory, fever-range hyperthermia may take advantage of tumor cell Hsps by inducing their release from tumor cells and augmenting DC priming against tumor antigens

In several models of hyperthermia, heat-treated tumors exhibited improved DC priming and generation of systemic immunity to tumor cell

hyperthermia alone can enhance antigen display by tumor cells, thus rendering them even more susceptible to programmed immune clearance

Fever-range hyperthermia may also induce Hsps

Hsps may exert an adjuvant effect by bolstering MHC class II and co-stimulatory molecule expression by DCs

thermal ablation of liver tumors in particular has demonstrated an ability to potentiate immune responses [57, 58] and elicit robust T-cell infiltrates at ablation sites

specific Hsp, Hsp70, directly inhibits apoptosis pathways in cancer cells, as demonstrated in human pancreatic, prostate and gastric cancer cells

Cross-priming is the ability of extracellular Hsps complexed to tumor peptides to be internalized and presented in the context of MHC class I molecules on APCs, thus allowing potent priming of CTLs against tumor antigens

It has been reported that Hsps are generated from necrotic tumor cell lysates, but not from tumor cells undergoing apoptosis

tumor cells exposed to hyperthermia in the heat shock range (42°C for 4h) prior to lysing, DC activation and cross-priming were significantly enhanced with the application of heat

Due to the ability of Hsps to activate DCs directly by chaperoning tumor antigens upon their release [28], it is possible that both local and regional immune stimulation can be achieved with hyperthermia.

support the use of hyperthermia as an inducer of Hsps to serve as ‘danger signals’, activating antitumor immune responses

whole-body hyperthermia not only augments immune responses, but also stimulates the migration of skin-derived DCs to draining lymph nodes

suggest a valuable role of hyperthermia in DC cancer vaccine strategies

In mice treated with fever-range whole-body hyperthermia, tumor growth was significantly inhibited and NK-cell infiltration increased

exposure to fever-range hyperthermia resulted in improved endogenous NK-cell cytotoxicity to several cancer types

improved activation and function of DCs and NK cells following hyperthermia

Hyperthermia increases the expression ICAM-1 a key adhesion molecule,

The combined effects of hyperthermia on lymphoid tissue endothelium and lymphocytes can promote immune surveillance and increase the probability of naive lymphocytes leaving the circulation and encountering their cognate antigen displayed by DCs in lymphoid organs.

In independent clinical studies, whole-body hyperthermia resulted in a transient decrease in circulating lymphocytes in patients with advanced cancer [12, 94, 99, 100], a finding which mirrored observations in animal models in which lymphocyte entry into lymph noeds was increased following hyperthermia treatment [93]. Enhanced recruitment of lymphocytes to lymphoid tissues may be exploited in the treatment of malignancies.

The initial tumor antigen presentation and initiation of clonal expansion of CTLs transpires in the lymph nodes and cannot take place outside this specialized compartment

the ability of DCs present in the lymph nodes to stimulate an anti-tumor immune response is critical

hyperthermia has been shown to improve immune surveillance by T-cell

and to increase DC trafficking to lymph nodes

NK cells are large granular lymphocytes that comprise approximately 10 % of circulating lymphocytes

all human NK cells express the CD56 antigen

treatment with various concentrations of IL‐2 in vivo may induce distinct functions within the NK cell compartment and, therefore, may have profound effects on NK cell‐mediated cytotoxicity

CD56bright

CD56dim

We show here that ADCC conducted by NK cells in vitro is enhanced by IL‐2 activation and is critically dependent on interactions between FcγRIII on NK cells and Herceptin‐coated tumor targets

administration of low‐dose IL‐2 to patients results in the marked expansion of a CD56+ population of immune effectors with the ability to lyse antibody‐coated cancer targets

NK cells represented only 7 % of lymphocytes prior to therapy but comprised over 50 % of the population after 10 weeks of low‐dose IL‐2

These data suggest that the enhanced ADCC seen following the expansion of NK cells with low‐dose IL‐2 is likely due to an increase in the overall number of NK cells

co‐administration of IL‐2 with rhu4D5 mAb will enhance activation of NK cell effector functions

Stimulation of NK cells with IL‐2 resulted in a significant increase in the lysis of rhu4D5‐coated targets

We have shown that costimulation with IL‐2 plus rhu4D5 results in significant production of IFN‐γ by NK cells with concomitant up‐regulation of cell‐surface activation and adhesion molecules

It has been previously demonstrated that continuous low‐dose IL‐2 can expand a CD56+ lymphocyte population, and we have now shown that this cell population is a potent mediator of ADCC against rhu4D5 mAb‐coated Her2 / neu+ targets

These results suggest that administration of low‐dose IL‐2 can be used to expand NK cell numbers, while higher doses may be used to enhance their cytolytic capacity in the setting of mAb therapy

we have demonstrated that NK cell lysis of Her2 / neu+ breast cancer cell lines in the presence of rhu4D5 mAb is markedly enhanced following stimulation with IL‐2

we have presented evidence that administration of low‐dose IL‐2 in vivo results in the expansion of a potent NK cell effector population

Our experiments suggest that NK cells costimulated with IL‐2 and immobilized IgG can secrete potent immunomodulatory cytokines which may serve to potentiate the anti‐tumor immune response.

the ketogenic diet may inhibit cancer progression in part by indirect dietary energy restriction

KD with HBO2T. Combining these therapies nearly doubled survival time in mice with metastatic cancer,

low carbohydrate or ketogenic diets promote weight loss in overweight individuals, they are also known to spare muscle wasting during conditions of energy restriction and starvation

dietary-induced therapeutic ketosis in a cancer patient would prevent muscle wasting similarly as it does with athletes undergoing intense exercise

when given as an adjuvant treatment to advanced cancer patients, the KD improves quality of life and enhances the efficacy of chemotherapy treatment in the clinic

Vitamin C may serve as a pro-drug for hydrogen peroxide delivery to extravascular tissues, but without the presence of hydrogen peroxide in blood

not all cancer cells were killed by ascorbic acid in vitro

Intravascular hemolysis was reported after massive vitamin C administration in people with glucose-6-phosphate dehydrogenase deficiency

Administration of high-dose vitamin C to patients with systemic iron overload may increase iron absorption and represents a contraindication

Ascorbic acid is metabolized to oxalate, and 2 cases of acute oxalate nephropathy were reported in patients with pre-existing renal insufficiency given massive intravenous doses of vitamin C

Rare cases of acute tumour hemorrhage and necrosis were reported in patients with advanced cancer within a few days of starting high-dose intravenous vitamin C therapy, although this was not independently verified by pathologic review

Huperzine A (a natural cholinesterase inhibitor) has been linked to improved memory performance in elderly people with benign forgetfulness, as well as patients with AD and vascular dementia

Cholinesterase inhibitors have been shown to have neuroprotective properties in patients with mild [37] as well as moderate-to-advanced AD